Mycobacterium smegmatis infection of a prosthetic total knee arthroplasty.

PubMed

Saffo, Zaid; Ognjan, Anthony

2016-01-01

The most common organisms causing prosthetic knee joint infections are staphylococci. However, arthroplasty infections with atypical microbial pathogens, such as Mycobacteria can occur. Due to the rarity of mycobacterial prosthetic joint infections, diagnosis, treatment, and management of these atypical infections represent a clinical challenge. A 71-year old female post-operative day 40 after a left total knee arthroplasty was hospitalized secondary to left knee pain and suspected arthroplasty infection. She had failed outpatient oral antimicrobial treatment for superficial stitch abscess; and outpatient IV/Oral antimicrobials for a clinical postoperative septic bursitis. Ultimately, resection arthroplasty with operative tissue acid fast bacterial cultures demonstrated growth of the Mycobacterium smegmatis group. Post-operatively, she completed a combination course of oral doxycycline and levofloxacin and successfully completed a replacement arthroplasty with clinical and microbial resolution of the infection. To our knowledge, literature review demonstrates three case of knee arthroplasty infection caused by the Mycobacterium smegmatis group. Correspondingly, optimal surgical procedures and antimicrobial management including antimicrobial selection, treatment duration are not well defined. Presently, the best treatment options consists of two step surgical management including prosthesis hardware removal followed by extended antimicrobial therapy, followed by consideration for re-implantation arthroplasty. Our case illustrates importance of considering atypical mycobacterial infections in post-operative arthroplasty infections not responding to traditional surgical manipulations and antimicrobials. For an arthroplasty infection involving the atypical Mycobacterium smegmatis group, two step arthroplasty revision, including arthroplasty resection, with a combination of oral doxycycline and levofloxacin can lead to successful infection resolution, allowing for a successful replacement arthroplasty.

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

PubMed Central

Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

2001-01-01

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis. PMID:11717265

Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.

PubMed

Mo, Yongkai; Quanquin, Natalie M; Vecino, William H; Ranganathan, Uma Devi; Tesfa, Lydia; Bourn, William; Derbyshire, Keith M; Letvin, Norman L; Jacobs, William R; Fennelly, Glenn J

2007-10-01

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant-albeit only 1.7-fold-increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120(h)(E)) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120(h)(E). M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

Uptake of Sulfate but Not Phosphate by Mycobacterium tuberculosis Is Slower than That for Mycobacterium smegmatis

PubMed Central

Song, Houhui

2012-01-01

Knowledge of the metabolic pathways used by Mycobacterium tuberculosis during infection is important for understanding its nutrient requirements and host adaptation. However, uptake, the first step in the utilization of nutrients, is poorly understood for many essential nutrients, such as inorganic anions. Here, we show that M. tuberculosis utilizes nitrate as the sole nitrogen source, albeit at lower efficiency than asparagine, glutamate, and arginine. The growth of the porin triple mutant M. smegmatis ML16 in media with limiting amounts of nitrate and sulfate as sole nitrogen and sulfur sources, respectively, was delayed compared to that of the wild-type strain. The uptake of sulfate was 40-fold slower than that of the wild-type strain, indicating that the efficient uptake of these anions is dependent on porins. The uptake by M. tuberculosis of sulfate and phosphate was approximately 40- and 10-fold slower than that of M. smegmatis, respectively, which is consistent with the slower growth of M. tuberculosis. However, the uptake of these anions by M. tuberculosis is orders of magnitude faster than diffusion through lipid membranes, indicating that unknown outer membrane proteins are required to facilitate this process. PMID:22194452

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

Genome-Wide Transcriptome Profiling of Mycobacterium smegmatis MC2 155 Cultivated in Minimal Media Supplemented with Cholesterol, Androstenedione or Glycerol

PubMed Central

Li, Qun; Ge, Fanglan; Tan, Yunya; Zhang, Guangxiang; Li, Wei

2016-01-01

Mycobacterium smegmatis strain MC2 155 is an attractive model organism for the study of M. tuberculosis and other mycobacterial pathogens, as it can grow well using cholesterol as a carbon resource. However, its global transcriptomic response remains largely unrevealed. In this study, M. smegmatis MC2 155 cultivated in androstenedione, cholesterol and glycerol supplemented media were collected separately for a RNA-Sequencing study. The results showed that 6004, 6681 and 6348 genes were expressed in androstenedione, cholesterol and glycerol supplemented media, and 5891 genes were expressed in all three conditions, with 237 specially expressed in cholesterol added medium. A total of 1852 and 454 genes were significantly up-regulated by cholesterol compared with the other two supplements. Only occasional changes were observed in basic carbon and nitrogen metabolism, while almost all of the genes involved in cholesterol catabolism and mammalian cell entry (MCE) were up-regulated by cholesterol, but not by androstenedione. Eleven and 16 gene clusters were induced by cholesterol when compared with glycerol or androstenedione, respectively. This study provides a comprehensive analysis of the cholesterol responsive transcriptome of M. smegmatis. Our results indicated that cholesterol induced many more genes and increased the expression of the majority of genes involved in cholesterol degradation and MCE in M. smegmatis, while androstenedione did not have the same effect. PMID:27164097

Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

PubMed

Ayala-Torres, Carlos; Novoa-Aponte, Lorena; Soto, Carlos Y

2015-07-01

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155

PubMed Central

2010-01-01

Background The usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis. Results A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. smegmatis. An enzymatic cell surface shaving method was used to determine the surface-exposed proteins. As a result, a total of 390 cell wall proteins and 63 surface-exposed proteins were identified. Further analysis of the 390 cell wall proteins provided the theoretical molecular mass and pI distributions and determined that 26 proteins are shared with the surface-exposed proteome. Detailed information about functional classification, signal peptides and number of transmembrane domains are given next to discussing the identified transcriptional regulators, transport proteins and the proteins involved in lipid metabolism and cell division. Conclusion In short, a comprehensive profile of the M. smegmatis cell wall subproteome is reported. The current research may help the identification of some valuable vaccine and drug target candidates and provide foundation for the future design of preventive, diagnostic, and therapeutic strategies against mycobacterial diseases. PMID:20412585

Rv2358 and FurB: Two Transcriptional Regulators from Mycobacterium tuberculosis Which Respond to Zinc

PubMed Central

Canneva, Fabio; Branzoni, Manuela; Riccardi, Giovanna; Provvedi, Roberta; Milano, Anna

2005-01-01

In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc2155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn2+ ions. PMID:16077132

LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis

PubMed Central

Li, Weihui; He, Zheng-Guo

2012-01-01

In a bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)/transcription factor binding screen, we identified Mycobacterium smegmatis Ms6479 as the first c-di-GMP-responsive transcriptional factor in mycobacteria. Ms6479 could specifically bind with c-di-GMP and recognize the promoters of 37 lipid transport and metabolism genes. c-di-GMP could enhance the ability of Ms6479 to bind to its target DNA. Furthermore, our results establish Ms6479 as a global activator that positively regulates the expression of diverse target genes. Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics. Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs. Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator. PMID:23047950

The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation

PubMed Central

Sala, Claudia; Forti, Francesca; Magnoni, Francesca; Ghisotti, Daniela

2008-01-01

Background In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA. Results In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing. Conclusion This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The furA-katG mRNA is transcribed from the furA promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA. PMID:18394163

Adaptation of Mycobacterium smegmatis to Stationary Phase

PubMed Central

Smeulders, Marjan J.; Keer, Jacquie; Speight, Richard A.; Williams, Huw D.

1999-01-01

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells. PMID:9864340

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

PubMed

Singh, Upasana; Akhtar, Shamim; Mishra, Abhishek; Sarkar, Dhiman

2011-02-01

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. Copyright © 2010 Elsevier B.V. All rights reserved.

Synthesis and biological activity of alkynoic acids derivatives against mycobacteria

PubMed Central

Vilchèze, Catherine; Leung, Lawrence W.; Bittman, Robert; Jacobs, William R.

2015-01-01

2-alkynoic acids have bactericidal activity against Mycobacterium smegmatis but their activity fall sharply as the length of the carbon chain increased. In this study, derivatives of 2- alkynoic acids were synthesized and tested against fast- and slow-growing mycobacteria. Their activity was first evaluated in M. smegmatis against their parental 2-alkynoic acids, as well as isoniazid, a first-line antituberculosis drug. The introduction of additional unsaturation or heteroatoms into the carbon chain enhanced the antimycobacterial activity of longer chain alkynoic acids (more than 19 carbons long). In contrast, although the modification of the carboxylic group did not improve the antimycobacterial activity, it significantly reduced the toxicity of the compounds against eukaryotic cells. Importantly, 4-(alkylthio)but-2-ynoic acids, had better bactericidal activity than the parental 2-alkynoic acids and on a par with isoniazid against the slow-grower Mycobacterium bovis BCG. These compounds had also low toxicity against eukaryotic cells, suggesting that they could be potential therapeutic agents against other types of topical mycobacterial infections causing skin diseases including Mycobacterium abscessus, Mycobacterium ulcerans, and Mycobacterium leprae. Moreover, they provide a possible scaffold for future drug development. PMID:26256431

Some South African Rubiaceae Tree Leaf Extracts Have Antimycobacterial Activity Against Pathogenic and Non-pathogenic Mycobacterium Species.

PubMed

Aro, Abimbola O; Dzoyem, Jean P; Hlokwe, Tiny M; Madoroba, Evelyn; Eloff, Jacobus N; McGaw, Lyndy J

2015-07-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds, which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa, and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium bovis BCG (Bacillus Calmette-Guérin) using a twofold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. Minimum inhibitory concentration values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied, and Oxyanthus speciosus had the most promising selectivity index values. Copyright © 2015 John Wiley & Sons, Ltd.

IRAK4 activity controls immune responses to intracellular bacteria Listeria monocytogenes and Mycobacterium smegmatis.

PubMed

Pattabiraman, Goutham; Murphy, Michael; Agliano, Federica; Karlinsey, Keaton; Medvedev, Andrei E

2018-05-11

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4 KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4 KI macrophages expressed decreased TNF-α, IL-6, IL-1β, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4 KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4 KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO. ©2018 Society for Leukocyte Biology.

cGAS-STING-TBK1-IRF3/7 induced interferon-β contributes to the clearing of non tuberculous mycobacterial infection in mice.

PubMed

Ruangkiattikul, Nanthapon; Nerlich, Andreas; Abdissa, Ketema; Lienenklaus, Stefan; Suwandi, Abdulhadi; Janze, Nina; Laarmann, Kristin; Spanier, Julia; Kalinke, Ulrich; Weiss, Siegfried; Goethe, Ralph

2017-10-03

Type I interferons (IFN-I), such as IFN-α and IFN-β are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-β than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-β in macrophages. Both bacteria induced IFN-β via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-β activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-β impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.

cGAS-STING-TBK1-IRF3/7 induced interferon-β contributes to the clearing of non tuberculous mycobacterial infection in mice

PubMed Central

Ruangkiattikul, Nanthapon; Nerlich, Andreas; Abdissa, Ketema; Lienenklaus, Stefan; Suwandi, Abdulhadi; Janze, Nina; Laarmann, Kristin; Spanier, Julia; Kalinke, Ulrich; Weiss, Siegfried; Goethe, Ralph

2017-01-01

Abstract Type I interferons (IFN-I), such as IFN-α and IFN-β are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-β than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-β in macrophages. Both bacteria induced IFN-β via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-β activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-β impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection. PMID:28422568

Disinfective process of strongly acidic electrolyzed product of sodium chloride solution against Mycobacteria.

PubMed

Yamamoto, Tomoyo Matsushita; Nakano, Takashi; Yamaguchi, Masaki; Shimizu, Mitsuhide; Wu, Hong; Aoki, Hiroaki; Ota, Rie; Kobayashi, Toyohide; Sano, Kouichi

2012-12-01

Electrolyzed acid water (EAW) has been studied for its disinfective potential against pathogenic microbes; however, the bactericidal process against Mycobacteria has not been clearly presented. In this study, to clarify the disinfective process against Mycobacteria, EAW-treated bacteria were examined against laboratory strains of Mycobacterium bovis (M. bovis), Mycobacterium smegmatis (M. smegmatis), and Mycobacterium terrae (M. terrae) by recovery culture and observation of morphology, enzymatic assay, and the detection of DNA. All experiments were performed with the use of EAW containing 30 ppm free chlorine that kills Mycobacteria, including three pathogenic clinical isolates of Mycobacterium tuberculosis (M. tuberculosis) and six isolates of other Mycobacteria, within 5 min. In morphology, the bacterial surface became rough, and a longitudinal concavity-like structure appeared. The intrabacterial enzyme of EAW-contacted bacteria was inactivated, but chromosomal DNA was not totally denatured. These results suggest that the bactericidal effect of EAW against Mycobacteria occurs by degradation of the cell wall, followed by denaturation of cytoplasmic proteins, but degeneration of the nucleic acid is not always necessary.

Structure and Function of AmtR in Mycobacterium smegmatis: Implications for Post-Transcriptional Regulation of Urea Metabolism through a Small Antisense RNA.

PubMed

Petridis, Michael; Vickers, Chelsea; Robson, Jennifer; McKenzie, Joanna L; Bereza, Magdalena; Sharrock, Abigail; Aung, Htin Lin; Arcus, Vickery L; Cook, Gregory M

2016-10-23

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ∆amtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N 4 -GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis-encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated P II protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis-encoded small antisense RNA. Copyright © 2016 Elsevier Ltd. All rights reserved.

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.

PubMed

McDonough, Justin A; Hacker, Kari E; Flores, Anthony R; Pavelka, Martin S; Braunstein, Miriam

2005-11-01

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.

The SigF Regulon in Mycobacterium smegmatis Reveals Roles in Adaptation to Stationary Phase, Heat, and Oxidative Stress▿ ‡

PubMed Central

Hümpel, Anja; Gebhard, Susanne; Cook, Gregory M.; Berney, Michael

2010-01-01

SigF is an alternative sigma factor that is highly conserved among species of the genus Mycobacterium. In this study we identified the SigF regulon in Mycobacterium smegmatis using whole-genome microarray and promoter consensus analyses. In total, 64 genes in exponential phase and 124 genes in stationary phase are SigF dependent (P < 0.01, >2-fold expression change). Our experimental data reveal the SigF-dependent promoter consensus GTTT-N(15-17)-GGGTA for M. smegmatis, and we propose 130 potential genes under direct control of SigF, of which more than 50% exhibited reduced expression in a ΔsigF strain. We previously reported an increased susceptibility of the ΔsigF strain to heat and oxidative stress, and our expression data indicate a molecular basis for these phenotypes. We observed SigF-dependent expression of several genes purportedly involved in oxidative stress defense, namely, a heme-containing catalase, a manganese-containing catalase, a superoxide dismutase, the starvation-induced DNA-protecting protein MsDps1, and the biosynthesis genes for the carotenoid isorenieratene. Our data suggest that SigF regulates the biosynthesis of the thermoprotectant trehalose, as well as an uptake system for osmoregulatory compounds, and this may explain the increased heat susceptibility of the ΔsigF strain. We identified the regulatory proteins SigH3, PhoP, WhiB1, and WhiB4 as possible genes under direct control of SigF and propose four novel anti-sigma factor antagonists that could be involved in the posttranslational regulation of SigF in M. smegmatis. This study emphasizes the importance of this sigma factor for stationary-phase adaptation and stress response in mycobacteria. PMID:20233930

Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

PubMed Central

Fan, Xiangyu; Teng, Tieshan; Wang, Honghai

2012-01-01

Mycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc2155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result. PMID:22923793

Biology of a novel mycobacteriophage, SWU1, isolated from Chinese soil as revealed by genomic characteristics.

PubMed

Fan, Xiangyu; Teng, Tieshan; Wang, Honghai; Xie, Jianping

2012-09-01

Mycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc(2)155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result.

Adhesion of Mycobacterium smegmatis to Charged Surfaces and Diagnostics Implications

NASA Astrophysics Data System (ADS)

Gorse, Diane; Dhinojwala, Ali; Moore, Francisco

Pulmonary tuberculosis (PTB) causes more than 1 million deaths annually. Smear microscopy is a primary rapid detection tool in areas where 95 % of PTB cases occur. This technique, in which the sputum of a symptomatic patient is stained and examined using a light microscope for Mycobacterium tuberculosis (MTB) shows sensitivity between 20 and 60 %. Insufficient bacterial isolation during sample preparation may be a reason for low sensitivity. We are optimizing a system to capture bacteria on the basis of electrostatic interactions to more thoroughly isolate bacteria from suspension and facilitate more accurate detection. Silica supports coated with positively-charged polyelectrolyte, poly(diallyldimethylammonium chloride), captured approximately 4.1 times more Mycobacterium smegmatis, a model organism for MTB, than was captured on negatively-charged silica substrates. Future experimentation will employ branched polymer systems and seek to justify the use of colloidal stability theories to describe initial capture. Supported by University of Akron, Department of Polymer Science, Department of Biology; LORD Corporation.

Bioactivity studies of extracts from Tridax procumbens.

PubMed

Taddei, A; Rosas-Romero, A J

2000-06-01

An updated review on the biological activity of Tridax procumbens is presented. A detailed biological screening comprised of gram-positive and gram-negative bacteria, yeasts and fungi using crude extracts of this plant was undertaken. The n-hexane extract of the flowers showed activity against Escherichia coli. The same extract of the whole aerial parts was active against Mycobacterium smegmatis, Escherichia coli, Salmonella group C and Salmonella paratyphi. The ethyl-acetate extract of the flowers was active against Bacillus cereus and Klebsiella sp. The aerial parts extract also showed activity only against Mycobacterium smegmatis and Staphylococcus aureus, while the aqueous extract showed no antimicrobial activity. None of the tested extracts was active against the yeasts, Candida albicans, Candida tropicalis and Rhodotorula rubra; or the fungi: Aspergillus flavus, Aspergillus niger, Mucor sp. and Trichophyton rubrum.

Peroxidatic Activity of Mycobacteria and Relation to Catalase

PubMed Central

Winder, Frank G.

1966-01-01

Winder, Frank G. (Trinity College, Dublin, Ireland). Peroxidatic activity of mycobacteria and relation to catalase. J. Bacteriol. 92:413–417. 1966.—Catalase from Mycobacterium smegmatis was purified about 50-fold. All fractions showed a ratio of peroxidatic activity to catalatic activity approximately the same as that of the crude extract, a ratio only about four times that given by catalase from Micrococcus lysodeikticus. This and other evidence strongly suggest that the peroxidatic activity of M. smegmatis is due to its catalase. Less complete evidence suggests that this is true in the case of Mycobacterium tuberculosis also. It is suggested that in the context of the mycobacteria the term “peroxidatic activity” should replace the term “peroxidase” unless evidence is found that a true peroxidase exists in these organisms. PMID:16562129

Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.

PubMed Central

Mukherjee, T; Basu, D; Mahapatra, S; Goffin, C; van Beeumen, J; Basu, J

1996-01-01

The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin. PMID:8947487

Non-tuberculous Mycobacteria in South African Wildlife: Neglected Pathogens and Potential Impediments for Bovine Tuberculosis Diagnosis.

PubMed

Gcebe, Nomakorinte; Hlokwe, Tiny M

2017-01-01

Non-tuberculous mycobacteria (NTM) are not only emerging and opportunistic pathogens of both humans and animals, but from a veterinary point of view some species induce cross-reactive immune responses that hamper the diagnosis of bovine tuberculosis (bTB) in both livestock and wildlife. Little information is available about NTM species circulating in wildlife species of South Africa. In this study, we determined the diversity of NTM isolated from wildlife species from South Africa as well as Botswana. Thirty known NTM species and subspecies, as well as unidentified NTM, and NTM closely related to Mycobacterium goodii/Mycobacterium smegmatis were identified from 102 isolates cultured between the years 1998 and 2010, using a combination of molecular assays viz PCR and sequencing of different Mycobacterial house-keeping genes as well as single nucleotide polymorphism (SNP) analysis. The NTM identified in this study include the following species which were isolated from tissue with tuberculosis- like lesions in the absence of Mycobacterium tuberculosis complex (MTBC) implying their potential role as pathogens of animals: Mycobacterium abscessus subsp. bolletii, Mycobacterium gastri, Mycobacterium species closely related to Mycobacterium goodii/Mycobacterium smegmatis, Mycobacterium brasiliensis, Mycobacterium sinense JMD 601, Mycobacterium avium subsp. avium, Mycobacterium sp. GR-2007, Mycobacterium bouchedurhonense , and Mycobacterium septicum / M. peregrinum. Mycobaterium brasiliensis, Mycobacterium gastri, Mycobacterium sp. GR-2007, and a potential novel Mycobacterium species closely related to Mycobacterium goodii were found for the first time in this study to be potential pathogens of animals. Mycobacterium simiae was isolated from a sample originating from a tuberculin skin test positive reactor, demonstrating its potential to elicit inappropriate immune responses in animals that may interfere with diagnosis of tuberculosis by immunology. Mycobacterium abscessus subsp. bolletti was the most frequently detected NTM identified in 37 of the 102 isolates. Other NTM species were also isolated from animals not showing any pathological changes. Knowledge gained in this study contribute to the understanding of NTM species circulating in wild animals in South Africa and the pathogenic potential of certain species, whose role in disease causation need to be examined, as well as to a certain extent the potential of M. simiae to hamper the diagnosis of bTB.

Non-tuberculous Mycobacteria in South African Wildlife: Neglected Pathogens and Potential Impediments for Bovine Tuberculosis Diagnosis

PubMed Central

Gcebe, Nomakorinte; Hlokwe, Tiny M.

2017-01-01

Non-tuberculous mycobacteria (NTM) are not only emerging and opportunistic pathogens of both humans and animals, but from a veterinary point of view some species induce cross-reactive immune responses that hamper the diagnosis of bovine tuberculosis (bTB) in both livestock and wildlife. Little information is available about NTM species circulating in wildlife species of South Africa. In this study, we determined the diversity of NTM isolated from wildlife species from South Africa as well as Botswana. Thirty known NTM species and subspecies, as well as unidentified NTM, and NTM closely related to Mycobacterium goodii/Mycobacterium smegmatis were identified from 102 isolates cultured between the years 1998 and 2010, using a combination of molecular assays viz PCR and sequencing of different Mycobacterial house-keeping genes as well as single nucleotide polymorphism (SNP) analysis. The NTM identified in this study include the following species which were isolated from tissue with tuberculosis- like lesions in the absence of Mycobacterium tuberculosis complex (MTBC) implying their potential role as pathogens of animals: Mycobacterium abscessus subsp. bolletii, Mycobacterium gastri, Mycobacterium species closely related to Mycobacterium goodii/Mycobacterium smegmatis, Mycobacterium brasiliensis, Mycobacterium sinense JMD 601, Mycobacterium avium subsp. avium, Mycobacterium sp. GR-2007, Mycobacterium bouchedurhonense, and Mycobacterium septicum/M. peregrinum. Mycobaterium brasiliensis, Mycobacterium gastri, Mycobacterium sp. GR-2007, and a potential novel Mycobacterium species closely related to Mycobacterium goodii were found for the first time in this study to be potential pathogens of animals. Mycobacterium simiae was isolated from a sample originating from a tuberculin skin test positive reactor, demonstrating its potential to elicit inappropriate immune responses in animals that may interfere with diagnosis of tuberculosis by immunology. Mycobacterium abscessus subsp. bolletti was the most frequently detected NTM identified in 37 of the 102 isolates. Other NTM species were also isolated from animals not showing any pathological changes. Knowledge gained in this study contribute to the understanding of NTM species circulating in wild animals in South Africa and the pathogenic potential of certain species, whose role in disease causation need to be examined, as well as to a certain extent the potential of M. simiae to hamper the diagnosis of bTB. PMID:28194371

Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis

PubMed Central

Vilchèze, Catherine; Morbidoni, Hector R.; Weisbrod, Torin R.; Iwamoto, Hiroyuki; Kuo, Mack; Sacchettini, James C.; Jacobs, William R.

2000-01-01

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42°C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH. PMID:10869086

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons.

PubMed

Galán, Beatriz; Uhía, Iria; García-Fernández, Esther; Martínez, Igor; Bahíllo, Esther; de la Fuente, Juan L; Barredo, José L; Fernández-Cabezón, Lorena; García, José L

2017-01-01

A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multi-step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc 2 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc 2 155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9α-hydroxylase (KshAB) and a ketosteroid Δ 1 -dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc 2 155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis

PubMed Central

Berger, Bradley J; Knodel, Marvin H

2003-01-01

Background Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. Results The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 μM, while the MIC for 8-azaguanine was >1.0 mM. Conclusion The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors. PMID:12809568

Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

PubMed Central

Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

2011-01-01

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

Synthesis and biological screening of 2'-aryl/benzyl-2-aryl-4-methyl-4',5-bithiazolyls as possible anti-tubercular and antimicrobial agents.

PubMed

Abhale, Yogita K; Sasane, Amit V; Chavan, Abhijit P; Deshmukh, Keshav K; Kotapalli, Sudha Sravanti; Ummanni, Ramesh; Sayyad, Sadikali F; Mhaske, Pravin C

2015-04-13

A series of 2'-aryl/benzyl-2-aryl-4-methyl-4',5-bithiazolyl derivatives, 25-64 were synthesized and evaluated for inhibitory activity against Mycobacterium smegmatis MC(2) 155 strain and antimicrobial activities against four pathogenic bacteria Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Proteus vulgaris. Among them, compounds 40, 49, 50, and 54 exhibited moderate to good inhibition on the growth of the bacteria Mycobacterium smegmatis at the concentration of 30 μM. Compounds 26, 40, 44, 54 and 56 exhibited moderate to good antibacterial activity. Compound 5-(2'-(4-fluorobenzyl)thiazol-4'-yl)-2-(4-fluorophenyl)-4-methyl-thiazole (54) exhibited both antitubercular as well as antimicrobial activity against all tested strains. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

PubMed

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Importance of Porins for Biocide Efficacy against Mycobacterium smegmatis▿

PubMed Central

Frenzel, Elrike; Schmidt, Stefan; Niederweis, Michael; Steinhauer, Katrin

2011-01-01

Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N′-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides. PMID:21398489

Mycobacterium tuberculosis Rv1152 is a Novel GntR Family Transcriptional Regulator Involved in Intrinsic Vancomycin Resistance and is a Potential Vancomycin Adjuvant Target

PubMed Central

Zeng, Jie; Deng, Wanyan; Yang, Wenmin; Luo, Hongping; Duan, Xiangke; Xie, Longxiang; Li, Ping; Wang, Rui; Fu, Tiwei; Abdalla, Abualgasim Elgaili; Xie, Jianping

2016-01-01

Novel factors involved in Mycobacteria antibiotics resistance are crucial for better targets to combat the ever-increasing drug resistant strains. Mycobacterium tuberculosis Rv1152, a novel GntR family transcriptional regulator and a promising vancomycin adjuvant target, was firstly characterized in our study. Overexpression of Rv1152 in Mycobacterium smegmatis decreased bacterial susceptibility to vancomycin. Moreover, a deficiency in MSMEG_5174, an Rv1152 homolog made M. smegmatis more sensitive to vancomycin, which was reverted by complementing the MSMEG_5174 deficiency with Rv1152 of M. tuberculosis. Rv1152 negatively regulated four vancomycin responsive genes, namely genes encoding the ribosome binding protein Hsp, small unit of sulfate adenylyltransferase CysD, L-lysine-epsilon aminotransferase Lat, and protease HtpX. Taken together, Rv1152 controls the expression of genes required for the susceptibility to vancomycin. This is the first report that links the GntR family transcriptional factor with vancomycin susceptibility. Inhibitors of Rv1152 might be ideal vancomycin adjuvants for controlling multi-drug resistant Mycobacterial infections. PMID:27349953

Synthesis of biocompatible nanoparticle drug complexes for inhibition of mycobacteria

NASA Astrophysics Data System (ADS)

Bhave, Tejashree; Ghoderao, Prachi; Sanghavi, Sonali; Babrekar, Harshada; Bhoraskar, S. V.; Ganesan, V.; Kulkarni, Anjali

2013-12-01

Tuberculosis (TB) is one of the most critical infectious diseases affecting the world today. Current TB treatment involves six months long daily administration of four oral doses of antibiotics. Due to severe side effects and the long treatment, a patient's adherence is low and this results in relapse of symptoms causing an alarming increase in the prevalence of multi-drug resistant (MDR) TB. Hence, it is imperative to develop a new drug delivery technology wherein these effects can be reduced. Rifampicin (RIF) is one of the widely used anti-tubercular drugs (ATD). The present study discusses the development of biocompatible nanoparticle-RIF complexes with superior inhibitory activity against both Mycobacterium smegmatis (M. smegmatis) and Mycobacterium tuberculosis (M. tuberculosis). Iron oxide nanoparticles (NPs) synthesized by gas phase condensation and NP-RIF complexes were tested against M. smegmatis SN2 strain as well as M. tuberculosis H37Rv laboratory strain. These complexes showed significantly better inhibition of M. smegmatis SN2 strain at a much lower effective concentration (27.5 μg ml-1) as compared to neat RIF (125 μg ml-1). Similarly M. tuberculosis H37Rv laboratory strain was susceptible to both nanoparticle-RIF complex and neat RIF at a minimum inhibitory concentration of 0.22 and 1 μg ml-1, respectively. Further studies are underway to determine the efficacy of NPs-RIF complexes in clinical isolates of M. tuberculosis as well as MDR isolates.

Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery.

PubMed

Namouchi, Amine; Cimino, Mena; Favre-Rochex, Sandrine; Charles, Patricia; Gicquel, Brigitte

2017-07-13

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations. We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process. We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process.

Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein

PubMed Central

Rawat, Mamta; Uppal, Mandeep; Newton, Gerald; Steffek, Micah; Fahey, Robert C.; Av-Gay, Yossef

2004-01-01

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner. PMID:15342574

Mycobacterium smegmatis proteoliposome induce protection in a murine progressive pulmonary tuberculosis model.

PubMed

Tirado, Yanely; Puig, Alina; Alvarez, Nadine; Borrero, Reinier; Aguilar, Alicia; Camacho, Frank; Reyes, Fatima; Fernandez, Sonsire; Perez, Jose Luis; Acevedo, Reynaldo; Mata Espinoza, Dulce; Payan, Jorge Alberto Barrios; Garcia, Maria de Los A; Kadir, Ramlah; Sarmiento, María E; Hernandez-Pando, Rogelio; Norazmi, Mohd-Nor; Acosta, Armando

2016-12-01

Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb. Copyright © 2016 Elsevier Ltd. All rights reserved.

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

PubMed Central

Speer, Alexander; Rowland, Jennifer L.; Haeili, Mehri; Niederweis, Michael

2013-01-01

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis. PMID:24013632

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis.

PubMed

Maiti, Krishnagopal; Syal, Kirtimaan; Chatterji, Dipankar; Jayaraman, Narayanaswamy

2017-10-05

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids-2,3-branched and 2,5-branched-were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non-pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 μg mL -1 in the case of the 2,5-branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 μg mL -1 ) potentiates the drug, making it three times more effective, with an improved MBIC of 30 μg mL -1 . These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

PubMed Central

Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K.; Ekins, Sean

2014-01-01

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules. PMID:25534741

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

PubMed

Horn, C; Namane, A; Pescher, P; Rivière, M; Romain, F; Puzo, G; Bârzu, O; Marchal, G

1999-11-05

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

PubMed

Sethi, Deepti; Mahajan, Sahil; Singh, Chaahat; Lama, Amrita; Hade, Mangesh Dattu; Gupta, Pawan; Dikshit, Kanak L

2016-02-05

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis

PubMed Central

LaMarca, B. Babbette D.; Zhu, Wenming; Arceneaux, Jean E. L.; Rowe Byers, B.; Lundrigan, Michael D.

2004-01-01

Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 μM Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an Rf of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an Rf of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin. PMID:14702306

Characterization of nanostructured surfaces generated by reconstitution of the porin MspA from Mycobacterium smegmatis.

PubMed

Wörner, Michael; Lioubashevski, Oleg; Basel, Matthew T; Niebler, Sandra; Gogritchiani, Eliso; Egner, Nicole; Heinz, Christian; Hoferer, Jürgen; Cipolloni, Michela; Janik, Katharine; Katz, Evgeny; Braun, Andre M; Willner, Itamar; Niederweis, Michael; Bossmann, Stefan H

2007-06-01

Nanostructures with long-term stability at the surface of gold electrodes are generated by reconstituting the porin MspA from Mycobacterium smegmatis into a specially designed monolayer of long-chain lipid surfactant on gold. Tailored surface coverage of gold electrodes with long-chain surfactants is achieved by electrochemically assisted deposition of organic thiosulfates (Bunte salts). The subsequent reconstitution of the octameric-pore MspA is guided by its extraordinary self-assembling properties. Importantly, electrochemical reduction of copper(II) yields copper nanoparticles within the MspA nanopores. Electrochemical impedance spectroscopy, reflection electron microscopy, and atomic force microscopy (AFM) show that: 1) the MspA pores within the self-assembled monolayer (SAM) are monodisperse and electrochemically active, 2) MspA reconstitutes in SAMs and with a 10-nm thickness, 3) AFM is a suitable method to detect pores within SAMs, and 4) the electrochemical reduction of Cu2+ to Cu0 under overpotential conditions starts within the MspA pores.

Purification of a d-Mannose Isomerase from Mycobacterium smegmatis1

PubMed Central

Hey-Ferguson, Ann; Elbein, Alan D.

1970-01-01

An enzyme, d-mannose ketol isomerase, catalyzing the isomerization of d-mannose and d-fructose was purified approximately 60-fold from cells of Mycobacterium smegmatis grown on mannose as the sole carbon source. This enzyme was shown to catalyze the conversion of d-mannose and d-lyxose to ketoses. The ketose produced from mannose was identified as fructose by chemical and chromatographic methods. The reaction was shown to be reversible, the equilibrium ratio of fructose to mannose being approximately 65 to 35. The pH optimum was about 7.5, and the Km for mannose was estimated to be 7 × 10−3m. Mannose isomerase activity was greatest in cells grown on mannose, whereas cells grown on fructose had about 30% as much activity. Very low levels of activity were detected in cells grown on other substrates. There was an immediate increase in enzyme activity on transfer of cells from nutrient broth to a mannose mineral salts medium. PMID:5438047

Characterization of differential pore-forming activities of ESAT-6 proteins from M. tuberculosis and M. smegmatis

PubMed Central

Peng, Xiuli; Jiang, Guozhong; Liu, Wei; Zhang, Qi; Qian, Wei; Sun, Jianjun

2016-01-01

Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) plays essential roles in pathogenesis. MtbESAT-6 exhibits a unique pore-forming activity (PFA) that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis (MsESAT-6). Here we characterized the differential PFAs and found that exchange of I25-H26/T25-A26 between two proteins reciprocally affected their PFAs. MtbESAT-6(IH/TA) had ~40% reduction, while MsESAT-6(TA/IH) fully acquired its activity similar to MtbESAT-6. Mutations of A17E, K38T, N67L or R74Q on MtbESAT-6(IH/TA) further reduced the activity, with MtbESAT-6(IH/TA-17) being the lowest. This study suggests I25-H26 as the pH-sensor essential for MsESAT-6 to fully acquire the activity, while multiple residues contributed to MtbESAT-6 PFA. PMID:26801203

Comparative bioactivation of the novel anti-tuberculosis agent PA-824 in Mycobacteria and a subcellular fraction of human liver

PubMed Central

Dogra, M; Palmer, BD; Bashiri, G; Tingle, MD; Shinde, SS; Anderson, RF; O'Toole, R; Baker, EN; Denny, WA; Helsby, NA

2011-01-01

BACKGROUND AND PURPOSE PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F420)-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis. EXPERIMENTAL APPROACH PA-824 (250 µM) was incubated with the 9000×g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis. KEY RESULTS PA-824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des-nitro product. Six of these products, but not the des-nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn-catalyzed des-nitrification and radiolytic reduction of PA-824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem-catalyzed product of imidazole ring hydration (M2). CONCLUSIONS AND IMPLICATIONS PA-824 was metabolized by des-nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA-824 was selectively bioactivated in M. tuberculosis and there was no evidence for ‘cross-activation’ by human enzymes. PMID:20955364

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

PubMed Central

Busby, Jason N.; Fritz, Georg; Moreland, Nicole J.; Cook, Gregory M.; Lott, J. Shaun; Baker, Edward N.

2014-01-01

Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains. PMID:25049090

Genome wide analysis of the complete GlnR nitrogen-response regulon in Mycobacterium smegmatis

PubMed Central

2013-01-01

Background Nitrogen is an essential element for bacterial growth and an important component of biological macromolecules. Consequently, responding to nitrogen limitation is critical for bacterial survival and involves the interplay of signalling pathways and transcriptional regulation of nitrogen assimilation and scavenging genes. In the soil dwelling saprophyte Mycobacterium smegmatis the OmpR-type response regulator GlnR is thought to mediate the transcriptomic response to nitrogen limitation. However, to date only ten genes have been shown to be in the GlnR regulon, a vastly reduced number compared to other organisms. Results We investigated the role of GlnR in the nitrogen limitation response and determined the entire GlnR regulon, by combining expression profiling of M. smegmatis wild type and glnR deletion mutant, with GlnR-specific chromatin immunoprecipitation and high throughput sequencing. We identify 53 GlnR binding sites during nitrogen limitation that control the expression of over 100 genes, demonstrating that GlnR is the regulator controlling the assimilation and utilisation of nitrogen. We also determine a consensus GlnR binding motif and identify key residues within the motif that are required for specific GlnR binding. Conclusions We have demonstrated that GlnR is the global nitrogen response regulator in M. smegmatis, directly regulating the expression of more than 100 genes. GlnR controls key nitrogen stress survival processes including primary nitrogen metabolism pathways, the ability to utilise nitrate and urea as alternative nitrogen sources, and the potential to use cellular components to provide a source of ammonium. These studies further our understanding of how mycobacteria survive nutrient limiting conditions. PMID:23642041

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

PubMed Central

Murdeshwar, Maya S.

2012-01-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme RelMsm. This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The relMsm knockout strain of M. smegmatis (ΔrelMsm) is expected to show a (p)ppGpp null [(p)ppGpp0] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRelMsm in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response. PMID:22636779

MS_RHII-RSD, a dual-function RNase HII-(p)ppGpp synthetase from Mycobacterium smegmatis.

PubMed

Murdeshwar, Maya S; Chatterji, Dipankar

2012-08-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria▿

PubMed Central

Furuya, Toshiki; Hirose, Satomi; Osanai, Hisashi; Semba, Hisashi; Kino, Kuniki

2011-01-01

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc2155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc2155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc2155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc2155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria. PMID:21183637

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes.

PubMed

Kapopoulou, Adamandia; Lew, Jocelyne M; Cole, Stewart T

2011-01-01

In this paper, we present the MycoBrowser portal (http://mycobrowser.epfl.ch/), a resource that provides both in silico generated and manually reviewed information within databases dedicated to the complete genomes of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum and Mycobacterium smegmatis. A central component of MycoBrowser is TubercuList (http://tuberculist.epfl.ch), which has recently benefited from a new data management system and web interface. These improvements were extended to all MycoBrowser databases. We provide an overview of the functionalities available and the different ways of interrogating the data then discuss how both the new information and the latest features are helping the mycobacterial research communities. Copyright © 2010 Elsevier Ltd. All rights reserved.

Multivalent Antimicrobial Polymer Nanoparticles Target Mycobacteria and Gram-Negative Bacteria by Distinct Mechanisms

PubMed Central

2017-01-01

Because of the emergence of antimicrobial resistance to traditional small-molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosis is a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coli versus Mycobacterium smegmatis (model of M. tuberculosis), leading to killing or growth inhibition, respectively. A convergent “grafting to” synthetic strategy was used to assemble a 50-member nanoparticle library, and using a high-throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared with the linear polymers, the nanoparticles displayed two- and eight-fold enhancements in antimicrobial activity against M. smegmatis and E. coli, respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. coli due to rapid disruption of the cell membranes. Conversely, against M. smegmatis the particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric antituberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesize is due to the unique cell envelope at the surface of these bacteria. PMID:29195272

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

PubMed

Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

2009-03-01

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

Three different [NiFe] hydrogenases confer metabolic flexibility in the obligate aerobe Mycobacterium smegmatis.

PubMed

Berney, Michael; Greening, Chris; Hards, Kiel; Collins, Desmond; Cook, Gregory M

2014-01-01

Mycobacterium smegmatis is an obligate aerobe that harbours three predicted [NiFe] hydrogenases, Hyd1 (MSMEG_2262–2263), Hyd2 (MSMEG_2720-2719) and Hyd3 (MSMEG_3931-3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd1 groups with hydrogenases that oxidize H2 produced by metabolic processes, and Hyd2 is homologous to a novel group of putative high-affinity hydrogenases. Hyd1 and Hyd2 respond to carbon and oxygen limitation, and, in the case of Hyd1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd3 are consistent with the enzyme evolving hydrogen. Hyd3 is controlled by DosR, a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd1 and Hyd2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M. smegmatis to oxidize hydrogen at high (Hyd1) and potentially tropospheric (Hyd2) concentrations, as well as recycle reduced equivalents by evolving hydrogen (Hyd3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.

Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants▿†

PubMed Central

Ta, Philong; Buchmeier, Nancy; Newton, Gerald L.; Rawat, Mamta; Fahey, Robert C.

2011-01-01

The mshA::Tn5 mutant of Mycobacterium smegmatis does not produce mycothiol (MSH) and was found to markedly overproduce both ergothioneine and an ∼15-kDa protein determined to be organic hydroperoxide resistance protein (Ohr). An mshA(G32D) mutant lacking MSH overproduced ergothioneine but not Ohr. Comparison of the mutant phenotypes with those of the wild-type strain indicated the following: Ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional MSH-dependent organic hydroperoxide peroxidase exists; and elevated isoniazid resistance in the mutant is associated with both Ohr and the absence of MSH. Purified Ohr showed high activity with linoleic acid hydroperoxide, indicating lipid hydroperoxides as the likely physiologic targets. The reduction of oxidized Ohr by NADH was shown to be catalyzed by lipoamide dehydrogenase and either lipoamide or DlaT (SucB). Since free lipoamide and lipoic acid levels were shown to be undetectable in M. smegmatis, the bound lipoyl residues of DlaT are the likely source of the physiological dithiol reductant for Ohr. The pattern of occurrence of homologs of Ohr among bacteria suggests that the ohr gene has been distributed by lateral transfer. The finding of multiple Ohr homologs with various sequence identities in some bacterial genomes indicates that there may be multiple physiologic targets for Ohr proteins. PMID:21335456

Type II flavohemoglobin of Mycobacterium smegmatis oxidizes d-lactate and mediate electron transfer.

PubMed

Thakur, Naveen; Kumar, Ashwani; Dikshit, Kanak L

2018-06-01

Two distantly related flavohemoglobins (FHbs), MsFHbI and MsFHbII, having crucial differences in their heme and reductase domains, co-exist in Mycobacterium smegmatis. Function of MsFHbI is associated with nitric-oxide detoxification but physiological relevance of MsFHbII remains unknown. This study unravels some unique spectral and functional characteristics of MsFHbII. Unlike conventional type I FHbs, MsFHbII lacks nitric-oxide dioxygenase and NADH oxidase activities but utilizes d-lactate as an electron donor to mediate electron transfer. MsFHbII carries a d-lactate dehydrogenase type FAD binding motif in its reductase domain and oxidizes d-lactate in a FAD dependent manner to reduce the heme iron, suggesting that the globin is acting as an electron acceptor. Importantly, expression of MsFHbII in Escherichia coli imparted protection under oxidative stress, suggesting its important role in stress management of its host. Since M. smegmatis lacks the gene encoding for d-lactate dehydrogenase and d-lactate is produced during aerobic metabolism and also as a by-product of lipid peroxidation, the ability of MsFHbII to metabolize d-lactate may provide it a unique ability to balance the oxidative stress generated due to accumulation of d-lactate in the cell and at the same time sequester electrons and pass it to the respiratory apparatus. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.

PubMed

Lee, Jason W; Parlane, Natalie A; Rehm, Bernd H A; Buddle, Bryce M; Heiser, Axel

2017-03-01

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli - Mycobacterium shuttle plasmids and expressed in trans Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens. IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens. Copyright © 2017 Lee et al.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine

PubMed Central

Lee, Jason W.; Parlane, Natalie A.; Rehm, Bernd H. A.; Buddle, Bryce M.

2017-01-01

ABSTRACT Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli-Mycobacterium shuttle plasmids and expressed in trans. Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A:E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens. IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens. PMID:28087528

Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria.

PubMed

De Maio, Flavio; Maulucci, Giuseppe; Minerva, Mariachiara; Anoosheh, Saber; Palucci, Ivana; Iantomasi, Raffaella; Palmieri, Valentina; Camassa, Serena; Sali, Michela; Sanguinetti, Maurizio; Bitter, Wilbert; Manganelli, Riccardo; De Spirito, Marco; Delogu, Giovanni

2014-01-01

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.

Protective effect of a lipid-based preparation from Mycobacterium smegmatis in a murine model of progressive pulmonary tuberculosis.

PubMed

García, Maria de los Angeles; Borrero, Reinier; Lanio, Maria E; Tirado, Yanely; Alvarez, Nadine; Puig, Alina; Aguilar, Alicia; Canet, Liem; Mata Espinoza, Dulce; Barrios Payán, Jorge; Sarmiento, María Elena; Hernández-Pando, Rogelio; Norazmi, Mohd-Nor; Acosta, Armando

2014-01-01

A more effective vaccine against tuberculosis (TB) is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb), the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms), could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL) or nonadjuvanted (LMs) showed significant reductions in bacterial load (P < 0.01) compared to the negative control group (animals immunized with phosphate buffered saline (PBS)). Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG). Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P < 0.01) and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB.

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins.

PubMed

Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C; Rutten, Victor

2016-01-01

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens.

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

PubMed Central

Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C.; Rutten, Victor

2016-01-01

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens. PMID:27375559

Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium tuberculosis.

PubMed

Shirude, Pravin S; Madhavapeddi, Prashanti; Tucker, Julie A; Murugan, Kannan; Patil, Vikas; Basavarajappa, Halesha; Raichurkar, Anandkumar V; Humnabadkar, Vaishali; Hussein, Syeed; Sharma, Sreevalli; Ramya, V K; Narayan, Chandan B; Balganesh, Tanjore S; Sambandamurthy, Vasan K

2013-03-15

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

Spectroscopic studies, antimicrobial activities and crystal structures of N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene

NASA Astrophysics Data System (ADS)

Ünver, Hüseyin; Yıldız, Mustafa; Dülger, Başaran; Özgen, Özen; Kendi, Engin; Durlu, Tahsin Nuri

2005-03-01

Schiff base N-(2-hydroxy-3-methoxybenzalidene)1-aminonaphthalene has been synthesized from the reaction of 2-hydroxy-3-methoxybenzaldehyde with 1-aminonaphthalene. The compound were characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR and UV-visible techniques. The UV-visible spectra of the Schiff base were studied in polar and nonpolar solvents in acidic and basic media. The structure of the compound has been examined cyrstallographically. There are two independent molecules in the asymmetric unit. It crystallizes in the monoclinic space group P21/c, with unit cell parameters: a=14, 602(2), b=5,800(1), c=16, 899(1) Å, V=1394.4(2) Å 3, Dx=1.321 g cm -3 and Z=4. The crystal structure was solved by direct methods and refined by full-matrix least squares to a find R=0.041 of for 1179 observed reflections. The title compound's antimicrobial activities also have been studied. The antimicrobial activities of the ligand has been screened in vitro against the organisms Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis CCM 2067, Bacillus cereus ATCC 7064 and Listeria monocytogenes ATCC 15313, the yeast cultures Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432.

Drying a tuberculosis vaccine without freezing.

PubMed

Wong, Yun-Ling; Sampson, Samantha; Germishuizen, Willem Andreas; Goonesekera, Sunali; Caponetti, Giovanni; Sadoff, Jerry; Bloom, Barry R; Edwards, David

2007-02-20

With the increasing incidence of tuberculosis and drug resistant disease in developing countries due to HIV/AIDS, there is a need for vaccines that are more effective than the present bacillus Calmette-Guérin (BCG) vaccine. We demonstrate that BCG vaccine can be dried without traditional freezing and maintained with remarkable refrigerated and room-temperature stability for months through spray drying. Studies with a model Mycobacterium (Mycobacterium smegmatis) revealed that by removing salts and cryoprotectant (e.g., glycerol) from bacterial suspensions, the significant osmotic pressures that are normally produced on bacterial membranes through droplet drying can be reduced sufficiently to minimize loss of viability on drying by up to 2 orders of magnitude. By placing the bacteria in a matrix of leucine, high-yield, free-flowing, "vial-fillable" powders of bacteria (including M. smegmatis and M. bovis BCG) can be produced. These powders show relatively minor losses of activity after maintenance at 4 degrees C and 25 degrees C up to and beyond 4 months. Comparisons with lyophilized material prepared both with the same formulation and with a commercial formulation reveal that the spray-dried BCG has better overall viability on drying.

The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode†

PubMed Central

Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

2013-01-01

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation. PMID:23735230

Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.

PubMed

Narayanan, Sai Shyam; Ramanujan, Ajeena; Krishna, Shyam; Nampoothiri, Kesavan Madhavan

2008-12-01

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.

Secondary metabolites from Tetracera potatoria stem bark with anti-mycobacterial activity.

PubMed

Fomogne-Fodjo, M C Y; Ndinteh, D T; Olivier, D K; Kempgens, P; van Vuuren, S; Krause, R W M

2017-01-04

Tetracera potatoria Afzel. Exg. Don (Dilleniaceae) is a medicinal plant used traditionally in Africa for the treatment of tuberculosis related ailments and respiratory infections. The antibacterial activity of the medium polar extracts of T. potatoria leaves and stem bark was recently reported against Mycobacterium smegmatis (MIC 25µg/mL) and M. aurum (65µg/mL), two fast-growing Mycobacterium strains used as model micro-organisms for the more pathogenic strain Mycobacterium tuberculosis (Fomogne-Fodjo et al., 2014). The aim of this study was consequently to isolate the compounds possibly contributing to this activity, and which may therefore be promising precursors to be used for the development of novel anti-TB drugs. T. potatoria medium polar extract [MeOH/DCM (1:1, v/v)] was fractionated sequentially with petroleum ether to which EtOAC and MeOH were gradually added to increase the polarity. The examination of T. potatoria extract and its fractions was guided by bioassays for anti-mycobacterial activity against M. smegmatis (ATCC 23246) and M. aurum (NCTC 10437) using the minimum inhibitory concentration (MIC) method. All the isolated compounds were structurally elucidated using spectroscopic techniques and evaluated for their anti-mycobacterial activity. Two novel secondary metabolites (1, 2) named tetraceranoate and N-hydroxy imidate-tetracerane, together with five known compounds [β-stigmasterol (3), stigmast-5-en-3β-yl acetate (4), betulinic acid (5), betulin (6) and lupeol (7)] were isolated and identified. Tetraceranoate exhibited the best activity against M. smegmatis with a minimum inhibitory concentration (MIC) of 7.8µg/mL, while β-stigmasterol, betulinic acid and betulin showed appreciable anti-mycobacterial activity against both strains (MIC 15µg/mL). Seven compounds were isolated from the medium polar extract [MeOH/DCM (1:1, v/v)] of T. potatoria stem bark. Only tetraceranoate one of the isolated compounds showed antibacterial activity against M. smegmatis having efficacy as high as rifampicin (one of a three drug regimen recommended in the initial phase short-course anti-tuberculosis therapy). Thus, tetraceranoate might be an interesting target for systematic testing of anti-TB treatment and management. This research supports the use of T. potatoria in African traditional medicine for the treatment of tuberculosis related symptoms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Thermal Inactivation of Mycobacterium avium subsp. paratuberculosis in Artificially Contaminated Milk by Direct Steam Injection

PubMed Central

Butot, Sophie; Jagadeesan, Balamurugan; Bakker, Douwe; Donaghy, John

2016-01-01

ABSTRACT The efficiency of direct steam injection (DSI) at 105°C for 3 s to inactivate Mycobacterium avium subsp. paratuberculosis in milk at a pilot-plant scale was investigated. Milk samples were artificially contaminated with M. avium subsp. paratuberculosis and also with cow fecal material naturally infected with M. avium subsp. paratuberculosis. We also tested milk artificially contaminated with Mycobacterium smegmatis as a candidate surrogate to compare thermal inactivation between M. smegmatis and M. avium subsp. paratuberculosis. Following the DSI process, no viable M. avium subsp. paratuberculosis or M. smegmatis was recovered using culture methods for both strains. For pure M. avium subsp. paratuberculosis cultures, a minimum reduction of 5.6 log10 was achieved with DSI, and a minimum reduction of 5.7 log10 was found with M. smegmatis. The minimum log10 reduction for wild-type M. avium subsp. paratuberculosis naturally present in feces was 3.3. In addition, 44 dairy and nondairy powdered infant formula (PIF) ingredients used during the manufacturing process of PIF were tested for an alternate source for M. avium subsp. paratuberculosis and were found to be negative by quantitative PCR (qPCR). In conclusion, the results obtained from this study indicate that a >7-fold-log10 reduction of M. avium subsp. paratuberculosis in milk can be achieved with the applied DSI process. IMPORTANCE M. avium subsp. paratuberculosis is widespread in dairy herds in many countries. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and infected animals can directly or indirectly (i.e., fecal contamination) contaminate milk. Despite much research and debate, there is no conclusive evidence that M. avium subsp. paratuberculosis is a zoonotic bacterium, i.e., one that causes disease in humans. The presence of M. avium subsp. paratuberculosis or its DNA has been reported in dairy products, including pasteurized milk, cheese, and infant formula. In light of this, it is appropriate to evaluate existing mitigation measures to inactivate M. avium subsp. paratuberculosis in dairy products. The work conducted in this study describes the efficacy of direct steam injection, a thermal process commonly used in the dairy industry, to eliminate M. avium subsp. paratuberculosis and a surrogate bacterium in milk, thus ensuring the absence of M. avium subsp. paratuberculosis in dairy products subject to these process conditions. PMID:26944840

Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress.

PubMed

Maheshwari, Jayapal Jeya; Dharmalingam, Kuppamuthu

2013-07-01

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

Phosphorylation of Mycobacterium tuberculosis ParB Participates in Regulating the ParABS Chromosome Segregation System

PubMed Central

Baronian, Grégory; Ginda, Katarzyna; Berry, Laurence; Cohen-Gonsaud, Martin; Zakrzewska-Czerwińska, Jolanta; Jakimowicz, Dagmara; Molle, Virginie

2015-01-01

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis. PMID:25807382

Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

PubMed

Baronian, Grégory; Ginda, Katarzyna; Berry, Laurence; Cohen-Gonsaud, Martin; Zakrzewska-Czerwińska, Jolanta; Jakimowicz, Dagmara; Molle, Virginie

2015-01-01

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

Assessing the inactivation of Mycobacterium avium subsp. paratuberculosis during composting of livestock carcasses.

PubMed

Tkachuk, Victoria L; Krause, Denis O; McAllister, Tim A; Buckley, Katherine E; Reuter, Tim; Hendrick, Steve; Ominski, Kim H

2013-05-01

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

PubMed Central

Tkachuk, Victoria L.; Krause, Denis O.; McAllister, Tim A.; Buckley, Katherine E.; Reuter, Tim; Hendrick, Steve

2013-01-01

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities. PMID:23503307

Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

PubMed Central

Yamazaki, Yoshitaka; Danelishvili, Lia; Wu, Martin; MacNab, Molly; Bermudez, Luiz E.

2006-01-01

Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm. PMID:16391123

An Unusual Mutation Results in the Replacement of Diaminopimelate with Lanthionine in the Peptidoglycan of a Mutant Strain of Mycobacterium smegmatis†

PubMed Central

Consaul, Sandra A.; Wright, Lori F.; Mahapatra, Sebabrata; Crick, Dean C.; Pavelka, Martin S.

2005-01-01

Mycobacterial peptidoglycan contains l-alanyl-d-iso-glutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the l-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate d-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to β-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine β-synthase, an enzyme that is a part of the cysteine biosynthetic pathway. PMID:15716431

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia.

PubMed

Berney, Michael; Greening, Chris; Conrad, Ralf; Jacobs, William R; Cook, Gregory M

2014-08-05

Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H2 when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD(+)/NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentatively-produced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments.

Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice.

PubMed

Sha, Shanshan; Shi, Xiaoxia; Deng, Guoying; Chen, Lina; Xin, Yi; Ma, Yufang

2017-04-01

Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc 2 155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc 2 155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination. Copyright © 2017 Elsevier GmbH. All rights reserved.

Mycobacterium tuberculosis toxin Rv2872 is an RNase involved in vancomycin stress response and biofilm development.

PubMed

Wang, Xiaoyu; Zhao, Xiaokang; Wang, Hao; Huang, Xue; Duan, Xiangke; Gu, Yinzhong; Lambert, Nzungize; Zhang, Ke; Kou, Zhenhao; Xie, Jianping

2018-06-11

Bacterial toxin-antitoxin (TA) systems are emerging important regulators of multiple cellular physiological events and candidates for novel antibiotic targets. To explore the role of Mycobacterium tuberculosis function, unknown toxin gene Rv2872 was heterologously expressed in Mycobacterium smegmatis (MS_Rv2872). Upon induction, MS_Rv2872 phenotype differed significantly from the control, such as increased vancomycin resistance, retarded growth, cell wall, and biofilm structure. This phenotype change might result from the RNase activity of Rv2872 as purified Rv2872 toxin protein can cleave the products of several key genes involved in abovementioned phenotypes. In summary, toxin Rv2872 was firstly reported to be a endonuclease involved in antibiotic stress responses, cell wall structure, and biofilm development.

Genome Sequence of JangDynasty, a Newly Isolated Mycobacteriophage

PubMed Central

Jang, Casey; Kalaj, Nancy; Hwang, Brian; Hughes, Lorelei; Yang, Connie; Pak, Thomas; Kim, John; Han, Dong Yoon; Tedjakusnadi, Jason; Fernandez, Nicholas; Dean, Natasha; Muthiah, Arun; Sutter, Nathaniel B.

2018-01-01

ABSTRACT JangDynasty is a bacteriophage that infects Mycobacterium smegmatis mc2155. It has a genome length of 70,883 bp, with 124 predicted open reading frames (ORFs), 42 of which have known functions. JangDynasty belongs to cluster O, and like other cluster O phages, it is a siphovirus with a prolate capsid. PMID:29798914

A novel host factor for integration of mycobacteriophage L5

PubMed Central

Pedulla, Marisa L.; Lee, Mong Hong; Lever, Dawn C.; Hatfull, Graham F.

1996-01-01

Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase–mIHF–attP intasome complexes. PMID:8986825

Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

PubMed Central

Viswanathan, Gopinath; Yadav, Sangya

2017-01-01

ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment. PMID:28438925

Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

DTIC Science & Technology

1990-05-01

Sta58 antigen and the Sta56 strain- GroES, C. burnetii HtpA, Mycobacterium tuberculosis 12- specific major antigen of R. tsutsugamushi (strain Karp...kb HindlIl fragment carrying the gene for the Sta58 tuberculosis, and Mycobacterium smegmatis (65-kDa anti- protein was subjected to DNA sequence...the Hsp6O and HsplO proteins. R. tsu., R. isutsugamushi; M. lep., Mvtcobacteriutn leprae : C. bur., C. burneiii; Synech.. Synechococcus strain 6301; T

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

PubMed

Kanno, Alex I; Goulart, Cibelly; Rofatto, Henrique K; Oliveira, Sergio C; Leite, Luciana C C; McFadden, Johnjoe

2016-04-01

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. Copyright © 2016 Kanno et al.

A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction.

PubMed

Dhouib, Rabeb; Laval, Françoise; Carrière, Frédéric; Daffé, Mamadou; Canaan, Stéphane

2010-09-01

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction▿ †

PubMed Central

Dhouib, Rabeb; Laval, Françoise; Carrière, Frédéric; Daffé, Mamadou; Canaan, Stéphane

2010-01-01

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids. PMID:20601476

Antiprotozoal and antimycobacterial activities of Persea americana seeds.

PubMed

Jiménez-Arellanes, Adelina; Luna-Herrera, Julieta; Ruiz-Nicolás, Ricardo; Cornejo-Garrido, Jorge; Tapia, Amparo; Yépez-Mulia, Lilián

2013-05-16

Persea americana seeds are widely used in traditional Mexican medicine to treat rheumatism, asthma, infectious processes as well as diarrhea and dysentery caused by intestinal parasites. The chloroformic and ethanolic extracts of P. americana seeds were prepared by maceration and their amoebicidal, giardicidal and trichomonicidal activity was evaluated. These extracts were also tested against Mycobacterium tuberculosis H37Rv, four mono-resistant and two multidrug resistant strains of M. tuberculosis as well as five non tuberculosis mycobacterium strains by MABA assay. The chloroformic and ethanolic extracts of P. americana seeds showed significant activity against E. histolytica, G. lamblia and T. vaginalis (IC50 <0.634 μg/ml). The chloroformic extract inhibited the growth of M. tuberculosis H37Rv, M. tuberculosis MDR SIN 4 isolate, three M. tuberculosis H37Rv mono-resistant reference strains and four non tuberculosis mycobacteria (M. fortuitum, M. avium, M. smegmatis and M. absessus) showing MIC values ≤50 μg/ml. Contrariwise, the ethanolic extract affected only the growth of two mono-resistant strains of M. tuberculosis H37Rv and M. smegmatis (MIC ≤50 μg/ml). The CHCl3 and EtOH seed extracts from P. americana showed amoebicidal and giardicidal activity. Importantly, the CHCl3 extract inhibited the growth of a MDR M. tuberculosis isolate and three out of four mono-resistant reference strains of M. tuberculosis H37Rv, showing a MIC = 50 μg/ml. This extract was also active against the NTM strains, M. fortuitum, M. avium, M. smegmatis and M. abscessus, with MIC values <50 μg/ml.

Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

PubMed Central

Slayden, R A; Lee, R E; Armour, J W; Cooper, A M; Orme, I M; Brennan, P J; Besra, G S

1996-01-01

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis. PMID:9124847

Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation*

PubMed Central

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-01-01

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically. PMID:23609448

Genetic Mimetics of Mycobacterium tuberculosis and Methicillin-Resistant Staphylococcus aureus as Verification Standards for Molecular Diagnostics.

PubMed

Machowski, Edith Erika; Kana, Bavesh Davandra

2017-12-01

Molecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective enrollment into treatment. In recognition of this, the World Health Organization approved the rollout of nucleic acid amplification technologies for identification of Mycobacterium tuberculosis using platforms such as GeneXpert MTB/RIF, the GenoType MTBDR plus line probe assay, and, more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampin resistance. However, their widespread use in health systems requires verification and quality assurance programs. To enable development of these, we report the construction of genetically modified strains of Mycobacterium smegmatis that mimic the profile of Mycobacterium tuberculosis on both the GeneXpert MTB/RIF and the MTBDR plus line probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampin-resistant M. tuberculosis , with mutations at positions 513, 516, 526, 531, and 533 in the rifampin resistance-determining region of the rpoB gene. Next, we extended this approach to other diseases and demonstrated that a Staphylococcus aureus gene sequence can be introduced into M. smegmatis to generate a positive response for the SCC mec probe in the GeneXpert SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin-resistant S. aureus These biomimetic strains are cost-effective, have low biohazard content, accurately mimic drug resistance, and can be produced with relative ease, thus illustrating their potential for widespread use as verification standards for diagnosis of a variety of diseases. Copyright © 2017 American Society for Microbiology.

Acetone leaf extracts of some South African trees with high activity against Escherichia coli also have good antimycobacterial activity and selectivity index.

PubMed

Elisha, Ishaku L; Botha, Francien S; Madikizela, Balungile; McGaw, Lyndy J; Eloff, Jacobus N

2017-06-19

Tuberculosis is a world-wide problem affecting humans and animals. There is increasing development of resistance of the pathogens to current antimycobacterial agents. Many authors have investigated activities of extracts and isolated compounds from plants. The traditional uses of plants have frequently been the criterion to select plants investigated. In this contribution, we investigate whether plant extracts with very good activity against Escherichia coli may also be active against mycobacteria. The antimycobacterial activity of eight South African tree leaf extracts with high activity against Escherichia coli were determined in vitro against Mycobacterium smegmatis, M. fortuitum and M. aurum, using a serial microdilution method. The cellular cytotoxicity was also determined by the MTT assay using Vero monkey kidney cells. The selectivity index was determined by dividing the cytotoxicity of extracts by MIC. The antimycobacterial activity of the extracts ranged from 0.02 to 2.5 mg/ml. Mycobacterium smegmatis was more sensitive to the extracts (Average MIC = 0.96 mg/ml) and Mycobacterium aurum was comparatively resistant (Average MIC = 2.04 mg/ml). The extracts of Cremaspora triflora had strong antimycobacterial activity with a MIC of 0.05 mg/ml that compared reasonably well with that of streptomycin (0.01 mg/ml) and rifampicin (0.03 mg/ml), p > 0.05. Cremaspora triflora had the best selectivity index of 2.87 against Mycobacterium fortuitum. The high activity of C. triflora extracts against the fast-growing mycobacteria and good cellular safety is promising. It may be interesting to investigate extracts against pathogenic M. tuberculosis, M. bovis and M. avium cultures and to isolate active antimycobacterial compounds.

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

PubMed

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-06-07

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165(T.).

PubMed

Sassi, Mohamed; Robert, Catherine; Raoult, Didier; Drancourt, Michel

2013-01-01

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165(T). The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10(6) M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.

Genome Sequences of Four Cluster P Mycobacteriophages.

PubMed

Doyle, Erin L; Fillman, Christy L; Reyna, Nathan S; Tobiason, Deborah M; Westholm, Daniel E; Askins, Jonathan L; Backus, Brittany P; Baker, Ashlynn C; Ballard, Harrison S; Bisesi, Paul J; Bond, Logan; Byrnes, Deanna; Carlstedt, Hannah; Dodson, Kinnon S; Fallert, Megan J; Foster, Kyla J; Games, Daniel N; Grams, Tristan R; Guild, Nancy A; Hurd, Autumn; Iwata, Nicholas; Kepler, Cassidy R; Krenzke, Lucinda R; Luekens, Kelly; Lewis, Jackie; McEntee, Cali; McGee, Justin C; Nalley, Noah; Plymale, Ruth C; Prochaska, Jade; Rogers, Reid G; Schipper, Jessica B; Snyder, Kelsey; Uhrich, Kali; Vermillion, Chelsey D; Vickers, Sarah K; Wenta, Meredyth D; Yates, Tyler Z; Young, Chas F; Stoner, Ty H; Pope, Welkin H; Jacobs-Sera, Deborah; Garlena, Rebecca A; Russell, Daniel A; Cresawn, Steven G; Hatfull, Graham F

2018-01-11

Four bacteriophages infecting Mycobacterium smegmatis mc 2 155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages. Copyright © 2018 Doyle et al.

Genome Sequence of JangDynasty, a Newly Isolated Mycobacteriophage.

PubMed

Jang, Casey; Kalaj, Nancy; Hwang, Brian; Hughes, Lorelei; Yang, Connie; Pak, Thomas; Kim, John; Han, Dong Yoon; Tedjakusnadi, Jason; Fernandez, Nicholas; Dean, Natasha; Muthiah, Arun; Sutter, Nathaniel B; Diaz, Arturo

2018-05-24

JangDynasty is a bacteriophage that infects Mycobacterium smegmatis mc 2 155. It has a genome length of 70,883 bp, with 124 predicted open reading frames (ORFs), 42 of which have known functions. JangDynasty belongs to cluster O, and like other cluster O phages, it is a siphovirus with a prolate capsid. Copyright © 2018 Jang et al.

Biosynthesis of Ergothioneine from Endogenous Hercynine in Mycobacterium smegmatis

PubMed Central

Genghof, Dorothy S.; Van Damme, Olga

1968-01-01

Ergothioneine was synthesized and accumulated in growing cultures of Mycobacterium smegmatis when the medium was adequately supplied with sulfur. In a low sulfur medium, the accumulation was sharply limited although growth of the organism was apparently normal. Synthesis of hercynine, the precursor of ergothioneine, was unaffected by low sulfur levels and was markedly increased by addition of l-histidine, the precursor of hercynine. Resting-cell pellicle experiments, performed with cells grown on the low sulfur high histidine medium, showed that ergothioniene was synthesized from endogenous hercynine, when cysteine or compounds readily converted to cysteine (such as cystine, lanthionine, cystathionine, and thiazolidine carboxylic acid) were added. Homocysteine and djenkolic acid allowed for minimal synthesis of betaine, whereas methionine, S-methylcysteine, sodium sulfate, and sodium thiosulfate were unable to donate sulfur for ergothioniene synthesis under the experimental conditions employed. Addition of cysteine to a resting pellicle preparation caused the formation of 100 to 200 μg of ergothioneine per g of dry cells in 2.5 to 3 hr. A modified procedure for isolating ergothioneine and hercynine, employing a 75% ethyl alcohol extraction of wet organisms, followed by a single alumina column separation of the compounds, is described. PMID:5644441

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

PubMed Central

Berney, Michael; Greening, Chris; Conrad, Ralf; Jacobs, William R.; Cook, Gregory M.

2014-01-01

Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H2 when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD+/NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentatively-produced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments. PMID:25049411

Global proteome and phosphoproteome dynamics indicate novel mechanisms of vitamin C induced dormancy in Mycobacterium smegmatis.

PubMed

Albeldas, Claudia; Ganief, Naadir; Calder, Bridget; Nakedi, Kehilwe C; Garnett, Shaun; Nel, Andrew J M; Blackburn, Jonathan M; Soares, Nelson C

2018-05-30

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO 2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level. Copyright © 2017 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis PPE44 (Rv2770c) is involved in response to multiple stresses and promotes the macrophage expression of IL-12 p40 and IL-6 via the p38, ERK, and NF-κB signaling axis.

PubMed

Yu, Zhaoxiao; Zhang, Chenhui; Zhou, Mingliang; Li, Qiming; Li, Hui; Duan, Wei; Li, Xue; Feng, Yonghong; Xie, Jianping

2017-09-01

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H 2 O 2 , SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage. Copyright © 2017. Published by Elsevier B.V.

Antiprotozoal and antimycobacterial activities of Persea americana seeds

PubMed Central

2013-01-01

Background Persea americana seeds are widely used in traditional Mexican medicine to treat rheumatism, asthma, infectious processes as well as diarrhea and dysentery caused by intestinal parasites. Methods The chloroformic and ethanolic extracts of P. americana seeds were prepared by maceration and their amoebicidal, giardicidal and trichomonicidal activity was evaluated. These extracts were also tested against Mycobacterium tuberculosis H37Rv, four mono-resistant and two multidrug resistant strains of M. tuberculosis as well as five non tuberculosis mycobacterium strains by MABA assay. Results The chloroformic and ethanolic extracts of P. americana seeds showed significant activity against E. histolytica, G. lamblia and T. vaginalis (IC50 <0.634 μg/ml). The chloroformic extract inhibited the growth of M. tuberculosis H37Rv, M. tuberculosis MDR SIN 4 isolate, three M. tuberculosis H37Rv mono-resistant reference strains and four non tuberculosis mycobacteria (M. fortuitum, M. avium, M. smegmatis and M. absessus) showing MIC values ≤50 μg/ml. Contrariwise, the ethanolic extract affected only the growth of two mono-resistant strains of M. tuberculosis H37Rv and M. smegmatis (MIC ≤50 μg/ml). Conclusions The CHCl3 and EtOH seed extracts from P. americana showed amoebicidal and giardicidal activity. Importantly, the CHCl3 extract inhibited the growth of a MDR M. tuberculosis isolate and three out of four mono-resistant reference strains of M. tuberculosis H37Rv, showing a MIC = 50 μg/ml. This extract was also active against the NTM strains, M. fortuitum, M. avium, M. smegmatis and M. abscessus, with MIC values <50 μg/ml. PMID:23680126

Design and synthesis of positional isomers of 5 and 6-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazoles as possible antimicrobial and antitubercular agents.

PubMed

Ranjith, P Karuvalam; Rajeesh, P; Haridas, Karickal R; Susanta, Nayak K; Row, Tayur N Guru; Rishikesan, R; Kumari, N Suchetha

2013-09-15

In this Letter, we report the structure-activity relationship (SAR) studies on series of positional isomers of 5(6)-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazoles derivatives 7(a-j) and 8(a-j) synthesized in good yields and characterized by (1)H NMR, (13)C NMR and mass spectral analyses. The crystal structure of 7a was evidenced by X-ray diffraction study. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, (Gram-positive), Escherichia coli and Klebsiella pneumoniae (Gram-negative), antifungal activity against Candida albicans, Aspergillus flavus and Rhizopus sp. and antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis, Mycobacterium fortuitum and MDR-TB strains. The synthesized compounds displayed interesting antimicrobial activity. The compounds 7b, 7e and 7h displayed significant activity against Mycobacterium tuberculosis H37Rv strain. Copyright © 2013 Elsevier Ltd. All rights reserved.

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

PubMed Central

Bhattarai, Hitesh; Gupta, Richa

2014-01-01

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. PMID:24957619

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn

PubMed Central

Anders, Kirk R.; Mavrodi, Dmitri V.; Vazquez, Edwin; Amoh, Nana Yaa A.; Baliraine, Frederick N.; Buchser, William J.; Cast, Thomas P.; Chamberlain, Carmen E.; Chung, Hui-Min; D’Angelo, William A.; Farris, Christian T.; Fernandez-Martinez, Mariceli; Fischman, Haley D.; Forsyth, Mark H.; Fortier, Anna G.; Gallo, Kara F.; Held, Greta J.; Lomas, Miguel A.; Maldonado-Vazquez, Natalia Y.; Moonsammy, Claudia H.; Namboote, Peace; Paudel, Sudip; Reyes, Gabriella M.; Rubin, Michael R.; Saha, Margaret S.; Stukey, Joseph; Tobias, Tristan D.; Garlena, Rebecca A.; Stoner, Ty H.; Russell, Daniel A.

2017-01-01

ABSTRACT We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites. PMID:29217790

Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith

2010-09-17

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG{_}6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidentlymore » inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG{_}6394, solved to 2.9 {angstrom} resolution, revealed an {alpha}/{beta} hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG{_}6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.« less

The trehalose-specific transporter LpqY-SugABC is required for antimicrobial and anti-biofilm activity of trehalose analogues in Mycobacterium smegmatis.

PubMed

Wolber, Jeffrey M; Urbanek, Bailey L; Meints, Lisa M; Piligian, Brent F; Lopez-Casillas, Irene C; Zochowski, Kailey M; Woodruff, Peter J; Swarts, Benjamin M

2017-10-10

Mycobacteria, including the bacterial pathogen that causes human tuberculosis, possess distinctive pathways for synthesizing and utilizing the non-mammalian disaccharide trehalose. Trehalose metabolism is essential for mycobacterial viability and has been linked to in vitro biofilm formation, which may bear relevance to in vivo drug tolerance. Previous research has shown that some trehalose analogues bearing modifications at the 6-position inhibit growth of various mycobacterial species. In this work, 2-, 5-, and 6-position-modified trehalose analogues were synthesized using our previously reported one-step chemoenzymatic method and shown to inhibit growth and biofilm formation in the two-to three-digit micromolar range in Mycobacterium smegmatis. The trehalose-specific ABC transporter LpqY-SugABC was essential for antimicrobial and anti-biofilm activity, suggesting that inhibition by monosubstituted trehalose analogues requires cellular uptake and does not proceed via direct action on extracellular targets such as antigen 85 acyltransferases or trehalose dimycolate hydrolase. Although the potency of the described compounds in in vitro growth and biofilm assays is moderate, this study reports the first trehalose-based mycobacterial biofilm inhibitors and reinforces the concept of exploiting unique sugar uptake pathways to deliver inhibitors and other chemical cargo to mycobacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability.

PubMed

Crellin, Paul K; Vivian, Julian P; Scoble, Judith; Chow, Frances M; West, Nicholas P; Brammananth, Rajini; Proellocks, Nicholas I; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C J; Britton, Warwick J; Coppel, Ross L; Rossjohn, Jamie; Beddoe, Travis

2010-09-24

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

Mycobacteria Modify Their Cell Size Control under Sub-Optimal Carbon Sources

PubMed Central

Priestman, Miles; Thomas, Philipp; Robertson, Brian D.; Shahrezaei, Vahid

2017-01-01

The decision to divide is the most important one that any cell must make. Recent single cell studies suggest that most bacteria follow an “adder” model of cell size control, incorporating a fixed amount of cell wall material before dividing. Mycobacteria, including the causative agent of tuberculosis Mycobacterium tuberculosis, are known to divide asymmetrically resulting in heterogeneity in growth rate, doubling time, and other growth characteristics in daughter cells. The interplay between asymmetric cell division and adder size control has not been extensively investigated. Moreover, the impact of changes in the environment on growth rate and cell size control have not been addressed for mycobacteria. Here, we utilize time-lapse microscopy coupled with microfluidics to track live Mycobacterium smegmatis cells as they grow and divide over multiple generations, under a variety of growth conditions. We demonstrate that, under optimal conditions, M. smegmatis cells robustly follow the adder principle, with constant added length per generation independent of birth size, growth rate, and inherited pole age. However, the nature of the carbon source induces deviations from the adder model in a manner that is dependent on pole age. Understanding how mycobacteria maintain cell size homoeostasis may provide crucial targets for the development of drugs for the treatment of tuberculosis, which remains a leading cause of global mortality. PMID:28748182

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

PubMed Central

Trojanowski, Damian; Ginda, Katarzyna; Pióro, Monika; Hołówka, Joanna; Skut, Partycja; Jakimowicz, Dagmara

2015-01-01

ABSTRACT It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. PMID:25691599

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn.

PubMed

Anders, Kirk R; Barekzi, Nazir; Best, Aaron A; Frederick, Gregory D; Mavrodi, Dmitri V; Vazquez, Edwin; Amoh, Nana Yaa A; Baliraine, Frederick N; Buchser, William J; Cast, Thomas P; Chamberlain, Carmen E; Chung, Hui-Min; D'Angelo, William A; Farris, Christian T; Fernandez-Martinez, Mariceli; Fischman, Haley D; Forsyth, Mark H; Fortier, Anna G; Gallo, Kara F; Held, Greta J; Lomas, Miguel A; Maldonado-Vazquez, Natalia Y; Moonsammy, Claudia H; Namboote, Peace; Paudel, Sudip; Polley, Sarah-Elizabeth M; Reyes, Gabriella M; Rubin, Michael R; Saha, Margaret S; Stukey, Joseph; Tobias, Tristan D; Garlena, Rebecca A; Stoner, Ty H; Cresawn, Steven G; Jacobs-Sera, Deborah; Pope, Welkin H; Russell, Daniel A; Hatfull, Graham F

2017-12-07

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites. Copyright © 2017 Anders et al.

CYTOPLASMIC ANTIGEN RELATIONSHIPS AMONG THE ACTINOMYCETALES

PubMed Central

Kwapinski, J. B.

1964-01-01

Kwapinski, J. B. (The University of New England, Armidale, N.S.W., Australia). Cytoplasmic antigen relationships among the Actinomycetales. J. Bacteriol. 87:1234–1237. 1964.—Cytoplasm obtained from 44 strains of the Actinomycetales was tested against the homologous and heterologous antisera in a diffusion precipitation test. A pattern of serological relationships among the cytoplasmic fractions was revealed, with Mycobacterium smegmatis occupying a central position in the antigenic evolution of these microorganisms. Images PMID:4959802

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C2 to C4 Alkanes and Alkenes

PubMed Central

Martin, Kiri E.; Ozsvar, Jazmin

2014-01-01

Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc2-155. Cells of mc2-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc2-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc2-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc2-155(pSmo) provides a new model for studying sMMO-like monooxygenases. PMID:25015887

Selection of a new Mycobacterium tuberculosis H37Rv aptamer and its application in the construction of a SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor.

PubMed

Zhang, XiaoQing; Feng, Ye; Yao, QiongQiong; He, Fengjiao

2017-12-15

A rapid and accurate detection method for Mycobacterium tuberculosis (M. tuberculosis) is essential for effectively treating tuberculosis. However, current detection methods cannot meet these clinical requirements because the methods are slow or of low specificity. Consequently, a new highly specific ssDNA aptamer against M. tuberculosis reference strain H37Rv was selected by using the whole-cell systematic evolution of ligands by exponential enrichment technique. The selected aptamer was used to construct a fast and highly specific H37Rv sensor. The probe was produced by immobilizing thiol-modified aptamer on an Au interdigital electrode (Au-IDE) of a multichannel series piezoelectric quartz crystal (MSPQC) through Au-S bonding, and then single-walled carbon nanotubes (SWCNTs) were bonded on the aptamer by π-π stacking. SWCNTs were used as a signal indicator because of their considerable difference in conductivity compared with H37Rv. When H37Rv is present, it replaces the SWCNTs because it binds to the aptamer much more strongly than SWCNTs do. The replacement of SWCNTs by H37Rv resulted in a large change in the electrical properties, and this change was detected by the MSPQC. The proposed sensor is highly selective and can distinguish H37Rv from Mycobacterium smegmatis (M. smegmatis) and Bacillus Calmette-Guerin vaccine (BCG). The detection time was 70min and the detection limit was 100cfu/mL. Compared with conventional methods, this new SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor was specific, rapid, and sensitive, and it holds great potential for the early detection of H37Rv in clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

PubMed

Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Two DD-carboxypeptidases from Mycobacterium smegmatis affect cell surface properties through regulation of peptidoglycan cross-linking and glycopeptidolipids.

PubMed

Pandey, Satya Deo; Pal, Shilpa; Kumar N, Ganesh; Bansal, Ankita; Mallick, Sathi; Ghosh, Anindya S

2018-05-07

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-DAP) and 4-3 cross-links (between D-ala and meso-DAP), though there is a predominance (60-80%) of 3-3 cross-links. The DD-CPases act on pentapeptides to generate tetrapeptides that are used by LD-transpeptidases as substrates to form 3-3 cross-links. Therefore, DD-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of DD-CPases in mycobacteria is relatively unexplored. Here, we deleted two DD-CPase genes, msmeg_2433 , and msmeg_2432 , both individually and in combination, from Mycobacterium smegmatis mc 2 155. Though the single DD-CPase deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double deletion mutant, viz. , a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced and susceptibility towards β-lactams and anti-tubercular agents was enhanced. Moreover, the existence of the double mutant within murine macrophages was better as compared to the parent. Interestingly, the complementation with any one of the DD-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3: 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation,, drug susceptibility and subsistence of the cells within macrophages. Importance The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The DD-CPases generate tetrapeptides by acting on the pentapeptides, and LD-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. Here, we showed that simultaneous deletions of two DD-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of Glycopeptidolipids (a significant surface lipid present in many non-tuberculous mycobacteria including Mycobacterium smegmatis ) and affect other physiological parameters like cell morphology, growth rate, biofilm formation, antibiotic susceptibility and existence within murine macrophages. Thus, unraveling the physiology of DD-CPases might help us design anti-mycobacterial therapeutics in future. Copyright © 2018 American Society for Microbiology.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

PubMed

Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

2017-06-01

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.

PubMed

Sherwood, Anna V; Frandsen, Jane K; Grundy, Frank J; Henkin, Tina M

2018-04-10

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNA Ile ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNA Ile and the other between the Stem IIA/B pseudoknot and the D loop of tRNA Ile These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

Evaluation of anti-bacterial and anti-oxidant potential of andrographolide and echiodinin isolated from callus culture of Andrographis paniculata Nees

PubMed Central

Arifullah, Mohmmed; Namsa, Nima Dandu; Mandal, Manabendra; Chiruvella, Kishore Kumar; Vikrama, Paritala; Gopal, Ghanta Rama

2013-01-01

Objective To evaluate the anti-bacterial and anti-oxidant activity of andrographolide (AND) and echiodinin (ECH) of Andrographis paniculata. Methods In this study, an attempt has been made to demonstrate the anti-microbial and anti-oxidant activity of isolated AND and ECH by broth micro-dilution method and 2,2-diphenyl-2-picryl-hydrazyl (DPPH) assay, respectively. Structure elucidation was determined by electro-spray ionization-MSD, NMR (1H and 13C) and IR spectra. Results AND was effective against most of the strains tested including Mycobacterium smegmatis, showing broad spectrum of growth inhibition activity with Minimum inhibitory concentration values against Staphylococcus aureus (100 µg/mL), Streptococcus thermophilus (350 µg/mL) Bacillus subtilis (100 µg/mL), Escherichia coli (50 µg/mL), Mycobacterium smegmatis (200 µg/mL), Klebsiella pneumonia (100 µg/mL), and Pseudomonas aeruginosa (200 µg/mL). ECH showed specific anti-bacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa at a concentration higher than 225 µg/mL. Both AND and ECH were not effective against the two yeast strains, Candida albicans and Saccharomyces cerevisiae tested in this study. Conclusion This preliminary study showed promising anti-bacterial activity and moderate free radical scavenging activity of AND and ECH, and it may provide the scientific rationale for its popular folklore medicines. PMID:23905016

One-plasmid tunable coexpression for mycobacterial protein–protein interaction studies

PubMed Central

Chang, Yong; Mead, David; Dhodda, Vinay; Brumm, Phil; Fox, Brian G

2009-01-01

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Δ9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications. PMID:19760663

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanki, Aditya K.; Boucau, Julie; Umesiri, Francis E.

Peptide-based 1,2-dicarbonyl compounds have emerged as potent inhibitors for serine proteases. Herein, we have designed and synthesized D-arabinose and D-trehalose-based esters, {alpha}-ketoesters and {alpha}-ketoamides, and evaluated their inhibitory activity against Mycobacterium tuberculosis (Mtb) antigen 85C (ag85C), an acyltransferase in the serine hydrolase superfamily. In addition the compounds were evaluated for the ability to inhibit the growth of Mycobacterium smegmatis ATCC 14468, a non-pathogenic surrogate for Mtb. Among the synthetic analogs evaluated only the methyl ester1 derived from D-arabinose was found to inhibit the acyltransferase activity of ag85C (IC{sub 50} = 25 mM). Based on this weak inhibitory activity it wasmore » not surprising that none of the compounds inhibits the growth of M. smegmatis. In spite of the weak inhibitory activity of 1, X-ray crystallography on crystals of ag85C soaked with 1 suggested the formation of a covalent ester adduct between 1 and the Ser124 side chain hydroxyl moiety found within the catalytic site of ag85C; however, some of the active site electron density appears to result from bound glycerol. The lack of activity associated with the {alpha}-ketoester and {alpha}-ketoamide derivatives of D-trehalose may be the result of intramolecular cyclization of the {alpha}-keto moiety with the nearby C-4/4' hydroxyls leading to the formation of stable bicyclo-ester and amide derivatives.« less

A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

PubMed Central

Liu, Jun; Nikaido, Hiroshi

1999-01-01

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a β-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the α-branch of mycolic acids, followed by the joining of these two branches. PMID:10097154

Direct cell-cell contact activates SigM to express the ESX-4 secretion system in Mycobacterium smegmatis.

PubMed

Clark, Ryan R; Judd, Julius; Lasek-Nesselquist, Erica; Montgomery, Sarah A; Hoffmann, Jennifer G; Derbyshire, Keith M; Gray, Todd A

2018-06-25

Conjugal cell-cell contact between strains of Mycobacterium smegmatis induces the esxUT transcript, which encodes the putative primary substrates of the ESAT-6 secretion system 4 (ESX-4) secretion system. This recipient response was required for conjugal transfer of chromosomal DNA from the donor strain. Here we show that the extracytoplasmic σ factor, SigM, is a cell contact-dependent activator of ESX-4 expression and is required for conjugal transfer of DNA in the recipient strain. The SigM regulon includes genes outside the seven-gene core esx4 locus that we show are also required for conjugation, and we show that some of these SigM-induced proteins likely function through ESX-4. A fluorescent reporter revealed that SigM is specifically activated in recipient cells in direct contact with donor cells. Coculture RNA-seq experiments indicated that SigM regulon induction occurred early and before transconjugants are detected. This work supports a model wherein donor contact with the recipient cell surface inactivates the transmembrane anti-SigM, thereby releasing SigM. Free SigM induces an extended ESX-4 secretion system, resulting in changes that facilitate chromosomal transfer. The contact-dependent inactivation of an extracytoplasmic σ-factor that tightly controls ESX-4 activity suggests a mechanism dedicated to detect, and appropriately respond to, external stimuli from mycobacteria.

Verification of Frequency in Species of Nontuberculous Mycobacteria in Kermanshah Drinking Water Supplies Using the PCR-Sequencing Method.

PubMed

Mohajeri, Parviz; Yazdani, Laya; Shahraki, Abdolrazagh Hashemi; Alvandi, Amirhoshang; Atashi, Sara; Farahani, Abbas; Almasi, Ali; Rezaei, Mansour

2017-04-01

Nontuberculous mycobacteria are habitants of environment, especially in aquatic systems. Some of them cause problems in immunodeficient patients. Over the last decade, 16S rRNA gene sequencing was established in 45 novel species of nontuberculous mycobacteria. Experiences revealed that this method underestimates the diversity, but does not distinguish between some of mycobacterium subsp. To recognize emerging rapidly growing mycobacteria and identify their subsp, rpoB gene sequencing has been developed. To better understand the transmission of nontuberculous mycobacterial species from drinking water and preventing the spread of illness with these bacteria, the aim of this study was to detect the presence of bacteria by PCR-sequencing techniques. Drinking water samples were collected from different areas of Kermanshah city in west of IRAN. After decontamination with cetylpyridinium chloride, samples were filtered with 0.45-micron filters, the filter transferred directly on growth medium waiting to appear in colonies, then DNA extraction and PCR were performed, and products were sent to sequencing. We found 35/110 (32%) nontuberculous mycobacterial species in drinking water samples, isolates included Mycobacterium goodii, Mycobacterium aurum, and Mycobacterium gastri with the most abundance (11.5%), followed by Mycobacterium smegmatis, Mycobacterium porcinum, Mycobacterium peregrinum, Mycobacterium mucogenicum, and Mycobacterium chelonae (8%). In this study, we recognized the evidence of contamination by nontuberculous mycobacteria in corroded water pipes. As a result of the high prevalence of these bacteria in drinking water in Kermanshah, this is important evidence of transmission through drinking water. This finding can also help public health policy makers control these isolates in drinking water supplies in Kermanshah.

Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages.

PubMed

Bakala N'goma, J C; Schué, M; Carrière, F; Geerlof, A; Canaan, S

2010-12-01

Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind. 2010 Elsevier B.V. All rights reserved.

Inhibition of Isolated Mycobacterium tuberculosis Fatty Acid Synthase I by Pyrazinamide Analogs▿

PubMed Central

Ngo, Silvana C.; Zimhony, Oren; Chung, Woo Jin; Sayahi, Halimah; Jacobs, William R.; Welch, John T.

2007-01-01

An analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA), has previously been shown to inhibit mycobacterial fatty acid synthase I (FASI). FASI has been purified from a recombinant strain of M. smegmatis (M. smegmatis Δfas1 attB::M. tuberculosis fas1). Following purification, FASI activity and inhibition were assessed spectrophotometrically by monitoring NADPH oxidation. The observed inhibition was both concentration and structure dependent, being affected by both substitution at the 5 position of the pyrazine nucleus and the nature of the ester or N-alkyl group. Under the conditions studied, both 5-Cl-PZA and PZA exhibited concentration and substrate dependence consistent with competitive inhibition of FASI with Kis of 55 to 59 μM and 2,567 to 2,627 μM, respectively. The results were validated utilizing a radiolabeled fatty acid synthesis assay. This assay showed that FASI was inhibited by PZA and pyrazinoic acid as well as by a series of PZA analogs. PMID:17485499

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

PubMed

Cao, Benjamin; Chen, Xingqiang; Yamaryo-Botte, Yoshiki; Richardson, Mark B; Martin, Kirstee L; Khairallah, George N; Rupasinghe, Thusita W T; O'Flaherty, Roisin M; O'Hair, Richard A J; Ralton, Julie E; Crellin, Paul K; Coppel, Ross L; McConville, Malcolm J; Williams, Spencer J

2013-03-15

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

PubMed Central

Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

2015-01-01

Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation in Mycobacterium smegmatis.

PubMed

Syal, Kirtimaan; Bhardwaj, Neerupma; Chatterji, Dipankar

2017-01-01

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using Rel MSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to Rel MSM by isothermal titration calorimetry. Enzyme kinetics was followed where K 0.5 was found to be increased with the concomitant reduction of V max value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Determining the mode of action of anti-mycobacterial C17 diyne natural products using expression profiling: evidence for fatty acid biosynthesis inhibition.

PubMed

Li, Haoxin; Cowie, Andrew; Johnson, John A; Webster, Duncan; Martyniuk, Christopher J; Gray, Christopher A

2016-08-11

The treatment of microbial infections is becoming increasingly challenging because of limited therapeutic options and the growing number of pathogenic strains that are resistant to current antibiotics. There is an urgent need to identify molecules with novel modes of action to facilitate the development of new and more effective therapeutic agents. The anti-mycobacterial activity of the C17 diyne natural products falcarinol and panaxydol has been described previously; however, their mode of action remains largely undetermined in microbes. Gene expression profiling was therefore used to determine the transcriptomic response of Mycobacterium smegmatis upon treatment with falcarinol and panaxydol to better characterize the mode of action of these C17 diynes. Our analyses identified 704 and 907 transcripts that were differentially expressed in M. smegmatis after treatment with falcarinol and panaxydol respectively. Principal component analysis suggested that the C17 diynes exhibit a mode of action that is distinct to commonly used antimycobacterial drugs. Functional enrichment analysis and pathway enrichment analysis revealed that cell processes such as ectoine biosynthesis and cyclopropane-fatty-acyl-phospholipid synthesis were responsive to falcarinol and panaxydol treatment at the transcriptome level in M. smegmatis. The modes of action of the two C17 diynes were also predicted through Prediction of Activity Spectra of Substances (PASS). Based upon convergence of these three independent analyses, we hypothesize that the C17 diynes inhibit fatty acid biosynthesis, specifically phospholipid synthesis, in mycobacteria. Based on transcriptomic responses, it is suggested that the C17 diynes act differently than other anti-mycobacterial compounds in M. smegmatis, and do so by inhibiting phospholipid biosynthesis.

Structure of the Ergothioneine-Biosynthesis Amidohydrolase EgtC.

PubMed

Vit, Allegra; Mashabela, Gabriel T; Blankenfeldt, Wulf; Seebeck, Florian P

2015-07-06

The ubiquitous sulfur metabolite ergothioneine is biosynthesized by oxidative attachment of a sulfur atom to the imidazole ring of Nα-trimethylhistidine. Most actinobacteria, including Mycobacterium tuberculosis, use γ-glutamyl cysteine as a sulfur donor. In subsequent steps the carbon scaffold of γ-glutamyl cysteine is removed by the glutamine amidohydrolase EgtC and the β-lyase EgtE. We determined the crystal structure of EgtC from Mycobacterium smegmatis in complex with its physiological substrate. The set of active site residues that define substrate specificity in EgtC are highly conserved, even in homologues that are not involved in ergothioneine production. This conservation is compounded by the phylogenetic distribution of EgtC-like enzymes indicates that their last common ancestor might have emerged for a purpose other than ergothioneine production. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thiol-modified gold nanoparticles for the inhibition of Mycobacterium smegmatis.

PubMed

Gifford, Jennifer C; Bresee, Jamee; Carter, Carly Jo; Wang, Guankui; Melander, Roberta J; Melander, Christian; Feldheim, Daniel L

2014-12-28

Antimicrobial drug discovery has slowed considerably over the last few decades. One major cause for concern is the lack of innovative approaches to treat infections caused by mycobacteria such as TB. Herein we demonstrate that our Small Molecule Variable Ligand Display (SMLVD) method for nanoparticle antibiotic discovery can be expanded around a ligand feed ratio parameter space to identify gold nanoparticle conjugates that are potent inhibitors of mycobacteria growth, with our most potent inhibitor able to reduce growth by five orders of magnitude at 8 μM.

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase

PubMed Central

Shleeva, Margarita O.; Trutneva, Kseniya A.; Demina, Galina R.; Zinin, Alexander I.; Sorokoumova, Galina M.; Laptinskaya, Polina K.; Shumkova, Ekaterina S.; Kaprelyants, Arseny S.

2017-01-01

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores. PMID:28424668

Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

PubMed Central

Tomioka, H

1983-01-01

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme. PMID:6885719

Host-directed strategies using lipid nanoparticles to reduce mycobacteria survival

NASA Astrophysics Data System (ADS)

Pereira, L.; Diogo, J.; Mateus, R.; Pimentel, M.; Videira, M.

2015-02-01

Antibiotic-resistant infections and the stagnations in the development of new drugs have increased the demand for new therapeutic approaches against Mycobacterium tuberculosis. Innovative systems that are able to target and eradicate the bacteria in the infected host cells may represent a therapeutic breakthrough while avoiding latency. The development of nanosystems aiming a controlled and targeted intracellular drug release, have proved to increase cytosolic therapeutic concentration while reducing undesired side effects. This work's main goal was to develop a host-directed strategy against mycobacterial infection through the design of a biocompatible nanocarrier for phage-derived protein delivery, using M. smegmatis as model. Since mycobacterial pathogenicity is strongly supported by the presence of lipids in the cell wall, their degradation induces bacterial destruction through cell wall hydrolysis. Phage-based lipolytic enzymes such as, LysB a mycolylarabinogalactan esterase, represent an appealing therapeutic approach. The herein proposed Ms6 LysB-containing lipid nanocarrier (SLN_LysB) explores the known advantages of nanomedicine-based systems for phagocytic cells selectively targeting thus allowing LysB intracellular accumulation and a more pronounced mycobacterial infection eradication. Adsorption efficiency value indicates the potential of this system as a protein nanocarrier. Moreover, promising outcomes were obtained in host-infected macrophages treated with SLN_LysB. The results show that the herein proposed strategy was more effective in inhibiting the growth of M. smegmatis than free LysB, which might be related to the nanocarrier internalization. Acting as effective protein nanocarriers, the protein-guided delivery in the infected phagocytic cells allows it to exert its hydrolytic action on the lipid layer of the Mycobacterium.

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

PubMed

Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

2018-05-01

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity.

PubMed

Fiołka, Marta J; Zagaja, Mirosław P; Piersiak, Tomasz D; Wróbel, Marek; Pawelec, Jarosław

2010-09-01

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa. Copyright 2010 Elsevier Inc. All rights reserved.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

PubMed

Ghosh, Gairika; Reddy, Jayavardhana; Sambhare, Susmit; Sen, Ranjan

2018-01-01

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis , Mycobacterium bovis , Mycobacterium tuberculosis , Xanthomonas campestris , Xanthomonas oryzae , Corynebacterium glutamicum , Vibrio cholerae , Salmonella enterica , and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis , M. bovis , X. campestris , and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions. IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens. Copyright © 2017 American Society for Microbiology.

Anti-mycobacterial activity of polyketides from Penicillium sp. endophyte isolated from Garcinia nobilis against Mycobacteriumsmegmatis.

PubMed

Jouda, Jean Bosco; Mawabo, Isabelle Kamga; Notedji, Augustin; Mbazoa, Céline Djama; Nkenfou, Jean; Wandji, Jean; Nkenfou, Céline Nguefeu

2016-06-01

According to estimates by the World Health Organization, there were 9.6 million new tuberculosis (TB) cases in 2014: 5.4 million among men, 3.2 million among women, and 1.0 million among children. There were also 1.5 million TB deaths. Although there are potent anti-TB molecules, the misuse of these drugs in addition to inconsistent or partial treatment have led to the development of multidrug-resistant TB and extensively drug-resistant TB. It is established that plants harbor microorganisms, collectively known as endophytes, which also produce metabolites. Exploring the as-yet untapped natural products from the endophytes increases the chances of finding novel and active compounds. The present study was aimed to investigate the antimycobacterial activity of the crude extract and compounds isolated from Penicillium sp. endophyte associated with Garcinia nobilis against Mycobacterium smegmatis. Liquid culture obtained from the fermentation of Penicillium sp. was extracted using ethylacetate and the liquid chromatography-mass spectrometry monitored fractionation of crude extracts yielded six compounds. Their structures were elucidated with spectroscopic analyses including two-dimensional nuclear magnetic resonance, high resolution mass spectrometry by dereplication using Antibase, and by comparison to literature data. All compounds and the crude extract from the liquid medium were evaluated for their antimycobacterial activity against M. smegmatis. In this study, the activity of penialidins A-C (1-3), citromycetin (4), p-hydroxy phenyl glyoxalaldoxime (5), and Brefeldin A (6) were tested against nonpathogenic M. smegmatis. Penialidin C was the most active compound with a minimum inhibitory concentration of 15.6μg/mL. Isolated compounds from Penicillium sp. harbored in G. nobilis exhibited promising antimycobacterial activity against M. smegmatis thus supporting the immensity of the potential of antimycobacterial drug discovery from endophytes from medicinal plants. Penialidin C could further be investigated for antimycobacterial drug development. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

MspA Nanopores from Subunit Dimers

PubMed Central

Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

2012-01-01

Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID:22719928

Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

PubMed

Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

2015-07-13

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

PubMed

Bansal, Ankita; Kar, Debasish; Murugan, Rajagopal A; Mallick, Sathi; Dutta, Mouparna; Pandey, Satya Deo; Chowdhury, Chiranjit; Ghosh, Anindya S

2015-05-01

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities. © 2015 The Authors.

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

PubMed Central

Syal, Kirtimaan; Flentie, Kelly; Bhardwaj, Neerupma; Maiti, Krishnagopal; Jayaraman, Narayanaswamy; Stallings, Christina L.

2017-01-01

ABSTRACT Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry. PMID:28396544

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria.

PubMed

Syal, Kirtimaan; Flentie, Kelly; Bhardwaj, Neerupma; Maiti, Krishnagopal; Jayaraman, Narayanaswamy; Stallings, Christina L; Chatterji, Dipankar

2017-06-01

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by Rel Msm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis , where inhibition in each case was observed. In vivo , (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry. Copyright © 2017 American Society for Microbiology.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis*

PubMed Central

Williams, Sunanda Margrett; Chandran, Anu V.; Vijayabaskar, Mahalingam S.; Roy, Sourav; Balaram, Hemalatha; Vishveshwara, Saraswathi; Vijayan, Mamannamana; Chatterji, Dipankar

2014-01-01

Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8–2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release. PMID:24573673

Mutational Analysis of a Role for Salicylic Acid in Iron Metabolism of Mycobacterium smegmatis

PubMed Central

Adilakshmi, Tadepalli; Ayling, Peter D.; Ratledge, Colin

2000-01-01

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc2155 and NCIMB 8548) and three mutant strains (mc21292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 μg/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc2155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 μg/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal. PMID:10629169

Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

PubMed Central

Rawat, Mamta; Newton, Gerald L.; Ko, Mary; Martinez, Gladys J.; Fahey, Robert C.; Av-Gay, Yossef

2002-01-01

Mycothiol (MSH; 1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1d-myo-inosityl 2-deoxy-α-d-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase (MshC) activities of the three mutant strains were ≤2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. PMID:12384335

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.

PubMed

Shleeva, Margarita O; Kondratieva, Tatyana K; Demina, Galina R; Rubakova, Elvira I; Goncharenko, Anna V; Apt, Alexander S; Kaprelyants, Arseny S

2017-01-01

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis ( Mtb ), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro , the AC-overexpressing pMind Rv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo , AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMind Rv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo .

The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca{sup 2+} binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeruva, Veena C., E-mail: veenachaitanya@ccmb.res.in; Savanagouder, Mamata; Khandelwal, Radhika

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome butmore » is unique in containing a βγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca{sup 2+} binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and {sup 45}CaCl{sub 2} overlay, we demonstrate that Ca{sup 2+} binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several βγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca{sup 2+} dependent variation in surface phenotype, revealing a functional role for Ca{sup 2+}in DesA1 activity. This study represents the first identification of a Ca{sup 2+} binding βγ-crystallin in M.tb, emphasizing the implicit role of Ca{sup 2+} in the pathogenesis of M.tb. - Highlights: • Mycobacterium tuberculosis DesA1 is an essential acyl-ACP desaturase. • DesA1 was identified to contain a βγ-crystallin Greek key signature motif. • Ca{sup 2+} binds to DesA1 with an affinity of 53 μM and induces changes in its conformation. • M. smegmatis overexpressing M.tb DesA1 shows a Ca{sup 2+} dependent phenotype. • Targetting the Ca{sup 2+} dependent function of DesA1 could be of therapeutic value.« less

ML1419c peptide immunization induces Mycobacterium leprae-specific HLA-A*0201-restricted CTL in vivo with potential to kill live mycobacteria.

PubMed

Geluk, Annemieke; van den Eeden, Susan J F; Dijkman, Karin; Wilson, Louis; Kim, Hee Jin; Franken, Kees L M C; Spencer, John S; Pessolani, Maria C V; Pereira, Geraldo M B; Ottenhoff, Tom H M

2011-08-01

MHC class I-restricted CD8(+) T cells play an important role in protective immunity against mycobacteria. Previously, we showed that p113-121, derived from Mycobacterium leprae protein ML1419c, induced significant IFN-γ production by CD8(+) T cells in 90% of paucibacillary leprosy patients and in 80% of multibacillary patients' contacts, demonstrating induction of M. leprae-specific CD8(+) T cell immunity. In this work, we studied the in vivo role and functional profile of ML1419c p113-121-induced T cells in HLA-A*0201 transgenic mice. Immunization with 9mer or 30mer covering the p113-121 sequence combined with TLR9 agonist CpG induced HLA-A*0201-restricted, M. leprae-specific CD8(+) T cells as visualized by p113-121/HLA-A*0201 tetramers. Most CD8(+) T cells produced IFN-γ, but distinct IFN-γ(+)/TNF-α(+) populations were detected simultaneously with significant secretion of CXCL10/IFN-γ-induced protein 10, CXCL9/MIG, and VEGF. Strikingly, peptide immunization also induced high ML1419c-specific IgG levels, strongly suggesting that peptide-specific CD8(+) T cells provide help to B cells in vivo, as CD4(+) T cells were undetectable. An additional important characteristic of p113-121-specific CD8(+) T cells was their capacity for in vivo killing of p113-121-labeled, HLA-A*0201(+) splenocytes. The cytotoxic function of p113-121/HLA-A*0201-specific CD8(+) T cells extended into direct killing of splenocytes infected with live Mycobacterium smegmatis expressing ML1419c: both 9mer and 30mer induced CD8(+) T cells that reduced the number of ML1419c-expressing mycobacteria by 95%, whereas no reduction occurred using wild-type M. smegmatis. These data, combined with previous observations in Brazilian cohorts, show that ML1419c p113-121 induces potent CD8(+) T cells that provide protective immunity against M. leprae and B cell help for induction of specific IgG, suggesting its potential use in diagnostics and as a subunit (vaccine) for M. leprae infection.

A Novel P1B-type Mn2+-transporting ATPase Is Required for Secreted Protein Metallation in Mycobacteria*

PubMed Central

Padilla-Benavides, Teresita; Long, Jarukit E.; Raimunda, Daniel; Sassetti, Christopher M.; Argüello, José M.

2013-01-01

Transition metals are central for bacterial virulence and host defense. P1B-ATPases are responsible for cytoplasmic metal efflux and play roles either in limiting cytosolic metal concentrations or in the maturation of secreted metalloproteins. The P1B-ATPase, CtpC, is required for Mycobacterium tuberculosis survival in a mouse model (Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989–12994). CtpC prevents Zn2+ toxicity, suggesting a role in Zn2+ export from the cytosol (Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M., Waddell, S. J., Foti, M., Lugo-Villarino, G., Gao, Q., Maridonneau-Parini, I., Butcher, P. D., Castagnoli, P. R., Gicquel, B., de Chastellièr, C., and Neyrolles, O. (2011) Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10, 248–259). However, key metal-coordinating residues and the overall structure of CtpC are distinct from Zn2+-ATPases. We found that isolated CtpC has metal-dependent ATPase activity with a strong preference for Mn2+ over Zn2+. In vivo, CtpC is unable to complement Escherichia coli lacking a functional Zn2+-ATPase. Deletion of M. tuberculosis or Mycobacterium smegmatis ctpC leads to cytosolic Mn2+ accumulation but no alterations in other metals levels. Whereas ctpC-deficient M. tuberculosis is sensitive to extracellular Zn2+, the M. smegmatis mutant is not. Both ctpC mutants are sensitive to oxidative stress, which might explain the Zn2+-sensitive phenotype of the M. tuberculosis ctpC mutant. CtpC is a high affinity/slow turnover ATPase, suggesting a role in protein metallation. Consistent with this hypothesis, mutation of CtpC leads to a decrease of Mn2+ bound to secreted proteins and of the activity of secreted Fe/Mn-superoxide dismutase, particularly in M. smegmatis. Alterations in the assembly of metalloenzymes involved in redox stress response might explain the sensitivity of M. tuberculosis ctpC mutants to oxidative stress and growth and persistence defects in mice infection models. PMID:23482562

New Isoflavonoids from the extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity.

PubMed

Coronado-Aceves, Enrique Wenceslao; Gigliarelli, Giulia; Garibay-Escobar, Adriana; Zepeda, Ramón Enrique Robles; Curini, Massimo; López Cervantes, Jaime; Inés Espitia-Pinzón, Clara Inés; Superchi, Stefano; Vergura, Stefania; Marcotullio, Maria Carla

2017-07-12

The evaluation of the antimycobacterial activity of extracts of medicinal plants used by Mayos against tuberculosis and respiratory problems, allowed the identification of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC (Fabaceae) as the best candidate to find new antimycobacterial compounds. To isolate and characterize the compounds of R. precatoria responsible for the inhibitory and bactericidal activity against Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis ATCC 700084. To determine antimycobacterial synergistic effect of pure compounds and their selectivity index towards Vero cells. A total of six flavonoids were purified by silica gel column chromatography. Structural elucidation of the isolated compounds was achieved by using 1D and 2D NMR spectroscopy techniques. The configuration at the C-3 chiral center was established by quantum mechanical calculation of the electronic circular dichroism (ECD) spectrum. In vitro inhibitory and bactericidal activity against M. tuberculosis and M. smegmatis were determined with the redox indicator Alamar Blue (resazurin). Synergy was determined by X/Y quotient. Cytotoxicity was measured by MTT assay. The isolated compounds were identified as precatorin A (1), precatorin B (2), precatorin C (3), lupinifolin (4), cajanone (5) and lupinifolinol (6). Compounds 1-3 are new. Compounds 1 to 5 inhibited the growth of M. tuberculosis (MIC ≥31.25µg/mL); compounds 1, 2, 4 and 5 killed the bacteria (MBC ≥31.25µg/mL) and also inhibited M. smegmatis (MIC ≥125µg/mL), while 1 and 4 also resulted bactericidal (MBC ≥125µg/mL). Compounds 4 and 5 presented synergistic effect (X/Y quotient value <0.5) at a concentration of 1/2 MIC of each compound in the combination. Cytotoxicity in murine macrophages (RAW 264.7 cells) gave IC 50 values of 13.3-46.98µM, for compounds 1-5. In this work we isolated two new isoflavanones (1 and 2), and one new isoflavone (3) with a weak antimycobacterial activity. The (3R) absolute configuration was assigned to 1 by computational analysis of its ECD spectrum and to 2 and 5 by similarity of their ECD spectra with that of 1. We are also reporting by first time, activity against virulent strain of M. tuberculosis for compounds 4 and 5 and their antimycobacterial synergistic effect. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Evolutionary interpretations of mycobacteriophage biodiversity and host-range through the analysis of codon usage bias.

PubMed

Esposito, Lauren A; Gupta, Swati; Streiter, Fraida; Prasad, Ashley; Dennehy, John J

2016-10-01

In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate students have isolated and sequenced the genomes of more than 1,150 mycobacteriophages, creating the largest database of sequenced bacteriophages able to infect a single host, Mycobacterium smegmatis , a soil bacterium. Genomic analysis indicates that these mycobacteriophages can be grouped into 26 clusters based on genetic similarity. These clusters span a continuum of genetic diversity, with extensive genomic mosaicism among phages in different clusters. However, little is known regarding the primary hosts of these mycobacteriophages in their natural habitats, nor of their broader host ranges. As such, it is possible that the primary host of many newly isolated mycobacteriophages is not M. smegmatis , but instead a range of closely related bacterial species. However, determining mycobacteriophage host range presents difficulties associated with mycobacterial cultivability, pathogenicity and growth. Another way to gain insight into mycobacteriophage host range and ecology is through bioinformatic analysis of their genomic sequences. To this end, we examined the correlations between the codon usage biases of 199 different mycobacteriophages and those of several fully sequenced mycobacterial species in order to gain insight into the natural host range of these mycobacteriophages. We find that UPGMA clustering tends to match, but not consistently, clustering by shared nucleotide sequence identify. In addition, analysis of GC content, tRNA usage and correlations between mycobacteriophage and mycobacterial codon usage bias suggests that the preferred host of many clustered mycobacteriophages is not M. smegmatis but other, as yet unknown, members of the mycobacteria complex or closely allied bacterial species.

Evolutionary interpretations of mycobacteriophage biodiversity and host-range through the analysis of codon usage bias

PubMed Central

Esposito, Lauren A.; Gupta, Swati; Streiter, Fraida; Prasad, Ashley

2016-01-01

In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate students have isolated and sequenced the genomes of more than 1,150 mycobacteriophages, creating the largest database of sequenced bacteriophages able to infect a single host, Mycobacterium smegmatis, a soil bacterium. Genomic analysis indicates that these mycobacteriophages can be grouped into 26 clusters based on genetic similarity. These clusters span a continuum of genetic diversity, with extensive genomic mosaicism among phages in different clusters. However, little is known regarding the primary hosts of these mycobacteriophages in their natural habitats, nor of their broader host ranges. As such, it is possible that the primary host of many newly isolated mycobacteriophages is not M. smegmatis, but instead a range of closely related bacterial species. However, determining mycobacteriophage host range presents difficulties associated with mycobacterial cultivability, pathogenicity and growth. Another way to gain insight into mycobacteriophage host range and ecology is through bioinformatic analysis of their genomic sequences. To this end, we examined the correlations between the codon usage biases of 199 different mycobacteriophages and those of several fully sequenced mycobacterial species in order to gain insight into the natural host range of these mycobacteriophages. We find that UPGMA clustering tends to match, but not consistently, clustering by shared nucleotide sequence identify. In addition, analysis of GC content, tRNA usage and correlations between mycobacteriophage and mycobacterial codon usage bias suggests that the preferred host of many clustered mycobacteriophages is not M. smegmatis but other, as yet unknown, members of the mycobacteria complex or closely allied bacterial species. PMID:28348827

Antimycobacterial action of a new glycolipid-peptide complex obtained from extracellular metabolites of Raoultella ornithinolytica.

PubMed

Fiołka, Marta J; Grzywnowicz, Krzysztof; Mendyk, Ewaryst; Zagaja, Mirosław; Szewczyk, Rafał; Rawski, Michał; Keller, Radosław; Rzymowska, Jolanta; Wydrych, Jerzy

2015-12-01

In this paper, an antimycobacterial component of extracellular metabolites of a gut bacterium Raoultella ornithinolytica from D. veneta earthworms was isolated and its antimycobacterial action was tested using Mycobacterium smegmatis. After incubation with the complex obtained, formation of pores and furrows in cell walls was observed using microscopic techniques. The cells lost their shape, stuck together and formed clusters. Surface-enhanced Raman spectroscopy analysis showed that, after incubation, the complex was attached to the cell walls of the Mycobacterium. Analyses of the component performed with Fourier transform infrared spectroscopy demonstrated high similarity to a bacteriocin nisin, but energy dispersive X-ray spectroscopy analysis revealed differences in the elemental composition of this antimicrobial peptide. The component with antimycobacterial activity was identified using mass spectrometry techniques as a glycolipid-peptide complex. As it exhibits no cytotoxicity on normal human fibroblasts, the glycolipid-peptide complex appears to be a promising compound for investigations of its activity against pathogenic mycobacteria. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

The PPE2 protein of Mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production

PubMed Central

Bhat, Khalid Hussain; Srivastava, Shruti; Kotturu, Sandeep Kumar; Ghosh, Sudip; Mukhopadhyay, Sangita

2017-01-01

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is one of the most successful pathogens of humans. It has evolved several adaptive skills and evasion mechanisms to hijack the immunologically educated host to suit its intracellular lifestyle. Here, we show that one of the unique PPE family member proteins of M. tuberculosis, PPE2, can limit nitric oxide (NO) production by inhibiting inos gene transcription. PPE2 protein has a leucine zipper DNA-binding motif and a functional nuclear localization signal. PPE2 was translocated into the macrophage nucleus via the classical importin α/β pathway where it interacted with a GATA-binding site overlapping with the TATA box of inos promoter and inhibited NO production. PPE2 prolonged intracellular survival of a surrogate bacterium M. smegmatis in vitro as well as in vivo. This information are likely to improve our knowledge of host-pathogen interactions during M. tuberculosis infection which is crucial for designing effective anti-TB therapeutics. PMID:28071726

Spectroscopic study, antimicrobial activity and crystal structures of N-(2-hydroxy-5-nitrobenzalidene)4-aminomorpholine and N-(2-hydroxy-1-naphthylidene)4-aminomorpholine

NASA Astrophysics Data System (ADS)

Yıldız, Mustafa; Ünver, Hüseyin; Dülger, Başaran; Erdener, Diğdem; Ocak, Nazan; Erdönmez, Ahmet; Durlu, Tahsin Nuri

2005-03-01

Schiff bases N-(2-hydroxy-3-nitrobenzalidene)4-aminomorpholine ( 1) and N-(2-hydroxy-1-naphthylidene)4-aminomorpholine ( 2) were synthesized from the reaction of 4-aminomorpholine with 2-hydroxy-5-nitrobenzaldehyde and 2-hydroxy-1-naphthaldehyde. Compounds 1 and 2 were characterized by elemental analysis, IR, 1H NMR, 13C NMR and UV-Visible techniques. The UV-Visible spectra of the Schiff bases with OH group in ortho position to the imino group were studied in polar and nonpolar solvents in acidic and basic media. The structures of compounds 1 and 2 have been examined cyrstallographically, for two compounds exist as dominant form of enol-imines in both the solutions and solid state. The title compounds 1 and 2 crystallize in the monoclinic space group P2 1/ c and P2 1/ n with unit cell parameters: a=8.410(1) and 11.911(3), b=6.350(9) and 4.860(9), c=21.728(3) and 22.381(6) Å, β=90.190(1) and 95.6(2)°, V=1160.6(3) and 1289.5(5) Å 3, Dx=1.438 and 1.320 g cm -3, respectively. The crystal structures were solved by direct methods and refined by full-matrix least squares. The antimicrobial activities of compounds 1 and 2 have also been studied. The antimicrobial activities of the ligands have been screened in vitro against the organisms Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis CCM 2067, Bacillus cereus ATCC 7064, Listeria monocytogenes ATCC 15313, Candida albicans ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora guilliermondii DSM 3432.

Mycobacterium abscessus Phospholipase C Expression Is Induced during Coculture within Amoebae and Enhances M. abscessus Virulence in Mice

PubMed Central

Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Canaan, Stéphane

2014-01-01

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria. PMID:25486995

Antimycobacterial Activity and Low Cytotoxicity of Leaf Extracts of Some African Anacardiaceae Tree Species.

PubMed

Kabongo-Kayoka, Prudence N; Eloff, Jacobus N; Obi, Chikwelu L; McGaw, Lyndy J

2016-12-01

Treatment of tuberculosis (TB) is a challenge because of multidrug-resistant and extremely drug-resistant strains of Mycobacterium tuberculosis. Plant species contain antimicrobial compounds that may lead to new anti-TB drugs. Previous screening of some tree species from the Anacardiaceae family revealed the presence of antimicrobial activity, justifying further investigations. Leaf extracts of 15 Anacardiaceae tree species were screened for antimycobacterial activity using a twofold serial microdilution assay against the pathogenic Mycobacterium bovis and multidrug resistant M. tuberculosis and rapidly growing mycobacteria, Mycobacterium smegmatis, Mycobacterium fortuitum and Mycobacterium aurum. The vaccine strain, M. bovis and an avirulent strain, H37Ra M. tuberculosis, were also used. Cytotoxicity was assessed using a colorimetric assay against Vero kidney, human hepatoma and murine macrophage cells. Four out of 15 crude acetone extracts showed significant antimycobacterial activity with minimum inhibitory concentration varying from 50 to 100 µg/mL. Searsia undulata had the highest activity against most mycobacteria, followed by Protorhus longifolia. M. fortuitum was the strongest predictor of activity against multidrug-resistant TB (correlation coefficient = 0.65). Bioautography against M. aurum and M. fortuitum worked well as indicators of the Rf values of active compounds yielding strong zones of inhibition. The leaf extracts of S. undulata and P. longifolia had more than ten different antimycobacterial compounds and had low cytotoxicity with LC 50 values above 100 µg/mL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

rpoB-Based Identification of Nonpigmented and Late-Pigmenting Rapidly Growing Mycobacteria

PubMed Central

Adékambi, Toïdi; Colson, Philippe; Drancourt, Michel

2003-01-01

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial β subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from “Mycobacterium houstonense.” Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM. PMID:14662964

Interaction between mycobacteria and mucus on a human respiratory tissue organ culture model with an air interface.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Wilson, R; Thornton, D J; Kirkham, S; Sheehan, J K

2004-01-01

Mycobacteria adhere specifically to extracellular matrix (ECM) and mucus with a fibrous, but not globular, appearance, in organ cultures of human respiratory mucosa examined by scanning electron microscopy. Previously, light microscopy sections made of tissue infected for 7 days demonstrated mycobacteria associated with mucus on the organ culture surface, and within submucosal glands in areas of damaged epithelium. The authors have now investigated the interactions between Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (MTB), and Mycobacterium smegmatis (MS) and mucus by preincubating bacteria with purified mucins MUC5AC and MUC5B prior to inoculation onto the organ culture mucosal surface. They have also measured mucin production by the organ culture after mycobacterial infection. Mucus did not cause clumping of mycobacteria. There was a significant (P=.03) increase in the amount of fibrous mucus, but not globular mucus, observed on tissue inoculated with mucins compared to controls. The number of bacteria adhering to ECM was markedly reduced after incubation with mucins, which could indicate a protective effect. Mycobacterial infection did not increase mucin production by the organ culture. Mycobacterial adherence to mucins may play a role in the pathogenicity of mycobacteria in diseases such as cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), in which there are changes in mucus composition and clearance.

A 38-Kilobase Pathogenicity Island Specific for Mycobacterium avium subsp. paratuberculosis Encodes Cell Surface Proteins Expressed in the Host

PubMed Central

Stratmann, Janin; Strommenger, Birgit; Goethe, Ralph; Dohmann, Karen; Gerlach, Gerald-F.; Stevenson, Karen; Li, Ling-ling; Zhang, Qing; Kapur, Vivek; Bull, Tim J.

2004-01-01

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe3+ and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. PMID:14977927

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductasemore » activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.« less

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis.

PubMed

Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C; Jacobs, William R; Kremer, Laurent

2010-12-01

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development. © 2010 Blackwell Publishing Ltd.

Deletion and overexpression studies on DacB2, a putative low molecular mass penicillin binding protein from Mycobacterium tuberculosis H(37)Rv.

PubMed

Bourai, Neema; Jacobs, William R; Narayanan, Sujatha

2012-02-01

Mycobacterium tuberculosis genome encodes several high and low molecular mass penicillin binding proteins. One such low molecular mass protein is DacB2 encoded by open reading frame Rv2911 of M. tuberculosis which is predicted to play a role in peptidoglycan synthesis. In this study we have tried to gain an insight into the role of this accessory cell division protein in mycobacterial physiology by performing overexpression and deletion studies. The overproduction of DacB2 in non-pathogenic, fast growing mycobacterium Mycobacterium smegmatis mc(2)155 resulted in reduced growth, an altered colony morphology, a defect in sliding motility and biofilm formation. A point mutant of DacB2 was made wherein the active site serine residue was mutated to cysteine to abolish the penicillin binding function of protein. The overexpression of mutant protein showed similar results indicating that the effects produced were independent of protein's penicillin binding function. The gene encoding DacB2 was deleted in M. tuberculosis by specialized transduction method. The deletion mutant showed reduced growth in Sauton's medium under acidic and low oxygen availability. The in vitro infection studies with THP-1 cells showed increased intracellular survival of dacB2 mutant compared to parent and complemented strains. The colony morphology and antibiotic sensitivity of mutant and wild-type strains were similar. Copyright © 2011 Elsevier Ltd. All rights reserved.

Domain Requirements for DNA Unwinding by Mycobacterial UvrD2, an Essential DNA Helicase†

PubMed Central

Sinha, Krishna Murari; Stephanou, Nicolas C.; Unciuleac, Mihaela-Carmen; Glickman, Michael S.; Shuman, Stewart

2008-01-01

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3′ to 5′ helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis. PMID:18702526

Identification of the anti‐mycobacterial functional properties of piperidinol derivatives

PubMed Central

Guy, Collette S; Tichauer, Esther; Kay, Gemma L; Phillips, Daniel J; Bailey, Trisha L; Harrison, James; Furze, Christopher M; Millard, Andrew D; Gibson, Matthew I; Pallen, Mark J

2017-01-01

Background and Purpose Tuberculosis (TB) remains a major global health threat and is now the leading cause of death from a single infectious agent worldwide. The current TB drug regimen is inadequate, and new anti‐tubercular agents are urgently required to be able to successfully combat the increasing prevalence of drug‐resistant TB. The purpose of this study was to investigate a piperidinol compound derivative that is highly active against the Mycobacterium tuberculosis bacillus. Experimental Approach The antibacterial properties of the piperidinol compound and its corresponding bis‐Mannich base analogue were evaluated against M. smegmatis and Gram‐negative organisms. Cytotoxicity studies were undertaken in order to determine the selectivity index for these compounds. Spontaneous resistant mutants of M. smegmatis were generated against the piperidinol and corresponding bis‐Mannich base lead derivatives and whole genome sequencing employed to determine the genetic modifications that lead to selection pressure in the presence of these compounds. Key Results The piperidinol and the bis‐Mannich base analogue were found to be selective for mycobacteria and rapidly kill this organism with a cytotoxicity selectivity index for mycobacteria of >30‐fold. Whole genome sequencing of M. smegmatis strains resistant to the lead compounds led to the identification of a number of single nucleotide polymorphisms indicating multiple targets. Conclusion and Implications Our results indicate that the piperidinol moiety represents an attractive compound class in the pursuit of novel anti‐tubercular agents. Linked Articles This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc PMID:28195652

Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro▿

PubMed Central

Shin, Sung Jae; Collins, Michael T.

2008-01-01

The in vitro susceptibility of human- and bovine-origin Mycobacterium paratuberculosis to the thioupurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) was established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn's disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP, even at high concentrations (≥64.0 μg/ml). Among rapidly growing mycobacteria, Mycobacterium phlei was susceptible to 6-MP and AZA whereas Mycobacterium smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-aminosalycilic acid [mesalamine]) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin, used as control drugs, were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6-MP and ciprofloxacin resulted in significantly higher CFU than use of ciprofloxacin alone. These data may partially explain the paradoxical response of Crohn's disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs, i.e., they do not worsen with anti-inflammatory treatment as would be expected with a microbiological etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn's disease: AZA drugs may confound interpretation of data on therapeutic responses for both antibiotic-treated and control groups. PMID:18070971

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids.

PubMed

Côtes, Karen; Dhouib, Rabeb; Douchet, Isabelle; Chahinian, Henri; de Caro, Alain; Carrière, Frédéric; Canaan, Stéphane

2007-12-15

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

PubMed

Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Herrmann, Jean-Louis; Canaan, Stéphane; Girard-Misguich, Fabienne

2015-02-01

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

PubMed Central

Côtes, Karen; Dhouib, Rabeb; Douchet, Isabelle; Chahinian, Henri; deCaro, Alain; Carrière, Frédéric; Canaan, Stéphane

2007-01-01

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units·mg−1 at 37 °C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids. PMID:17784850

Antimycobacterial terpenoids from Juniperus communis L. (Cuppressaceae).

PubMed

Gordien, Andréa Y; Gray, Alexander I; Franzblau, Scott G; Seidel, Véronique

2009-12-10

Juniperus communis is a plant which has been reported as a traditional cure for tuberculosis (TB) and other respiratory diseases. The aim of this study was to isolate and identify the constituents responsible for the activity of the n-hexane extract of Juniperus communis roots against Mycobacterium tuberculosis H(37)Rv and Juniperus communis aerial parts against Mycobacterium aurum. Subsequently, it was to evaluate the activity of the pure isolated compounds against (i) drug-resistant Mycobacterium tuberculosis variants, (ii) non-replicating Mycobacterium tuberculosis and (iii) a range of non-tuberculous mycobacteria (NTM). The antimycobacterial activity of Juniperus communis extracts, fractions and constituents was determined against Mycobacterium tuberculosis H(37)Rv, and against rifampicin-, isoniazid-, streptomycin- and moxifloxacin-resistant variants, using the microplate broth Alamar Blue assay (MABA) method. Isolated constituents were tested against non-replicating Mycobacterium tuberculosis H(37)Rv, using the low oxygen recovery assay (LORA), and against NTM (Mycobacterium aurum, Mycobacterium phlei, Mycobacterium fortuitum and Mycobacterium smegmatis), using a broth microdilution method. Cytotoxicity studies were performed using mammalian Vero cells. The antimycobacterial activity of Juniperus communis was attributed to a sesquiterpene identified as longifolene (1) and two diterpenes, characterised as totarol (2) and trans-communic acid (3). All compounds were identified following analysis of their spectroscopic data (1D- and 2D-NMR, MS) and by comparison with the literature and commercial authentic standards when available. Revised assignments for 3 are reported. Totarol showed the best activity against Mycobacterium tuberculosis H(37)Rv (MIC of 73.7 microM). It was also most active against the isoniazid-, streptomycin-, and moxifloxacin-resistant variants (MIC of 38.4, 83.4 and 60 microM, respectively). Longifolene and totarol were most active against the rifampicin-resistant variant (MICs of 24 and 20.2 microM, respectively). Totarol showed the best activity in the LORA assay (MIC of 81.3 microM) and against all NTM species (MICs in the range of 7-14 microM). Trans-communic acid showed good activity against Mycobacterium aurum (MIC of 13.2 microM). The low selectivity indices (SI) obtained following cytotoxicity studies indicated that the isolated terpenoids were relatively toxic towards mammalian cells. This is the first report of the isolation of (1) and (2) from Juniperus communis roots, and of (3) from the aerial parts. The antimycobacterial activity of (1) and (3), and the activity of (2) against Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium phlei, is reported for the first time. The effect of totarol on drug-resistant variants and non-replicating Mycobacterium tuberculosis has never been published. The presence of antimycobacterial terpenoids in Juniperus communis aerial parts and roots justifies, to some extent, the ethnomedicinal use of this species as a traditional anti-TB remedy.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

PubMed

Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart

2016-05-15

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Macrophage sphingolipids are essential for the entry of mycobacteria.

PubMed

Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha

2018-07-01

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Prophage-mediated defense against viral attack and viral counter-defense

PubMed Central

Dedrick, Rebekah M.; Jacobs-Sera, Deborah; Guerrero Bustamante, Carlos A.; Garlena, Rebecca A.; Mavrich, Travis N.; Pope, Welkin H.; Reyes, Juan C Cervantes; Russell, Daniel A.; Adair, Tamarah; Alvey, Richard; Bonilla, J. Alfred; Bricker, Jerald S.; Brown, Bryony R.; Byrnes, Deanna; Cresawn, Steven G.; Davis, William B.; Dickson, Leon A.; Edgington, Nicholas P.; Findley, Ann M.; Golebiewska, Urszula; Grose, Julianne H.; Hayes, Cory F.; Hughes, Lee E.; Hutchison, Keith W.; Isern, Sharon; Johnson, Allison A.; Kenna, Margaret A.; Klyczek, Karen K.; Mageeney, Catherine M.; Michael, Scott F.; Molloy, Sally D.; Montgomery, Matthew T.; Neitzel, James; Page, Shallee T.; Pizzorno, Marie C.; Poxleitner, Marianne K.; Rinehart, Claire A.; Robinson, Courtney J.; Rubin, Michael R.; Teyim, Joseph N.; Vazquez, Edwin; Ware, Vassie C.; Washington, Jacqueline; Hatfull, Graham F.

2017-01-01

Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution. PMID:28067906

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution

PubMed Central

Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Peebles, Craig L.; Al-Atrache, Zein; Alcoser, Turi A.; Alexander, Lisa M.; Alfano, Matthew B.; Alford, Samantha T.; Amy, Nichols E.; Anderson, Marie D.; Anderson, Alexander G.; Ang, Andrew A. S.; Ares, Manuel; Barber, Amanda J.; Barker, Lucia P.; Barrett, Jonathan M.; Barshop, William D.; Bauerle, Cynthia M.; Bayles, Ian M.; Belfield, Katherine L.; Best, Aaron A.; Borjon, Agustin; Bowman, Charles A.; Boyer, Christine A.; Bradley, Kevin W.; Bradley, Victoria A.; Broadway, Lauren N.; Budwal, Keshav; Busby, Kayla N.; Campbell, Ian W.; Campbell, Anne M.; Carey, Alyssa; Caruso, Steven M.; Chew, Rebekah D.; Cockburn, Chelsea L.; Cohen, Lianne B.; Corajod, Jeffrey M.; Cresawn, Steven G.; Davis, Kimberly R.; Deng, Lisa; Denver, Dee R.; Dixon, Breyon R.; Ekram, Sahrish; Elgin, Sarah C. R.; Engelsen, Angela E.; English, Belle E. V.; Erb, Marcella L.; Estrada, Crystal; Filliger, Laura Z.; Findley, Ann M.; Forbes, Lauren; Forsyth, Mark H.; Fox, Tyler M.; Fritz, Melissa J.; Garcia, Roberto; George, Zindzi D.; Georges, Anne E.; Gissendanner, Christopher R.; Goff, Shannon; Goldstein, Rebecca; Gordon, Kobie C.; Green, Russell D.; Guerra, Stephanie L.; Guiney-Olsen, Krysta R.; Guiza, Bridget G.; Haghighat, Leila; Hagopian, Garrett V.; Harmon, Catherine J.; Harmson, Jeremy S.; Hartzog, Grant A.; Harvey, Samuel E.; He, Siping; He, Kevin J.; Healy, Kaitlin E.; Higinbotham, Ellen R.; Hildebrandt, Erin N.; Ho, Jason H.; Hogan, Gina M.; Hohenstein, Victoria G.; Holz, Nathan A.; Huang, Vincent J.; Hufford, Ericka L.; Hynes, Peter M.; Jackson, Arrykka S.; Jansen, Erica C.; Jarvik, Jonathan; Jasinto, Paul G.; Jordan, Tuajuanda C.; Kasza, Tomas; Katelyn, Murray A.; Kelsey, Jessica S.; Kerrigan, Larisa A.; Khaw, Daryl; Kim, Junghee; Knutter, Justin Z.; Ko, Ching-Chung; Larkin, Gail V.; Laroche, Jennifer R.; Latif, Asma; Leuba, Kohana D.; Leuba, Sequoia I.; Lewis, Lynn O.; Loesser-Casey, Kathryn E.; Long, Courtney A.; Lopez, A. Javier; Lowery, Nicholas; Lu, Tina Q.; Mac, Victor; Masters, Isaac R.; McCloud, Jazmyn J.; McDonough, Molly J.; Medenbach, Andrew J.; Menon, Anjali; Miller, Rachel; Morgan, Brandon K.; Ng, Patrick C.; Nguyen, Elvis; Nguyen, Katrina T.; Nguyen, Emilie T.; Nicholson, Kaylee M.; Parnell, Lindsay A.; Peirce, Caitlin E.; Perz, Allison M.; Peterson, Luke J.; Pferdehirt, Rachel E.; Philip, Seegren V.; Pogliano, Kit; Pogliano, Joe; Polley, Tamsen; Puopolo, Erica J.; Rabinowitz, Hannah S.; Resiss, Michael J.; Rhyan, Corwin N.; Robinson, Yetta M.; Rodriguez, Lauren L.; Rose, Andrew C.; Rubin, Jeffrey D.; Ruby, Jessica A.; Saha, Margaret S.; Sandoz, James W.; Savitskaya, Judith; Schipper, Dale J.; Schnitzler, Christine E.; Schott, Amanda R.; Segal, J. Bradley; Shaffer, Christopher D.; Sheldon, Kathryn E.; Shepard, Erica M.; Shepardson, Jonathan W.; Shroff, Madav K.; Simmons, Jessica M.; Simms, Erika F.; Simpson, Brandy M.; Sinclair, Kathryn M.; Sjoholm, Robert L.; Slette, Ingrid J.; Spaulding, Blaire C.; Straub, Clark L.; Stukey, Joseph; Sughrue, Trevor; Tang, Tin-Yun; Tatyana, Lyons M.; Taylor, Stephen B.; Taylor, Barbara J.; Temple, Louise M.; Thompson, Jasper V.; Tokarz, Michael P.; Trapani, Stephanie E.; Troum, Alexander P.; Tsay, Jonathan; Tubbs, Anthony T.; Walton, Jillian M.; Wang, Danielle H.; Wang, Hannah; Warner, John R.; Weisser, Emilie G.; Wendler, Samantha C.; Weston-Hafer, Kathleen A.; Whelan, Hilary M.; Williamson, Kurt E.; Willis, Angelica N.; Wirtshafter, Hannah S.; Wong, Theresa W.; Wu, Phillip; Yang, Yun jeong; Yee, Brandon C.; Zaidins, David A.; Zhang, Bo; Zúniga, Melina Y.; Hendrix, Roger W.; Hatfull, Graham F.

2011-01-01

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists. PMID:21298013

Functional dissection of protein domains involved in the immunomodulatory properties of PE_PGRS33 of Mycobacterium tuberculosis.

PubMed

Zumbo, Antonella; Palucci, Ivana; Cascioferro, Alessandro; Sali, Michela; Ventura, Marcello; D'Alfonso, Pamela; Iantomasi, Raffaella; Di Sante, Gabriele; Ria, Francesco; Sanguinetti, Maurizio; Fadda, Giovanni; Manganelli, Riccardo; Delogu, Giovanni

2013-12-01

PE_PGRSs are a large family of proteins identified in Mycobacterium tuberculosis complex and in few other pathogenic mycobacteria. The PE domain of PE_PGRS33 mediates localization of the protein on the mycobacterial cell surface, where the PGRS domain is available to interact with host components. In this study, PE_PGRS33 and its functional deletion mutants were expressed in M. smegmatis, and in vitro and in vivo assays were used to dissect the protein domains involved in the immunomodulatory properties of the protein. We demonstrate that PE_PGRS33-mediated secretion of TNF-α by macrophages occurs by extracellular interaction with TLR2. Our results also show that while the PGRS domain of the protein is required for triggering TNF-α secretion, mutation in the PE domain affects the pro-inflammatory properties of the protein. These results indicate that PE_PGRS33 is a protein with immunomodulatory activity and that protein stability and localization on the mycobacterial surface can affect these properties. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria

PubMed Central

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S.; Bhatt, Apoorva

2012-01-01

Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. PMID:22520756

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

PubMed

Pope, Welkin H; Jacobs-Sera, Deborah; Russell, Daniel A; Peebles, Craig L; Al-Atrache, Zein; Alcoser, Turi A; Alexander, Lisa M; Alfano, Matthew B; Alford, Samantha T; Amy, Nichols E; Anderson, Marie D; Anderson, Alexander G; Ang, Andrew A S; Ares, Manuel; Barber, Amanda J; Barker, Lucia P; Barrett, Jonathan M; Barshop, William D; Bauerle, Cynthia M; Bayles, Ian M; Belfield, Katherine L; Best, Aaron A; Borjon, Agustin; Bowman, Charles A; Boyer, Christine A; Bradley, Kevin W; Bradley, Victoria A; Broadway, Lauren N; Budwal, Keshav; Busby, Kayla N; Campbell, Ian W; Campbell, Anne M; Carey, Alyssa; Caruso, Steven M; Chew, Rebekah D; Cockburn, Chelsea L; Cohen, Lianne B; Corajod, Jeffrey M; Cresawn, Steven G; Davis, Kimberly R; Deng, Lisa; Denver, Dee R; Dixon, Breyon R; Ekram, Sahrish; Elgin, Sarah C R; Engelsen, Angela E; English, Belle E V; Erb, Marcella L; Estrada, Crystal; Filliger, Laura Z; Findley, Ann M; Forbes, Lauren; Forsyth, Mark H; Fox, Tyler M; Fritz, Melissa J; Garcia, Roberto; George, Zindzi D; Georges, Anne E; Gissendanner, Christopher R; Goff, Shannon; Goldstein, Rebecca; Gordon, Kobie C; Green, Russell D; Guerra, Stephanie L; Guiney-Olsen, Krysta R; Guiza, Bridget G; Haghighat, Leila; Hagopian, Garrett V; Harmon, Catherine J; Harmson, Jeremy S; Hartzog, Grant A; Harvey, Samuel E; He, Siping; He, Kevin J; Healy, Kaitlin E; Higinbotham, Ellen R; Hildebrandt, Erin N; Ho, Jason H; Hogan, Gina M; Hohenstein, Victoria G; Holz, Nathan A; Huang, Vincent J; Hufford, Ericka L; Hynes, Peter M; Jackson, Arrykka S; Jansen, Erica C; Jarvik, Jonathan; Jasinto, Paul G; Jordan, Tuajuanda C; Kasza, Tomas; Katelyn, Murray A; Kelsey, Jessica S; Kerrigan, Larisa A; Khaw, Daryl; Kim, Junghee; Knutter, Justin Z; Ko, Ching-Chung; Larkin, Gail V; Laroche, Jennifer R; Latif, Asma; Leuba, Kohana D; Leuba, Sequoia I; Lewis, Lynn O; Loesser-Casey, Kathryn E; Long, Courtney A; Lopez, A Javier; Lowery, Nicholas; Lu, Tina Q; Mac, Victor; Masters, Isaac R; McCloud, Jazmyn J; McDonough, Molly J; Medenbach, Andrew J; Menon, Anjali; Miller, Rachel; Morgan, Brandon K; Ng, Patrick C; Nguyen, Elvis; Nguyen, Katrina T; Nguyen, Emilie T; Nicholson, Kaylee M; Parnell, Lindsay A; Peirce, Caitlin E; Perz, Allison M; Peterson, Luke J; Pferdehirt, Rachel E; Philip, Seegren V; Pogliano, Kit; Pogliano, Joe; Polley, Tamsen; Puopolo, Erica J; Rabinowitz, Hannah S; Resiss, Michael J; Rhyan, Corwin N; Robinson, Yetta M; Rodriguez, Lauren L; Rose, Andrew C; Rubin, Jeffrey D; Ruby, Jessica A; Saha, Margaret S; Sandoz, James W; Savitskaya, Judith; Schipper, Dale J; Schnitzler, Christine E; Schott, Amanda R; Segal, J Bradley; Shaffer, Christopher D; Sheldon, Kathryn E; Shepard, Erica M; Shepardson, Jonathan W; Shroff, Madav K; Simmons, Jessica M; Simms, Erika F; Simpson, Brandy M; Sinclair, Kathryn M; Sjoholm, Robert L; Slette, Ingrid J; Spaulding, Blaire C; Straub, Clark L; Stukey, Joseph; Sughrue, Trevor; Tang, Tin-Yun; Tatyana, Lyons M; Taylor, Stephen B; Taylor, Barbara J; Temple, Louise M; Thompson, Jasper V; Tokarz, Michael P; Trapani, Stephanie E; Troum, Alexander P; Tsay, Jonathan; Tubbs, Anthony T; Walton, Jillian M; Wang, Danielle H; Wang, Hannah; Warner, John R; Weisser, Emilie G; Wendler, Samantha C; Weston-Hafer, Kathleen A; Whelan, Hilary M; Williamson, Kurt E; Willis, Angelica N; Wirtshafter, Hannah S; Wong, Theresa W; Wu, Phillip; Yang, Yun jeong; Yee, Brandon C; Zaidins, David A; Zhang, Bo; Zúniga, Melina Y; Hendrix, Roger W; Hatfull, Graham F

2011-01-27

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

PubMed

Pope, Welkin H; Mavrich, Travis N; Garlena, Rebecca A; Guerrero-Bustamante, Carlos A; Jacobs-Sera, Deborah; Montgomery, Matthew T; Russell, Daniel A; Warner, Marcie H; Hatfull, Graham F

2017-08-15

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution. Copyright © 2017 Pope et al.

Evaluation of the RT-LAMP and LAMP methods for detection of Mycobacterium tuberculosis.

PubMed

Wu, Dandan; Kang, Jiwen; Li, Baosheng; Sun, Dianxing

2018-05-01

The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop-mediated isothermal amplification (LAMP) cannot differentiate live Mtb. This study compared reverse transcription (RT)-LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non-Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT-LAMP. In the clinical samples. The rates of positivity for Mtb of the SCM, LAMP, and RT-LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT-LAMP and SCM, but RT-LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods. The specificities of the methods were similar. The sensitivity of RT-LAMP was ~10-fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT-LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT-LAMP, the latter is the most suitable for wide use in the lower-level hospitals and clinics of China for detecting Mtb in sputum samples. © 2017 Wiley Periodicals, Inc.

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

PubMed

Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

2015-02-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

PubMed Central

Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

2014-01-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling.

PubMed

Balážová, Tereza; Makovcová, Jitka; Šedo, Ondrej; Slaný, Michal; Faldyna, Martin; Zdráhal, Zbyněk

2014-04-01

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints. However, cell-wall characteristics of mycobacterial species, and their well known stability, complicate MALDI-TOF MS profiling analysis. In this study, we tested two recently published protocols for inactivation and disruption of mycobacteria, and we also examined the influence of different culture conditions (four culture media and five cultivation times) on mass spectral quality and the discriminatory power of the method. We found a significant influence of sample pretreatment method and culture medium on species identification and differentiation for a total of 10 strains belonging to Mycobacterium phlei and Mycobacterium smegmatis. Optimum culture conditions yielding the highest identification success rate against the BioTyper database (Bruker Daltonics) and permitting the possibility of automatic acquisition of mass spectra were found to be distinct for the two mycobacterial species examined. Similarly, individual changes in growth conditions had diverse effects on the two species. For these reasons, thorough control over cultivation conditions should always be employed to maximize the performance and discriminatory power of MALDI-TOF MS profiling, and cultivation conditions must be optimized separately for individual groups of mycobacterial species/strains. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Anti-mycobacteria potential and synergistic effects of combined crude extracts of selected medicinal plants used by Bapedi traditional healers to treat tuberculosis related symptoms in Limpopo Province, South Africa.

PubMed

Komape, Nancy Patience Motlalepula; Bagla, Victor Patrick; Kabongo-Kayoka, Prudence; Masoko, Peter

2017-02-24

Tuberculosis is an infectious communicable disease and the causative agent of the disease has over the years developed resistance to streamline chemotherapeutic agents with dire consequences and there is a need for development of new and more potent alternatives. Constituents of leaves material of Combretum heroroense, Citrus lemon and Apodytes dimidiata were serially extracted using solvents of varying polarity. TLC finger print profile of the different extracts were determined by spraying eluted plates with vanillin sulphuric acid and 2, 2- diphenylpicryl hydrazyl (DPPH) for the presence of antioxidant constituents. Presence of different phytochemicals was determined using standard chemical test. Bioautography was used to determine the number of compounds present in sub-fractions active against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values extract and sub-fractions were determined using serial microplate dilution method against M. smegmatis (ATCC 1441), M. tuberculosis (ATCC H37Rv) and multi-drug resistant TB (MDR-TB) field strain. Synergy of the crude extracts of the three plants was determined using microplate dilution method against M. smegmatis. Mass extracted by different solvents was less than 6% dry weight for all the plants. Phlobatannins were not detected in A. dimidiata, C. heroroense and C. lemon as well as cardiac glycosides in C. lemon and A. dimidiata, and saponins in C. heroroense. Sub-fractions of the different plants were shown to contain constituents with antioxidant activity with the highest number detected in C. heroroense. Bioautography results reveal the presence of a compound(s) in the ethyle acetate sub-fraction of C. heroroense and butanol, methanol/water, ethyl acetate and water no.2 subfractions of A. dimidiata, active against M. smegmatis that were not shown to have antioxidant capacity. MIC results for different crude extracts of the three plants against M. smegmatis ranges from 0.1 to 3 mg/ml. The average MIC for the synergistic effect of the plants ranged from 0.04 mg/ml to 1.25 mg/ml. An activity greater than that obtained for the reference drugs was shown for the butanol and hexane fractions of A. dimidiata (0.47 mg/ml) against the field strain of MDR-TB while that obtained for the M.TB (ATCC H37Rv) was 0.31 mg/ml. A significant finding shown in this study reveals the potent anti-mycobacteria potential of sub-fractions of A. dimidiata against MDR-TB field strain that can lead to the isolation of compounds that can be used to counter resistant strains of tuberculosis.

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria.

PubMed

Artsatbanov, V Yu; Vostroknutova, G N; Shleeva, M O; Goncharenko, A V; Zinin, A I; Ostrovsky, D N; Kapreliants, A S

2012-04-01

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

PubMed

Larimer, Curtis; Islam, Mohammad Shyful; Ojha, Anil; Nettleship, Ian

2014-08-01

Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain.

Clinical and Taxonomic Status of Pathogenic Nonpigmented or Late-Pigmenting Rapidly Growing Mycobacteria

PubMed Central

Brown-Elliott, Barbara A.; Wallace, Richard J.

2002-01-01

The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing. PMID:12364376

Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria.

PubMed

Brown-Elliott, Barbara A; Wallace, Richard J

2002-10-01

The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing.

Antimycobacterial potency and cytotoxicity study of three medicinal plants.

PubMed

Tsouh Fokou, Patrick Valere; Appiah-Opong, Regina; Yeboah-Manu, Dorothy; Kissi-Twum, Abena Adomah; Yamthe, Lauve Rachel Tchokouaha; Mokale Kognou, Aristide Laurel; Addo, Phyllis; Boyom, Fabrice Fekam; Nyarko, Alexander Kwadwo

2016-12-01

Mycobacterial infections including tuberculosis, leprosy, and buruli ulcer are among the most prevalent, debilitating, and deadly tropical diseases, especially in Sub-Saharan Africa. The development of drug resistance to the currently available drugs and the poor compliance emphasize the need for new chemotherapeutic agents. This study was designed to evaluate the in vitro activity of Cleistopholis patens, Annona reticulata, and Greenwayodendron suaveolens against Mycobacterium smegmatis. The safety on normal liver cells was also assessed. The crude extracts, fractions, and subfractions were tested against M. smegmatis and for cell cytotoxicity on WRL-68, normal human hepatocyte using microdilution resazurin-based assays. The phytochemical screening was performed using standard methods. Most of the extracts, fractions, and subfractions inhibited the growth of M. smegmatis with minimum inhibitory concentration (MIC) values ranging from 6.25μg/mL to 125μg/mL. The subfractions P12 and P29 from G. suaveolens twig were more potent with MIC values of 6.25μg/mL and 25μg/mL, respectively. Fruit crude extract and root CH 2 Cl 2 fraction from A. reticulata also showed activity with MIC values of 50μg/mL and 25μg/mL, respectively. Crude extracts from the twig and stem bark of C. patens displayed inhibition at MIC values of 125μg/mL and 100μg/mL, respectively. Majority of active extracts showed no cell cytotoxicity, except the extract from C. patens with IC 50 ranging from 41.40μg/mL to 93.78μg/mL. The chemical investigation of the promising extracts revealed the presence of phenols, alkaloids, glycosides, triterpenes, and acetogenins. The results achieved from this preliminary antimycobacterial drug discovery study supported the traditional claims of C. patens, A. reticulata, and G. suaveolens in the treatment of mycobacterial infections. Meanwhile, further fractionation is required to characterize the active ingredients. Copyright © 2016.

MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.

PubMed

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S; Bhatt, Apoorva

2012-04-20

Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. Copyright © 2012 Elsevier Ltd. All rights reserved.

The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

PubMed

López, Marcela; Quitian, Laudy-Viviana; Calderón, Martha-Nancy; Soto, Carlos-Y

2018-04-01

P 1B -type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P 1B -ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ Cd 2+ and Pb 2+ stimulate the ATPase activity of the putative P 1B -type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd 2+ and Cu 2+ . As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max /K m ratio 7.4-fold higher for Cd 2+ than for Cu 2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd 2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.

Rapid presumptive identification of the Mycobacterium tuberculosis-bovis complex by radiometric determination of heat stable urease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gandy, J.H.; Pruden, E.L.; Cox, F.R.

1983-12-01

Simple and rapid Bactec methodologies for the determination of neat (unaltered) and heat stable urease activity of mycobacteria are presented. Clinical isolates (63) and stock cultures (32)--consisting of: M. tuberculosis (19), M. bovis (5), M. kansasii (15), M. marinum (4), M. simiae (3), M. scrofulaceum (16), M. gordonae (6), M. szulgai (6), M. flavescens (1), M. gastri (1), M. intracellulare (6), M. fortuitum-chelonei complex (12), and M. smegmatis (1)--were tested for neat urease activity by Bactec radiometry. Mycobacterial isolates (50-100 mg wet weight) were incubated at 35 degrees C for 30 minutes with microCi14C-urea. Urease-positive mycobacteria gave Bactec growth indexmore » (GI) values greater than 100 units, whereas urease-negative species gave values less than 10 GI units. Eighty-three isolates possessing neat urease activity were heated at 80 degrees C for 30 minutes followed by incubation at 35 degrees C for 30 minutes with 1 microCi14C-urea. Mycobacterium tuberculosis-bovis complex demonstrated heat-stable urease activity (GI more than 130 units) and could be distinguished from mycobacteria other than tuberculosis (MOTT), which gave GI values equal to or less than 40 units.« less

Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

PubMed Central

Saito, H; Tomioka, H; Watanabe, T; Yoneyama, T

1983-01-01

Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters. Images PMID:6826523

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

PubMed Central

Smith, Laura J.; Stapleton, Melanie R.; Fullstone, Gavin J. M.; Crack, Jason C.; Thomson, Andrew J.; Le Brun, Nick E.; Hunt, Debbie M.; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S.; Green, Jeffrey

2010-01-01

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was severely inhibited, irrespective of the presence or absence of the CRP protein Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442

Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages▿

PubMed Central

Korch, Shaleen B.; Contreras, Heidi; Clark-Curtiss, Josephine E.

2009-01-01

Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages. PMID:19114484

Characterization of a chromosomal toxin-antitoxin, Rv1102c-Rv1103c system in Mycobacterium tuberculosis.

PubMed

Han, Jeong-Sun; Lee, Jae Jin; Anandan, Tripti; Zeng, Minghui; Sripathi, Srinivas; Jahng, Wan Jin; Lee, Sang Hee; Suh, Joo-Won; Kang, Choong-Min

2010-09-24

Toxin-antitoxin systems, ubiquitous in prokaryotic genomes, have been proposed to play an important role in several stress responses. While Mycobacterium tuberculosis contains more than 80 putative TA loci, the roles they play in this pathogen are yet to be studied. Here, we characterize a chromosomal Rv1102c-Rv1103c TA system in M. tuberculosis. We found that the Rv1102c toxin interacts with the Rv1103c antitoxin in a pull-down assay and the yeast two-hybrid system. Rv1102c cleaved the era mRNA in Escherichia coli, and cleavage was inhibited by co-expression of Rv1103c. Heterologous expression of Rv1102c led to growth arrest in E. coli, which was fully recovered only when Rv1103c was co-expressed in cis with Rv1102c, suggesting that the production and assembly of Rv1102c and Rv1103c are tightly linked. Our additional results indicate that translational coupling of the Rv1102c and Rv1103c genes is important for Rv1102c-Rv1103c binding. Finally, we discovered that the expression of Rv1102c induced growth arrest and increased the level of persister cells in Mycobacterium smegmatis. These results suggest that the Rv1102c-Rv1103c TA system could play a role in M. tuberculosis pathogenesis via generating bacilli that survive in the face of multidrug therapy. Copyright © 2010. Published by Elsevier Inc.

Mycobacterium tuberculosis PE13 (Rv1195) manipulates the host cell fate via p38-ERK-NF-κB axis and apoptosis.

PubMed

Li, Hui; Li, Qiming; Yu, Zhaoxiao; Zhou, Mingliang; Xie, Jianping

2016-07-01

PE/PPE family proteins are mycobacteria unique molecules, named after their N-terminal conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains. Mycobacterium tuberculosis (Mtb) PE family gene encoded cell surface proteins are previously reported to be involved in virulence and interaction with host. To explore the role of a novel PE member (PE13, Rv1195), M. smegmatis was used as surrogate host. The study showed that Rv1195 was a cell wall associated protein. Rv1195 can enhance the survival of recombinants under stress conditions such as H2O2, SDS, low pH. This is largely due to the upregulated transcription of Rv1195, since diverse stresses can increase the promoter activity of Rv1195 gene, consistent with enhanced survival within macrophages. Ms_Rv1195 infection also increased the production of interlukin-6 (IL-6) and IL-1β from macrophages, while decreased the secretion of suppressor of cytokine signaling 3 (SOCS3) in comparison with the vector-only control. The cell death was also precipitated by the Ms_Rv1195 infection. Inhibitors treatment showed that the p38-ERK-NF-κB axis was involved in the Rv1195 triggered change of IL-6 and IL-1β expression. In summary, we showed that PE13 (Rv1195) is a new PE family member actively engaged in the interaction between Mycobacterium and host, signaling through p38-ERK-NF-κB axis and apoptosis.

Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico

PubMed Central

Alvarado-Esquivel, Cosme; García-Corral, Nora; Carrero-Dominguez, David; Enciso-Moreno, José Antonio; Gurrola-Morales, Teodoro; Portillo-Gómez, Leopoldo; Rossau, Rudi; Mijs, Wouter

2009-01-01

Background Little information is available on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites in humans. This study used molecular methods to determine the Mycobacterium species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease. Methods Bacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the Mycobacterium species present. DNA samples positive for M. tuberculosis complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the rpoB gene associated with rifampicin resistance. Results Of the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for Mycobacterium species by PCR or line probe assay, respectively. In tissues, M. tuberculosis complex and M. genus were found in lymph nodes, and M. genus was found in brain and vagina specimens. In body fluids, M. tuberculosis complex was found in synovial fluid. M. gordonae, M. smegmatis, M. kansasii, M. genus, M. fortuitum/M. peregrinum complex and M. tuberculosis complex were found in urine. M. chelonae/M. abscessus was found in pericardial fluid and M. kansasii was found in gastric aspirate. Two of M. tuberculosis complex isolates were also PCR and LiPA positive for the rpoB gene. These two isolates were from lymph nodes and were sensitive to rifampicin. Conclusion 1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found. PMID:19272158

Low Molecular Weight Glucosamine/L-lactide Copolymers as Potential Carriers for the Development of a Sustained Rifampicin Release System: Mycobacterium Smegmatis as a Tuberculosis Model

NASA Astrophysics Data System (ADS)

Ragusa, Jorge Alejandro

Tuberculosis, a highly contagious disease, ranks as the second leading cause of death from an infectious disease, and remains a major global health problem. In 2013, 9 million new cases were diagnosed and 1.5 million people died worldwide from tuberculosis. This dissertation aims at developing a new, ultrafine particle-based efficient antibiotic delivery system for the treatment of tuberculosis. The carrier material to make the rifampicin (RIF)-loaded particles is a low molecular weight star-shaped polymer produced from glucosamine (molecular core building unit) and L-lactide (GluN-LLA). Stable particles with a very high 50% drug loading capacity were made via electrohydrodynamic atomization. Prolonged release (>14 days) of RIF from these particles is demonstrated. Drug release data fits the Korsmeyer-Peppas equation, which suggests the occurrence of a modified diffusion-controlled RIF release mechanism, and is also supported by differential scanning calorimetry and drug leaching tests. Cytotoxicity tests on Mycobacterium smegmatis showed that antibiotic-free GluN-LLA and polylactides (PLA) (reference material) particles did not show any significant anti-bacterial activity. The minimum inhibitory concentration and minimum bactericidal concentration values obtained for RIF-loaded particles showed 2- to 4-fold improvements in the anti-bacterial activity relative to the free drug. Cytotoxicity tests on macrophages indicated an increment in cell death as particle dose increased, but was not significantly affected by material type or particle size. Confocal microscopy was used to track internalization and localization of particles in the macrophages. GluN-LLA particles led to higher uptakes than the PLA particles. In addition, after phagocytosis, the GluN-LLA particles stayed in the cytoplasm and the particles showed a favorable long term drug release effect in killing intracellular bacteria compared to free RIF. The studies presented and discussed in this dissertation suggest that these drug carrier materials are potentially very attractive candidates for the development of high-payload, sustained-release antibiotic/resorbable polymer particle systems for treating bacterial lung infections.

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil

PubMed Central

2013-01-01

Background Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli. Results Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp. Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds. Conclusion The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products. PMID:23618368

Nisin depletes ATP and proton motive force in mycobacteria.

PubMed

Chung, H J; Montville, T J; Chikindas, M L

2000-12-01

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Direct observation of gold nanoparticle assemblies with the porin MspA on mica.

PubMed

Basel, Matthew T; Dani, Raj Kumar; Kang, Myungshim; Pavlenok, Mikhail; Chikan, Viktor; Smith, Paul E; Niederweis, Michael; Bossmann, Stefan H

2009-02-24

The octameric porin MspA from Mycobacterium smegmatis is sufficiently stable to form a nonmembrane-supported stand-alone porin on mica surfaces. About 98% of all MspA octamers were found to stand upright on mica, with their periplasmic loop regions bound to the hydrophilic mica surface. Both, small (d = 3.7 nm) and large (d = 17 nm) gold nanoparticles bind to MspA, however, in different positions: small gold nanoparticles bind within the MspA pore, whereas the large gold nanoparticles bind to the upper region of MspA. These experiments demonstrate that gold nanoparticles can be positioned at different, well-defined distances from the underlying surface using the MspA pore as a template. These findings represent a significant step toward the use of electrically insulating stable proteins in combination with metal nanoparticles in nanodevices.

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by Apolipoprotein N-Acyltransferase BCG_2070c

PubMed Central

2013-01-01

Background Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. Results We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Conclusions Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria. PMID:24093492

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

PubMed

Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

2013-10-05

Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains.

PubMed

Lopes Ferreira, Nicolas; Mathis, Hugues; Labbé, Diane; Monot, Frédéric; Greer, Charles W; Fayolle-Guichard, Françoise

2007-06-01

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C2 to C16). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C1 to C16) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.

Identification of compounds that decrease numbers of Mycobacteria in human macrophages in the presence of serum amyloid P.

PubMed

Xiang, Wang; Cox, Nehemiah; Gomer, Richard H

2017-09-01

Mϕs are a heterogeneous population of cells and include classically activated Mϕs (M1) and alternatively activated Mϕs (M2). Mϕs can change from M1 to M2 and vice versa in response to environmental stimuli. Serum amyloid P (SAP) is a constitutive plasma protein that polarizes Mϕs to an M2 phenotype, and part of this effect is mediated through FcγRI receptors. In an effort to find ways to alter Mϕs phenotypes, we screened for compounds that can block the SAP-FcγRI interaction. From a screen of 3000 compounds, we found 12 compounds that reduced the ability of fluorescently labeled human SAP to bind cells expressing human FcγRI. Based on cell surface marker expression, 8 of the compounds inhibited the effect of SAP on skewing human Mϕs to an M2 phenotype and in the presence of SAP polarized Mϕs to an M1 phenotype. In diseases, such as tuberculosis, M1s are more effective at killing bacteria than M2s. SAP potentiated the numbers of the mycobacterial strains Mycobacterium smegmatis and Mycobacterium tuberculosis in Mϕs. When added along with SAP, 2 of the compounds reduced intracellular Mycobacterium numbers. Together, these results indicate that the blocking of SAP effects on Mϕs can skew these cells toward an M1 phenotype, and this may be useful in treating diseases, such as tuberculosis. © Society for Leukocyte Biology.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

PubMed Central

Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R.; Pyle, Margaret M.; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A.; Ioerger, Thomas R.; Fortune, Sarah M.; Derbyshire, Keith M.; Wade, Joseph T.; Gray, Todd A.

2015-01-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression. PMID:26536359

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

PubMed

Shell, Scarlet S; Wang, Jing; Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R; Pyle, Margaret M; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A; Ioerger, Thomas R; Fortune, Sarah M; Derbyshire, Keith M; Wade, Joseph T; Gray, Todd A

2015-11-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil.

PubMed

Sgarioni, Sônia Aparecida; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Leite, Clarice Queico Fujimura; de Prince, Karina Andrade; de Andrade Leite, Sergio Roberto; Filho, Dirceu Vedovello; Siqueira, Vera Lucia Dias; Caleffi-Ferracioli, Katiany Rizzieri; Cardoso, Rosilene Fressatti

2014-01-01

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

PubMed Central

Sgarioni, Sônia Aparecida; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Leite, Clarice Queico Fujimura; de Prince, Karina Andrade; de Andrade Leite, Sergio Roberto; Filho, Dirceu Vedovello; Siqueira, Vera Lucia Dias; Caleffi-Ferracioli, Katiany Rizzieri; Cardoso, Rosilene Fressatti

2014-01-01

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5–10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil. PMID:25242962

Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

PubMed

Furuya, Toshiki; Nakao, Tomomi; Kino, Kuniki

2015-10-01

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

PubMed Central

Williams, Kerstin J.; Bennett, Mark H.; Barton, Geraint R.; Jenkins, Victoria A.; Robertson, Brian D.

2013-01-01

Summary PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation. PMID:23352854

Post-translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis*

PubMed Central

Vergnolle, Olivia; Xu, Hua; Tufariello, JoAnn M.; Favrot, Lorenza; Malek, Adel A.; Jacobs, William R.; Blanchard, John S.

2016-01-01

Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection. PMID:27566542

The diacylglycerol acyltransferase Rv3371 of Mycobacterium tuberculosis is required for growth arrest and involved in stress-induced cell wall alterations.

PubMed

Rastogi, Shivangi; Singh, Amit Kumar; Chandra, Gyan; Kushwaha, Pragati; Pant, Garima; Singh, Kavita; Mitra, Kalyan; Sashidhara, Koneni V; Krishnan, Manju Y

2017-05-01

Triacylglycerol (TAG) is important to mycobacteria both as cell envelope component and energy reservoir. Mycobacterium tuberculosis (Mtb) genome encodes at least 15 putative TAG synthase (tgs)s. We report that one of these genes, Rv3371, specific to pathogenic mycobacteria, when expressed in M. smegmatis leads to modifications in colony morphotype, bacterial architecture, cell surface properties and elevated TAG levels. Rv3371 was found to largely localize in the cell membrane. The Rv3371 promoter is minimally active during exponential growth in vitro, however, is up-regulated under stationary phase, hypoxia, nutrient starvation, nitrosative stress, low iron, in IFN-γ activated macrophages and infected mice. The low iron-induced expression of Rv3371 is likely due to the de-repression by Rv1404, which is probably activated by ideR. An Rv3371 deletion mutant of Mtb showed impaired non-replicating persistence in vitro and altered sensitivity to anti-mycobacterial drugs. In low iron medium, the Rv3371 deletion mutant showed reduced formation of TAG containing extracellular vesicles. Therefore Rv3371 is likely involved in Mtb growth arrest and cell wall alterations during persistence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87).

PubMed

Pan, Qian; Zhao, Feng-Lan; Ye, Bang-Ce

2018-02-05

Enhanced intracellular survival (Eis) proteins were found to enhance the intracellular survival of mycobacteria in macrophages by acetylating aminoglycoside antibiotics to confer resistance to these antibiotics and by acetylating DUSP16/MPK-7 to suppress host innate immune defenses. Eis homologs composing of two GCN5 N-acetyltransferase regions and a sterol carrier protein fold are found widely in gram-positive bacteria. In this study, we found that Eis proteins have an unprecedented ability to acetylate many arylalkylamines, are a novel type of arylalkylamine N-acetyltransferase AANAT (EC 2.3.1.87). Sequence alignment and phyletic distribution analysis confirmed Eis belongs to a new aaNAT-like cluster. Among the cluster, we studied three typical Eis proteins: Eis_Mtb from Mycobacterium tuberculosis, Eis_Msm from Mycobacterium smegmatis, and Eis_Sen from Saccharopolyspora erythraea. Eis_Mtb prefers to acetylate histamine and octopamine, while Eis_Msm uses tyramine and octopamine as substrates. Unlike them, Eis_Sen exihibits good catalytic efficiencies for most tested arylalkylamines. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions, future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis, and reveal potential clinical implications for many gram-positive pathogens.

Antimicrobial and anti-inflammatory activities of Pleurostylia capensis Turcz (Loes) (celastraceae).

PubMed

Razwinani, Mapula; Tshikalange, Thilivhali Emmanuel; Motaung, Shirley C K M

2014-01-01

Pleurostylia capensis is a large tree that can reach the maximum height of 20 m long, and it have been traditionally used as cosmetic, for steam bath, ritual body wash, and as a purgative to treat symptoms of witchcraft. Using ethanol, chloroform, dichloromethane (DCM), ethyl acetate (EA), and water extracts, leaves, bark and roots of Pleurostylia capensis were investigated scientifically for their effectiveness in antimicrobial, antioxidant and anti-inflammatory activities using standard methods. The extracts were evaluated for antimicrobial activity against Gram positive (Staphylococcus aureus, Bacillus cereus, and Mycobacterium smegmatis), Gram negative (Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Streptococcus pyogenes, Pseudomonas aeruginosa and Salmonella typhimurium), and Candida albicans. The antioxidant activity was investigated using 2, 2-diphenlyl-1-picrylhadrazyl (DPPH), free radical scavenging assay. The anti-inflammatory activity of P. capensis extracts was evaluated against both cyclooxygenase enzymes (COX 1 and 2). The ethyl acetate extracts of P. capensis showed a strong antimicrobial activity against B. cereus, K. pneumonia, S. pyogenes, and M. smegmatis with MIC value of 0.39 and 0.78 mg/ml. While the ethanol bark extract was most active against M. smegmatis with MIC value of 0.78 mg/ml; the least potent activity was observed with dichloromethane, chloroform and water extracts, with an MIC value ranging from 1.56 mg/ml to 50.0 mg/ml. The plant extracts proved to be good antioxidant agent, whereas extracts of ethanol were the most active, with IC50 ranging from 1.00 to 1.74 µg/ml, which is lower, and in close range to Vitamin C (1.40 µg/ml). Its moderation to potent inhibitory activity was observed in all extracts. Ethanol and dichloromethane extracts were among the most potent when compared to water and petroleum ether extracts. The water extracts showed to be nontoxic on the Hek cell line with an IC50 value of 204.0, and 207.3 µg/ml (roots and bark) respectively. The dichloromethane, ethyl acetate, chloroform and ethanol extracts showed to be toxic on the Hek cell, with IC50 range from 5.94 to 42.91µg/ml. The results obtained indicate the effectiveness of these plants.

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Antimycobacterial constituents from Juniperus procera, Ferula communis and Plumbago zeylanica and their in vitro synergistic activity with isonicotinic acid hydrazide.

PubMed

Mossa, Jaber S; El-Feraly, Farouk S; Muhammad, Ilias

2004-11-01

The synergistic activity of antimycobacterial constituents from Saudi plants was evaluated in combination with isonicotinic acid hydrazide (INH) against four atypical organisms, namely, Mycobacterium intracellulare, M. smegmatis, M. xenopei and M. chelonei. The potency of INH was increased four-fold, using an in vitro checkerboard method, against each mycobacteria when tested with a subtoxic concentration of the totarol, isolated from J. procera. The MIC values of totarol, ferulenol (from Ferula communis) and plumbagin (from Plumbago zeylanica) were thus lowered from 1.25-2.5 to 0.15-0.3 microg/mL due to synergism with INH. When tested against the resistant strain of M. tuberculosis H37Rv, plumbagin and 7beta-hydroxyabieta-8,13-dien-11,12-dione exhibited inhibitory activity at <12.5 microg/mL, while others were inactive at this concentration. Copyright 2004 John Wiley & Sons, Ltd.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment.

PubMed

Gomez, James E; Kaufmann-Malaga, Benjamin B; Wivagg, Carl N; Kim, Peter B; Silvis, Melanie R; Renedo, Nikolai; Ioerger, Thomas R; Ahmad, Rushdy; Livny, Jonathan; Fishbein, Skye; Sacchettini, James C; Carr, Steven A; Hung, Deborah T

2017-02-21

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

Direct Observation of Gold Nanoparticle Assemblies with the Porin MspA on Mica

PubMed Central

Basel, Matthew T.; Dani, Raj Kumar; Kang, Myungshim; Pavlenok, Mikhail; Chikan, Viktor; Smith, Paul E.; Niederweis, Michael

2009-01-01

The octameric porin MspA from Mycobacterium smegmatis is sufficiently stable to form a non-membrane-supported stand-alone porin on Mica surfaces. About 98% of all MspA octamers were found to stand upright on Mica, with their periplasmic loop regions bound to the hydrophilic Mica surface. Both, small (d = 3.7 nm) and large (d = 17 nm) gold nanoparticles bind to MspA, however in different positions: small gold nanoparticles bind within the MspA pore, whereas the large gold nanoparticles bind to the upper region of MspA. These experiments demonstrate that gold nanoparticles can be positioned at different, well-defined distances from the underlying surface using the MspA pore as a template. These findings represent a significant step towards the use of electrically insulating stable proteins in combination with metal nanoparticles in nanodevices. PMID:19236086

Antimicrobial Activity and Phytochemical Screening of Buchenavia tetraphylla (Aubl.) R. A. Howard (Combretaceae: Combretoideae)

PubMed Central

de Oliveira, Ygor Lucena Cabral; Nascimento da Silva, Luís Cláudio; da Silva, Alexandre Gomes; Macedo, Alexandre José; de Araújo, Janete Magali; Correia, Maria Tereza dos Santos; da Silva, Márcia Vanusa

2012-01-01

This study evaluated the antimicrobial and hemolytic activities and phytochemical constituents of hydroalcoholic extract and its fractions from Buchenavia tetraphylla leaves. Cyclohexane (BTCF), ethyl acetate (BTEF), and n-butanol-soluble (BTSBF) and non-soluble (BTNBF) fractions were obtained from a liquid-liquid partition of hydroalcoholic extract (BTHE) from B. tetraphylla leaves. The hemolytic activity of active fractions was checked. The BTHE inhibited the growth of Micrococcus luteus (MIC: 0.10 mg/mL), Pseudomonas aeruginosa (MIC: 0.20 mg/mL), Mycobacterium smegmatis (MIC: 0.39 mg/mL), Proteus vulgaris, and Staphylococcus aureus (MIC: 0.78 mg/mL for both). The more active fractions were BTCF and BTBSF. BTCF showed better potential to inhibit M. luteus (0.10 mg/mL), P. aeruginosa (0.20 mg/mL), S. enteritidis (0.39 mg/mL), and S. aureus (1.56 mg/mL). BTBSF showed the best results for M. luteus (0.10 mg/mL), M. smegmatis, B. subtilis (0.39 mg/mL for both), and P. vulgaris (0.10 mg/mL). The HC50 were greater than observed MIC: 20.30, 4.70 and 2.53 mg/mL, respectively, to BTBF, BTHE and BTCF, which. The phytochemical analysis detected the presence of flavanoids, triterpene, carbohydrate, and tannin. Our work showed for the first time the broad-spread antimicrobial activity of B. tetraphylla, which has nonhemolytic action, creating a new perspective on the interesting association of traditional and scientific knowledge. PMID:23365533

F420H2-Dependent Degradation of Aflatoxin and other Furanocoumarins Is Widespread throughout the Actinomycetales

PubMed Central

Lapalikar, Gauri V.; Taylor, Matthew C.; Warden, Andrew C.; Scott, Colin; Russell, Robyn J.; Oakeshott, John G.

2012-01-01

Two classes of F420-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F420 is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F420H2 to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F420 to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products. PMID:22383957

F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the actinomycetales.

PubMed

Lapalikar, Gauri V; Taylor, Matthew C; Warden, Andrew C; Scott, Colin; Russell, Robyn J; Oakeshott, John G

2012-01-01

Two classes of F(420)-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F(420) is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F(420)H(2) to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F(420) to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products.

Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D

PubMed Central

Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S.

2013-01-01

Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3’-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotide and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologs of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both, in an M. smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5’-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. PMID:23198659

[Frontier of mycobacterium research--host vs. mycobacterium].

PubMed

Okada, Masaji; Shirakawa, Taro

2005-09-01

During the past decade, we have observed advance in tuberculosis research including novel vaccines, innate immunity (TLR), SNIP analysis and molecular mechanism of drug resistance. Worldwide genome project enabled the whole genome sequence of host resistant against tuberculosis as well as the whole genome sequence of M. tuberculosis H37Rv. DNA technology has also provided a great impact on the development of novel vaccine against TB. In this symposium, we have invited leading researchers in the field of the frontier study of Mycobacterium research in order to provide general overview of the cutting edge of frontier research. Molecular mechanism of drug resistance of M. tuberculosis has been clarified. On the other hand, molecular mechanism of host-defence (insusceptibility of host) against M. tuberculosis has not yet elucidated. Dr. Taro Shirakawa (Kyoto University) reviewed the susceptibility genes of host in TB infection and presented candidate genes associated with multi-drug resistant tuberculosis. Dr. Naoto Keicho (International Medical Center of Japan) tried to identify host genetic factors involved in susceptibility to pulmonary Mycobacterium avium complex (MAC) infection by candidate gene approach and genome-wide approach. In Japan, Dr. Masaji Okada (National Hospital Organization Kinki-Chuo Chest Medical Center) has been engaged actively in the development of new tuberculosis vaccines (HVJ-liposome/Hsp65 DNA + IL-12 DNA vaccine and recombinant 72f BCG vaccine). He showed basic strategy for construction of new candidate vaccines and also showed significant efficacy on the protection of tuberculosis infection using cynomolgus monkeys, which are very similar to human tuberculosis. Dr. Hatsumi Taniguchi (University of Occupational and Environmental Health) presented that M. tuberculosis mIHF and the neighbor genes went into a dormacy-like state of M. smegmatis in J774 macrophage cells. This study might provide a weapon for elucidating the mechanism of dormacy of M. tuberculosis and the development of novel therapy. Dr. Chiyoji Abe (Nippon Becton Dickinson Co.) reviewed the molecular basis of the resistance to anti-tuberculosis drugs. Most cases of resistance are related to simple nucleotide substitutions rather than to acquisition of new elements. Dr. Kiyoshi Takeda (Kyushu University) showed interesting finding. He analyzed whether Toll-like receptor (TLR)-mediated activation of innate immunity in host defense against mycobacterial infection. MyD88/TRIF double defi-indicating that innate immunity is involved in anti-mycobacterial infection. (1) SNP (single nucleotide polymorphism) analysis in association with Mycobacterium tuberculosis: Taro SHIRAKAWA (Department of Health Promotion & Human Behavior, Kyoto University Medical School, and RIKEN SRC Center) Candidate gene approach was made on 18 SNPs in 11 genes in association with M. tuberculosis. Patients with multi-drug resistance against M. tuberculosis are also subjected. SNPs in NRAMP1 gene were associated with the disease, and drug resistance, its mechanisms remain unknown. (2) Search for genes susceptible to pulmonary Mycobacterium avium complex infection: Naoto KEICHO (Department of Respiratory Diseases, Research Institute, International Medical Center of Japan) Interaction among pathogens and host factors is important for development of infectious diseases. We are trying to identify host genetic factors involved in susceptibility to nonimmunocompromized pulmonary Mycobacterium avium complex (MAC) infection by candidate gene approach and genome-wide approach. Elucidation of functional significance of susceptibility gene polymorphisms will lead to a new strategy for control and prevention of the disease. (3) T cell immunity against Tuberculosis in host and the establishment of novel vaccine: Masaji OKADA (Clinical Research Center, National Hospital Organization Kinki-Chuo Chest Medical Center) T cell (CTL, Th1) immunity including granulysin play an important role in host defense against tuberculosis (TB) in human. Patients with TB or Multi-drug resistant TB showed suppression of all these immunities. HVJ-liposome/Hsp65 DNA + IL-12 DNA vaccination was 100 fold more efficient than BCG on the elimination of Mycobacterium tuberculosis (M.TB) in the BALB/c mice. Cytotoxic T cells activity against M. TB was augmented. By using these new vaccines (Hsp 65 DNA + IL-12 DNA, recombinant 72f BCG) and the cynomolgus monkey models which are very similar to human tuberculosis, the prophylactic effect of vaccines was observed. Thus, these novel vaccines should provide a useful tool for the prevention of human TB infection. (4) Mycobacterium tuberculosis mIHF and the neighbor genes go into a dormancy-like state of M. smegmatis J15CS in J774 cells: Hatsumi TANIGUCHI (Department of Microbiology, School of Medicine, University of Occupational and Environmental Health) Mycobacterium smegmatis J15CS transformants harboring the mIHF gene or the mIHF-gmk-Rv1390 genes showed no difference in in vitro growth and acid-fastness. However, transformants harboring mIHF-gmk-Rv1390 formed short-rod cell morphology and decreased acid-fastness in the mouse macrophage-like cell line J774 compared to those of the other transformants, and the nuclei of the infected J774 cells also changed. Nevertheless, the colony forming units were similar. These indicate that mIHF and the neighbor genes of M. tuberculosis might regulate a growth of mycobacteria in macrophages. (5) Molecular basis of the resistance to anti-tuberculosis drugs: Chiyoji ABE (Nippon Becton Dickinson Company, Ltd.) Considerable progress has been made toward understanding the molecular basis of the resistance to anti-tuberculosis drugs. Most cases of resistance are related usually to simple nucleotide substitutions rather than to acquisition of new elements. Multi-drug resistant isolates of Mycobacterium tuberculosis arise a consequence of sequential accumulation of mutation conferring resistance to single therapeutic agents. The basis of resistance is not able to be explained yet in a substantial percentage of strains for other anti-tuberculosis drugs than rifampin and pyrazinamide. Further studies are required to fully understand the molecular mechanisms of resistance. (6) Toll-like receptors in anti-mycobacterial immune responses: Kiyoshi TAKEDA (Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University) Toll-like receptors (TLRs) play an essential role in the recognition of specific patterns of microbial components. TLRs mediate activation of innate immunity and further development of antigen-specific adaptive immunity. In TLR signaling pathways, Toll/IL-1 receptor (TIR) domain-containing adaptors, such as MyD88, TIRAP, TRIF, and TRAM, have been shown to play pivotal roles. Thus, the molecular mechanisms for TLR-mediated activation of innate immunity have been largely understood. We analyzed whether TLR-mediated activation of innate immunity is involved in host defense against mycobacterial infection. MyD88/TRIF double deficient mice, in which TLR-dependent activation of innate immunity is abolished, showed high sensitivity to mycobacterial infection, indicating that innate immunity is critically involved in anti-mycobacterial responses.

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Wilson, R

2000-10-01

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

PubMed Central

Ligon, Lauren S.; Rigel, Nathan W.; Romanchuk, Artur; Jones, Corbin D.

2013-01-01

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins. PMID:23913320

Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing.

PubMed

Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

2016-01-01

TLR2-dependent cellular signaling in Mycobacterium tuberculosis -infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2 -/- mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4 + T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 {angstrom} resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase foldmore » and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. While the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the 'x' of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between {beta}5 and {alpha}11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or {beta}-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.« less

Topical application of zinc oxide nanoparticles reduces bacterial skin infection in mice and exhibits antibacterial activity by inducing oxidative stress response and cell membrane disintegration in macrophages.

PubMed

Pati, Rashmirekha; Mehta, Ranjit Kumar; Mohanty, Soumitra; Padhi, Avinash; Sengupta, Mitali; Vaseeharan, Baskarlingam; Goswami, Chandan; Sonawane, Avinash

2014-08-01

Here we studied immunological and antibacterial mechanisms of zinc oxide nanoparticles (ZnO-NPs) against human pathogens. ZnO-NPs showed more activity against Staphylococcus aureus and least against Mycobacterium bovis-BCG. However, BCG killing was significantly increased in synergy with antituberculous-drug rifampicin. Antibacterial mechanistic studies showed that ZnO-NPs disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity and down-regulate the transcription of oxidative stress-resistance genes in bacteria. ZnO-NP treatment also augmented the intracellular bacterial killing by inducing reactive oxygen species production and co-localization with Mycobacterium smegmatis-GFP in macrophages. Moreover, ZnO-NPs disrupted biofilm formation and inhibited hemolysis by hemolysin toxin producing S. aureus. Intradermal administration of ZnO-NPs significantly reduced the skin infection, bacterial load and inflammation in mice, and also improved infected skin architecture. We envision that this study offers novel insights into antimicrobial actions of ZnO-NPs and also demonstrates ZnO-NPs as a novel class of topical anti-infective agent for the treatment of skin infections. This in-depth study demonstrates properties of ZnO nanoparticles in infection prevention and treatment in several skin infection models, dissecting the potential mechanisms of action of these nanoparticles and paving the way to human applications. Copyright © 2014 Elsevier Inc. All rights reserved.

Stable Regulation of Cell Cycle Events in Mycobacteria: Insights From Inherently Heterogeneous Bacterial Populations.

PubMed

Logsdon, Michelle M; Aldridge, Bree B

2018-01-01

Model bacteria, such as E. coli and B. subtilis , tightly regulate cell cycle progression to achieve consistent cell size distributions and replication dynamics. Many of the hallmark features of these model bacteria, including lateral cell wall elongation and symmetric growth and division, do not occur in mycobacteria. Instead, mycobacterial growth is characterized by asymmetric polar growth and division. This innate asymmetry creates unequal birth sizes and growth rates for daughter cells with each division, generating a phenotypically heterogeneous population. Although the asymmetric growth patterns of mycobacteria lead to a larger variation in birth size than typically seen in model bacterial populations, the cell size distribution is stable over time. Here, we review the cellular mechanisms of growth, division, and cell cycle progression in mycobacteria in the face of asymmetry and inherent heterogeneity. These processes coalesce to control cell size. Although Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) utilize a novel model of cell size control, they are similar to previously studied bacteria in that initiation of DNA replication is a key checkpoint for cell division. We compare the regulation of DNA replication initiation and strategies used for cell size homeostasis in mycobacteria and model bacteria. Finally, we review the importance of cellular organization and chromosome segregation relating to the physiology of mycobacteria and consider how new frameworks could be applied across the wide spectrum of bacterial diversity.

Identification of New Molecular Entities (NMEs) as Potential Leads against Tuberculosis from Open Source Compound Repository.

PubMed

Kotapalli, Sudha Sravanti; Nallam, Sri Satya Anila; Nadella, Lavanya; Banerjee, Tanmay; Rode, Haridas B; Mainkar, Prathama S; Ummanni, Ramesh

2015-01-01

The purpose of this study was to provide a number of diverse and promising early-lead compounds that will feed into the drug discovery pipeline for developing new antitubercular agents. The results from the phenotypic screening of the open-source compound library against Mycobacterium smegmatis and Mycobacterium bovis (BCG) with hit validation against M. tuberculosis (H37Rv) have identified novel potent hit compounds. To determine their druglikeness, a systematic analysis of physicochemical properties of the hit compounds has been performed using cheminformatics tools. The hit molecules were analysed by clustering based on their chemical finger prints and structural similarity determining their chemical diversity. The hit compound library is also filtered for druglikeness based on the physicochemical descriptors following Lipinski filters. The robust filtration of hits followed by secondary screening against BCG, H37Rv and cytotoxicity evaluation has identified 12 compounds with potential against H37Rv (MIC range 0.4 to 12.5 μM). Furthermore in cytotoxicity assays, 12 compounds displayed low cytotoxicity against liver and lung cells providing high therapeutic index > 50. To avoid any variations in activity due to the route of chemical synthesis, the hit compounds were re synthesized independently and confirmed for their potential against H37Rv. Taken together, the hits reported here provides copious potential starting points for generation of new leads eventually adds to drug discovery pipeline against tuberculosis.

Copper resistance is essential for virulence of Mycobacterium tuberculosis

PubMed Central

Wolschendorf, Frank; Ackart, David; Shrestha, Tej B.; Hascall-Dove, Laurel; Nolan, Scott; Lamichhane, Gyanu; Wang, Ying; Bossmann, Stefan H.; Basaraba, Randall J.; Niederweis, Michael

2011-01-01

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy. PMID:21205886

Post-translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis.

PubMed

Vergnolle, Olivia; Xu, Hua; Tufariello, JoAnn M; Favrot, Lorenza; Malek, Adel A; Jacobs, William R; Blanchard, John S

2016-10-14

Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD + -dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping.

PubMed

Hotra, Adam; Suter, Manuel; Biuković, Goran; Ragunathan, Priya; Kundu, Subhashri; Dick, Thomas; Grüber, Gerhard

2016-05-01

The F1 FO -ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the α3 :β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H(+) pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop-encoding sequence (γ166-179 ) in the genome of M. smegmatis. ATP hydrolysis-driven H(+) pumping was observed in IMVs containing the Δγ166-179 mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Δγ166-179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H(+) pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode. © 2016 Federation of European Biochemical Societies.

Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

PubMed Central

2011-01-01

Background The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells. PMID:21762531

Trehalose Polyphleates, External Cell Wall Lipids in Mycobacterium abscessus, Are Associated with the Formation of Clumps with Cording Morphology, Which Have Been Associated with Virulence

PubMed Central

Llorens-Fons, Marta; Pérez-Trujillo, Míriam; Julián, Esther; Brambilla, Cecilia; Alcaide, Fernando; Byrd, Thomas F.; Luquin, Marina

2017-01-01

Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. In searching for the virulence factors responsible for this difference, R morphotypes have been found to form large aggregates (clumps) that, after being phagocytozed, result in macrophage death. Furthermore, the aggregates released to the extracellular space by damaged macrophages grow, forming unphagocytosable structures that resemble cords. In contrast, bacilli of the S morphotype, which do not form aggregates, do not damage macrophages after phagocytosis and do not form cords. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains, and no differences were observed between morphotypes. Furthermore, cords formed by R morphotypes were disrupted with petroleum ether (PE), and the extracted lipids were analyzed by thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. Substantial amounts of trehalose polyphleates (TPP) were recovered as major lipids from PE extracts, and images obtained by transmission electron microscopy suggested that these lipids are localized to the external surfaces of cords and R bacilli. The structure of M. abscessus TPP was revealed to be similar to those previously described in Mycobacterium smegmatis. Although the exact role of TPP is unknown, our results demonstrated that TPP are not toxic by themselves and have a function in the formation of clumps and cords in M. abscessus, thus playing an important role in the pathogenesis of this species. PMID:28790995

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity

PubMed Central

Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F; Abbazia, Patrick; Ababio, Amma; Adam, Naazneen

2015-01-01

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery. DOI: http://dx.doi.org/10.7554/eLife.06416.001 PMID:25919952

Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms.

PubMed

Ziemski, Michal; Jomaa, Ahmad; Mayer, Daniel; Rutz, Sonja; Giese, Christoph; Veprintsev, Dmitry; Weber-Ban, Eilika

2018-05-29

Cdc48 is a AAA+ ATPase that plays an essential role for many cellular processes in eukaryotic cells. An archaeal homologue of this highly conserved enzyme was shown to directly interact with the 20S proteasome. Here, we analyze the occurrence and phylogeny of a Cdc48 homologue in Actinobacteria and assess its cellular function and possible interaction with the bacterial proteasome. Our data demonstrate that Cdc48-like protein of actinobacteria (Cpa) forms hexameric rings and that the oligomeric state correlates directly with the ATPase activity. Furthermore, we show that the assembled Cpa rings can physically interact with the 20S core particle. Comparison of the Mycobacterium smegmatis wild-type with a cpa knockout strain under carbon starvation uncovers significant changes in the levels of around 500 proteins. Pathway mapping of the observed pattern of changes identifies ribosomal proteins as a particular hotspot, pointing amongst others toward a role of Cpa in ribosome adaptation during starvation. © 2018, Ziemski et al.

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

DOE PAGES

Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; ...

2017-07-13

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the a CTD may play a role in Mtb transcription regulation. Here, our results reveal the structure of an Actinobacteria-unique insert ofmore » the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σ A, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.« less

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

PubMed

Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F

2015-04-28

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

Steroid 11-Alpha-Hydroxylation by the Fungi Aspergillus nidulans and Aspergillus ochraceus.

PubMed

Ríos, Lidia Ortega-de Los; Luengo, José M; Fernández-Cañón, José M

2017-01-01

Steroids are a group of natural compounds derived from the cyclopentane-perhydro-phenantrene nucleus that have a great interest for the pharmaceutical industries as a consequence of their physiological effects. Among their functions are anti-inflammatory, immunosuppressive, or contraceptive activities. Nowadays, microbial transformation of steroid precursors is winning relevance opposite to the chemical synthesis, since it allows for decreasing time, expenses, and environmental pollution. Pharmaceutical industry tends to use cholesterol and phytosterols as starting materials due to their low cost. Aspergillus ochraceus and Aspergillus nidulans, a fungus whose biochemistry and genetics are well known, have been chosen because of their capacity of 11-α-hydroxylation over some steroids which confers on them their anti-inflammatory properties. We have cloned the genes encoding the 11-α-hydroxylase enzymatic activities with the aim to introduce them in other microorganisms, such as Mycobacterium smegmatis, used in the industry to split the side chain of phytosterols, and thus creating recombinant microorganisms able to generate useful steroids from cheap precursors in just one-step fermentation.

Experimental and modeling studies of sorption of ceria nanoparticle on microbial biofilms.

PubMed

Jing, Hengye; Mezgebe, Bineyam; Aly Hassan, Ashraf; Sahle-Demessie, Endalkachew; Sorial, George A; Bennett-Stamper, Christina

2014-06-01

This study focuses on the interaction of ceria nanoparticles (CeO2-NPs) with Pseudomonas fluorescens and Mycobacterium smegmatis biofilms. Confocal laser microscopy and transmission electron microscopy determined the distribution of NPs in the complex structures of biofilm at molecular levels. Visual data showed that most of the adsorption takes place on the bacterial cell walls and spores. The interaction of nanoparticles (NPs) with biofilms reached equilibrium after the initial high adsorption rate regardless of biofilm heterogeneity and different nanoparticle concentrations in the bulk liquid. Physical processes may dominate this sorption phenomenon. Pseudo first order sorption kinetics was used to estimate adsorption and desorption rate of CeO2-NPs onto biofilms. When biofilms got exposed to CeO2-NPs, a self-protecting mechanism was observed. Cells moved away from the bulk solution in the biofilm matrix, and portions of biofilm outer layer were detached, hence releasing some CeO2-NPs back to the bulk phase. Published by Elsevier Ltd.

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAPmore » β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.« less

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the a CTD may play a role in Mtb transcription regulation. Here, our results reveal the structure of an Actinobacteria-unique insert ofmore » the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σ A, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.« less

Antioxidant, electrochemical, thermal, antimicrobial and alkane oxidation properties of tridentate Schiff base ligands and their metal complexes

NASA Astrophysics Data System (ADS)

Ceyhan, Gökhan; Çelik, Cumali; Uruş, Serhan; Demirtaş, İbrahim; Elmastaş, Mahfuz; Tümer, Mehmet

2011-10-01

In this study, two Schiff base ligands (HL 1 and HL 2) and their Cu(II), Co(II), Ni(II), Pd(II) and Ru(III) metal complexes were synthesized and characterized by the analytical and spectroscopic methods. Alkane oxidation activities of the metal complexes were studied on cyclohexane as substrate. The ligands and their metal complexes were evaluated for their antimicrobial activity against Corynebacterium xerosis, Bacillus brevis, Bacillus megaterium, Bacillus cereus, Mycobacterium smegmatis, Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis (as Gram-positive bacteria) and Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Yersinia enterocolitica, Klebsiella fragilis, Saccharomyces cerevisiae, and Candida albicans (as Gram-negative bacteria). The antioxidant properties of the Schiff base ligands were evaluated in a series of in vitro tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH rad ) free radical scavenging and reducing power activity of superoxide anion radical generated non-enzymatic systems. Electrochemical and thermal properties of the compounds were investigated.

Crystal Structure of CCM3, a Cerebral Cavernous Malformation Protein Critical for Vascular Integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, X.; Zhang, R; Zhang, H

CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 {angstrom} crystal structure of CCM3. This structure shows an all {alpha}-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAT-homology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1)more » abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM.« less

Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage

PubMed Central

Purwantini, Endang; Mukhopadhyay, Biswarup

2009-01-01

In mycobacteria, F420, a deazaflavin derivative, acts as a hydride transfer coenzyme for an F420-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F420 (F420H2) are unknown. In this work, F420H2 was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO2, produced from NO and O2, was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F420H2 reduced NO2 to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO2 than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO2. Hence, by converting NO2 back to NO with F420H2, M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F420, was ≈4-fold more sensitive to NO2 than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of γ-tocopherol, which reduces NO2 to NO and protects human cells from NO2-induced carcinogenesis. PMID:19325122

Encapsulation of zinc-rifampicin complex into transferrin-conjugated silver quantum-dots improves its antimycobacterial activity and stability and facilitates drug delivery into macrophages

PubMed Central

Pati, Rashmirekha; Sahu, Rojalin; Panda, Jagannath; Sonawane, Avinash

2016-01-01

In order to improve the chemotherapy of tuberculosis, there is an urgent need to enhance the efficacy of existing agents and also to develop more efficient drug delivery systems. Here, we synthesized a novel anti-TB drug complex consisting of zinc and rifampicin (Zn-RIF), and encapsulated it into transferrin-conjugated silver quantum-dots (Zn-RIF-Tf-QD) to improve delivery in macrophages. Successful synthesis of Zn-RIF and Zn-RIF-Tf-QD was confirmed by UV/Vis-spectroscopy, TEM, FTIR, photoluminescence, XRD, XPS, and NMR. The sizes of silver QDs and transferrin-conjugated QDs were found to be in the range of 5–20 nm. Activity assays showed that Zn-RIF-Tf-QD exhibited 10-fold higher antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis-BCG as compared to Zn-RIF, RIF and Zn. Immunofluorescence studies showed that Zn-RIF-Tf-QD-conjugates were actively endocytosed by macrophages and dendritic cells, but not by lung epithelial cells. Treatment with Zn-RIF-Tf-QD efficiently killed mycobacteria residing inside macrophages without exhibiting cytotoxicity and genotoxicity. Moreover, the conjugates remained stable for upto 48 h, were taken up into the late endosomal compartment of macrophages, and released the drug in a sustainable manner. Our data demonstrate that Zn-RIF-Tf-QDs have a great potential as anti-TB drugs. In addition, transferrin-conjugated QDs may constitute an effective drug delivery system for tuberculosis therapy. PMID:27113139

Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis.

PubMed

Syal, Kirtimaan; Joshi, Himanshu; Chatterji, Dipankar; Jain, Vikas

2015-10-01

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra- and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response. © 2015 FEBS.

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV)

PubMed Central

Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B.; Thompson, Charles J.

2013-01-01

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7’s ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the −35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7–SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases. PMID:23990327

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

PubMed

Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B; Thompson, Charles J

2013-12-01

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

PubMed

Agnew, Christopher R J; Warrilow, Andrew G S; Burton, Nicholas M; Lamb, David C; Kelly, Steven L; Brady, R Leo

2012-01-01

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 μM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

Cluster K Mycobacteriophages: Insights into the Evolutionary Origins of Mycobacteriophage TM4

PubMed Central

Pope, Welkin H.; Ferreira, Christina M.; Jacobs-Sera, Deborah; Benjamin, Robert C.; Davis, Ariangela J.; DeJong, Randall J.; Elgin, Sarah C. R.; Guilfoile, Forrest R.; Forsyth, Mark H.; Harris, Alexander D.; Harvey, Samuel E.; Hughes, Lee E.; Hynes, Peter M.; Jackson, Arrykka S.; Jalal, Marilyn D.; MacMurray, Elizabeth A.; Manley, Coreen M.; McDonough, Molly J.; Mosier, Jordan L.; Osterbann, Larissa J.; Rabinowitz, Hannah S.; Rhyan, Corwin N.; Russell, Daniel A.; Saha, Margaret S.; Shaffer, Christopher D.; Simon, Stephanie E.; Sims, Erika F.; Tovar, Isabel G.; Weisser, Emilie G.; Wertz, John T.; Weston-Hafer, Kathleen A.; Williamson, Kurt E.; Zhang, Bo; Cresawn, Steven G.; Jain, Paras; Piuri, Mariana; Jacobs, William R.; Hendrix, Roger W.; Hatfull, Graham F.

2011-01-01

Five newly isolated mycobacteriophages –Angelica, CrimD, Adephagia, Anaya, and Pixie – have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them – with the exception of TM4 – form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species. PMID:22053209

Characterization of ML0314c of Mycobacterium leprae and deciphering its role in the immune response in leprosy patients.

PubMed

Kaur, Gurkamajit; Sharma, Aashish; Narang, Tarun; Dogra, Sunil; Kaur, Jagdeep

2018-02-15

Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an α/β hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45°C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60°C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients. Copyright © 2017 Elsevier B.V. All rights reserved.

An Enlarged, Adaptable Active Site in CYP164 Family P450 Enzymes, the Sole P450 in Mycobacterium leprae

PubMed Central

Agnew, Christopher R. J.; Warrilow, Andrew G. S.; Burton, Nicholas M.; Lamb, David C.; Kelly, Steven L.

2012-01-01

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin “open” conformation of the enzyme (Kd [dissociation constant] of 0.1 μM), with binding to the low-spin “closed” form being significantly hindered (Kd of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy. PMID:22037849

Comparative genomic and phylogenetic approaches to characterize the role of genetic recombination in mycobacterial evolution.

PubMed

Smith, Silvia E; Showers-Corneli, Patrice; Dardenne, Caitlin N; Harpending, Henry H; Martin, Darren P; Beiko, Robert G

2012-01-01

The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.

Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice

PubMed Central

Choi, Jaesung P.; Foley, Matthew; Zhou, Zinan; Wong, Weng-Yew; Gokoolparsadh, Naveena; Arthur, J. Simon C.; Li, Dean Y.; Zheng, Xiangjian

2016-01-01

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases. PMID:27513872

High-throughput sequencing of the entire genomic regions of CCM1/KRIT1, CCM2 and CCM3/PDCD10 to search for pathogenic deep-intronic splice mutations in cerebral cavernous malformations.

PubMed

Rath, Matthias; Jenssen, Sönke E; Schwefel, Konrad; Spiegler, Stefanie; Kleimeier, Dana; Sperling, Christian; Kaderali, Lars; Felbor, Ute

2017-09-01

Cerebral cavernous malformations (CCM) are vascular lesions of the central nervous system that can cause headaches, seizures and hemorrhagic stroke. Disease-associated mutations have been identified in three genes: CCM1/KRIT1, CCM2 and CCM3/PDCD10. The precise proportion of deep-intronic variants in these genes and their clinical relevance is yet unknown. Here, a long-range PCR (LR-PCR) approach for target enrichment of the entire genomic regions of the three genes was combined with next generation sequencing (NGS) to screen for coding and non-coding variants. NGS detected all six CCM1/KRIT1, two CCM2 and four CCM3/PDCD10 mutations that had previously been identified by Sanger sequencing. Two of the pathogenic variants presented here are novel. Additionally, 20 stringently selected CCM index cases that had remained mutation-negative after conventional sequencing and exclusion of copy number variations were screened for deep-intronic mutations. The combination of bioinformatics filtering and transcript analyses did not reveal any deep-intronic splice mutations in these cases. Our results demonstrate that target enrichment by LR-PCR combined with NGS can be used for a comprehensive analysis of the entire genomic regions of the CCM genes in a research context. However, its clinical utility is limited as deep-intronic splice mutations in CCM1/KRIT1, CCM2 and CCM3/PDCD10 seem to be rather rare. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development.

PubMed

He, Yun; Zhang, Haifeng; Yu, Luyang; Gunel, Murat; Boggon, Titus J; Chen, Hong; Min, Wang

2010-04-06

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

Provider Experiences with Chronic Care Management (CCM) Services and Fees: A Qualitative Research Study.

PubMed

O'Malley, Ann S; Sarwar, Rumin; Keith, Rosalind; Balke, Patrick; Ma, Sai; McCall, Nancy

2017-12-01

Support for ongoing care management and coordination between office visits for patients with multiple chronic conditions has been inadequate. In January 2015, Medicare introduced the Chronic Care Management (CCM) payment policy, which reimburses providers for CCM activities for Medicare beneficiaries occurring outside of office visits. To explore the experiences, facilitators, and challenges of practices providing CCM services, and their implications going forward. Semi-structured telephone interviews from January to April 2016 with 71 respondents. Sixty billing and non-billing providers and practice staff knowledgeable about their practices' CCM services, and 11 professional society representatives. Practice respondents noted that most patients expressed positive views of CCM services. Practice respondents also perceived several patient benefits, including improved adherence to treatment, access to care team members, satisfaction, care continuity, and care coordination. Facilitators of CCM provision included having an in-practice care manager, patient-centered medical home recognition, experience developing care plans, patient trust in their provider, and supplemental insurance to cover CCM copayments. Most billing practices reported few problems obtaining patients' consent for CCM, though providers felt that CMS could better facilitate consent by marketing CCM's goals to beneficiaries. Barriers reported by professional society representatives and by billing and non-billing providers included inadequacy of CCM payments to cover upfront investments for staffing, workflow modification, and time needed to manage complex patients. Other barriers included inadequate infrastructure for health information exchange with other providers and limited electronic health record capabilities for documenting and updating care plans. Practices owned by hospital systems and large medical groups faced greater bureaucracy in implementing CCM than did smaller, independent practices. Improving providers' experiences with and uptake of CCM will require addressing several challenges, including the upfront investment for CCM set-up and the time required to provide CCM to more complex patients.

Stabiliztin of VEGFR2 Signaling by Cerebral Cavernous Malformation 3 is Critical for Vascular Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y He; H Zhang; L Yu

2011-12-31

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an earlymore » embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.« less

Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

PubMed Central

Feltcher, Meghan E.; Gibbons, Henry S.; Ligon, Lauren S.

2013-01-01

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm. PMID:23204463

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

PubMed Central

Kieser, Karen J.; Baer, Christina E.; Barczak, Amy K.; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah M.; Rubin, Eric J.

2015-01-01

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress. PMID:26114871

Synthesis and biological evaluation of benzo[d]isothiazole, benzothiazole and thiazole Schiff bases.

PubMed

Vicini, Paola; Geronikaki, Athina; Incerti, Matteo; Busonera, Bernadetta; Poni, Graziella; Cabras, Carla Alba; La Colla, Paolo

2003-11-03

Three new series of benzo[d]isothiazole, benzothiazole and thiazole Schiff bases were synthesized and tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle infectious diseases (AIDS, hepatitis B and C, tuberculosis, bovine viral diarrhoea) or against drug-resistant cancers (leukaemia, carcinoma, melanoma, MDR tumors) for which no definitive cure or efficacious vaccine is available at present. In particular, these compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNA(+) viruses Yellow fever virus (YFV) and Bovine viral diarrhoea virus (BVDV), both belonging to Flaviviridae. Title compounds were also tested against representatives of Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Salmonella spp.), various atypic mycobacterial strains (Mycobacterium fortuitum and Mycobacterium smegmatis), yeast (Candida albicans) and mould (Aspergillus fumigatus). None of the compounds showed antiviral or antimicrobial activity. The benzo[d]isothiazole compounds showed a marked cytotoxicity (CC(50)=4-9 microM) against the human CD4(+) lymphocytes (MT-4) that were used to support HIV-1 growth. For this reason, the most cytotoxic compounds of this series were evaluated for their antiproliferative activity against a panel of human cell lines derived from haematological and solid tumors. The results highlighted that all the benzo[d]isothiazole derivatives inhibited the growth of leukaemia cell lines, whereas only one of the above mentioned compounds (1e) showed antiproliferative activity against two solid tumor-derived cell lines.

Child contact management in high tuberculosis burden countries: A mixed-methods systematic review.

PubMed

Szkwarko, Daria; Hirsch-Moverman, Yael; Du Plessis, Lienki; Du Preez, Karen; Carr, Catherine; Mandalakas, Anna M

2017-01-01

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Considering the World Health Organization recommendation to implement child contact management (CCM) for TB, we conducted a mixed-methods systematic review to summarize CCM implementation, challenges, predictors, and recommendations. We searched the electronic databases of PubMed/MEDLINE, Scopus, and Web of Science for studies published between 1996-2017 that reported CCM data from high TB-burden countries. Protocol details for this systematic review were registered on PROSPERO: International prospective register of systematic reviews (#CRD42016038105). We formulated a search strategy to identify all available studies, published in English that specifically targeted a) population: child contacts (<15 years) exposed to TB in the household from programmatic settings in high burden countries (HBCs), b) interventions: CCM strategies implemented within the CCM cascade, c) comparisons: CCM strategies studied and compared in HBCs, and d) outcomes: monitoring and evaluation of CCM outcomes reported in the literature for each CCM cascade step. We included any quantitative, qualitative, mixed-methods study design except for randomized-controlled trials, editorials or commentaries. Thirty-seven studies were reviewed. Child contact losses varied greatly for screening, isoniazid preventive therapy initiation, and completion. CCM challenges included: infrastructure, knowledge, attitudes, stigma, access, competing priorities, and treatment. CCM recommendations included: health system strengthening, health education, and improved preventive therapy. Identified predictors included: index case and clinic characteristics, perceptions of barriers and risk, costs, and treatment characteristics. CCM lacks standardization resulting in common challenges and losses throughout the CCM cascade. Prioritization of a CCM-friendly healthcare environment with improved CCM processes and tools; health education; and active, evidence-based strategies can decrease barriers. A focused approach toward every aspect of the CCM cascade will likely diminish losses throughout the CCM cascade and ultimately decrease TB related morbidity and mortality in children.

Calcium absorption from apple and orange juice fortified with calcium citrate malate (CCM).

PubMed

Andon, M B; Peacock, M; Kanerva, R L; De Castro, J A

1996-06-01

Determine calcium (Ca) absorption from Ca fortified orange and apple juice. Absorbability was assessed by measuring 45Ca absorption in healthy women (mean age 57 years, n = 57/group) and whole body 47Ca retention in adult female beagle dogs (n = 6/group) and young adult male rats (n = 6/group). Women received 6.24 mmol (250 mg) Ca as calcium citrate malate fortified orange juice (CCM-OJ) or apple juice (CCM-AJ). Dogs received 3.12 mmol (125 mg) Ca as CCM-OJ or CCM-AJ. Rats were administered 0.15 mmol (6 mg) Ca as either milk, CCM-OJ, or CCM-AJ. Additional 47Ca whole body retention experiments in rats measured the effects of differences in the carbohydrate and organic acid contents of the juices on Ca absorption. Mean +/- SEM percent Ca fractional absorption was greater (p < 0.003) in women who consumed CCM-AJ (42 +/- 2%) than those who consumed CCM-OJ (36 +/- 1%). Ca retention in dogs was 15 +/- 1% for CCM-OJ and 29 +/- 2% for CCM-AJ (p < 0.001). Ca retention was significantly different (p < 0.05) in rats administered milk (42 +/- 2%), CCM-OJ (52 +/- 2%), or CCM-AJ (61 +/- 2%). By manipulating the carbohydrate and organic acid concentrations of test solutions to mimic the composition of Ca fortified juices, we found that the greater fructose and lower organic acid content of apple juice accounted for its greater Ca absorbability. CCM fortified versions of orange and apple juice have high Ca absorbability and are potentially important vehicles for increasing dietary Ca intake. The greater Ca absorption from CCM-AJ compared with CCM-OJ is accounted for by differences in the carbohydrate and organic acid content of the juices. These data suggest that by modifying common beverage ingredients, products with even greater Ca absorbability could be formulated.

Child contact management in high tuberculosis burden countries: A mixed-methods systematic review

PubMed Central

Du Plessis, Lienki; Du Preez, Karen; Carr, Catherine; Mandalakas, Anna M.

2017-01-01

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Considering the World Health Organization recommendation to implement child contact management (CCM) for TB, we conducted a mixed-methods systematic review to summarize CCM implementation, challenges, predictors, and recommendations. We searched the electronic databases of PubMed/MEDLINE, Scopus, and Web of Science for studies published between 1996–2017 that reported CCM data from high TB-burden countries. Protocol details for this systematic review were registered on PROSPERO: International prospective register of systematic reviews (#CRD42016038105). We formulated a search strategy to identify all available studies, published in English that specifically targeted a) population: child contacts (<15 years) exposed to TB in the household from programmatic settings in high burden countries (HBCs), b) interventions: CCM strategies implemented within the CCM cascade, c) comparisons: CCM strategies studied and compared in HBCs, and d) outcomes: monitoring and evaluation of CCM outcomes reported in the literature for each CCM cascade step. We included any quantitative, qualitative, mixed-methods study design except for randomized-controlled trials, editorials or commentaries. Thirty-seven studies were reviewed. Child contact losses varied greatly for screening, isoniazid preventive therapy initiation, and completion. CCM challenges included: infrastructure, knowledge, attitudes, stigma, access, competing priorities, and treatment. CCM recommendations included: health system strengthening, health education, and improved preventive therapy. Identified predictors included: index case and clinic characteristics, perceptions of barriers and risk, costs, and treatment characteristics. CCM lacks standardization resulting in common challenges and losses throughout the CCM cascade. Prioritization of a CCM-friendly healthcare environment with improved CCM processes and tools; health education; and active, evidence-based strategies can decrease barriers. A focused approach toward every aspect of the CCM cascade will likely diminish losses throughout the CCM cascade and ultimately decrease TB related morbidity and mortality in children. PMID:28763500

The 15-lipoxygenase inhibitory, antioxidant, antimycobacterial activity and cytotoxicity of fourteen ethnomedicinally used African spices and culinary herbs.

PubMed

Dzoyem, Jean Paul; Kuete, Victor; McGaw, Lyndy J; Eloff, Jacobus N

2014-10-28

Culinary herbs and spices are widely used ethnomedically across Africa. They are traditionally employed in the treatment of several ailments including inflammation disorders, pain alleviation and infectious diseases. Pharmacological studies are necessary to provide a scientific basis to substantiate their traditional use and safety. In this study, the 15-lipoxygenase inhibitory, antioxidant, antimycobacterial and the cytotoxic activities, total phenolic and flavonoid contents of fourteen edible plants were investigated. The 15-lipoxygenase inhibitory activity was evaluated by the ferrous oxidation-xylenol orange (FOX) assay method. The antioxidant activity was determined using free-radical scavenging assays. The antimycobacterial activity was determined by a broth microdilution method against three species of mycobacteria: Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium fortuitum using tetrazolium violet as growth indicator. The cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. All the extracts tested had some 15-lipoxygenase inhibitory activity ranging from 32.9 to 78.64%. Adansonia digitata (fruit) had the highest antioxidant capacity (IC₅₀ values of 8.15 μg/mL and 9.16 μg/mL in the DPPH and ABTS assays respectively; TEAC of 0.75 in the FRAP assay) along with the highest amount of total phenolics (237.68 mg GAE/g) and total flavonoids (16.14 mg E/g). There were good correlations between DPPH and ABTS values (R(2) 0.98) and between total phenolics and total flavonoids (R(2) 0.94). Tamarindus indica had significant antimycobacterial activity against Mycobacterium aurum (MIC 78 μg/mL). As could be expected with edible plants, all the extracts had a relatively low cytotoxicity with LC₅₀ values higher than 102 μg/mL with the exception of the two Aframomum species (33 and 74 μg/mL). This study provides scientific support for some of the the traditional uses and the pharmacological activities of the culinary herbs and spices investigated. The results suggest that increasing intake of some of these herbs may be useful in preventing or reducing the progression of lifestyle-related diseases. The diversity of the pharmacological activities of the extract from the fruit of Adansonia digitata suggested that this plant might be valuable for application in human and animal health. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Uptake and metabolism of fluorescent steroids by mycobacterial cells.

PubMed

Faletrov, Yaroslav; Brzostek, Anna; Plocinska, Renata; Dziadek, Jarosław; Rudaya, Elena; Edimecheva, Irina; Shkumatov, Vladimir

2017-01-01

Fluorescent steroids BODIPY-cholesterol (BPCh) and 7-nitrobenzoxadiazole-4-amino-(NBD)-labeled 22-NBD-chelesterol (22NC) as well as synthesized 20-(NBD)-pregn-5-en-3β-ol (20NP) were found to undergo bioconversions by Mycobacterium tuberculosis H 37 Rv and M. smegmatis mc 2 155. The major fluorescent products were determined to be 4-en-3-one derivatives of the compounds. Degradation of NBD fluorophore was also detected in the cases of 22NC and 20NP, but neither NBD degradation nor steroidal part modification were observed for the synthesized 3-(NBD)-cholestane. Mycobacterial 3β-hydroxysteroid dehydrogenases were concluded to be responsible for the formation of the 4-en-3-one derivatives. All the compounds tested were found to cause staining both membrane lipids and cytosolic lipid droplets when incubated with mycobacteria in different manner, demonstrating ability of the steroids to reside in the compartments. The findings reveal a potential of the compounds for monitoring of steroid interactions with mycobacteria and provide information for design of new probes for this purpose. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

PubMed Central

2018-01-01

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration. PMID:29503973

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malformations.

PubMed

Scimone, Concetta; Bramanti, Placido; Alafaci, Concetta; Granata, Francesca; Piva, Francesco; Rinaldi, Carmela; Donato, Luigi; Greco, Federica; Sidoti, Antonina; D'Angelo, Rosalia

2017-02-01

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH.

PubMed

Allen, James W A; Harvat, Edgar M; Stevens, Julie M; Ferguson, Stuart J

2006-09-04

C-type cytochromes are characterized by post-translational covalent attachment of heme to thiols that occur in a Cys-Xxx-Xxx-Cys-His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well-studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His-Xxx-Xxx-Xxx-Tyr motif, is replaced by a cysteine residue that occurs in a Cys-Xxx-Xxx-Xxx-Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY-type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY-type CcmE.

75 FR 31419 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-03

... Person Interview (PI) and CCM PFU have been added. These are to be conducted as part of a CCM evaluation... CCM PI OMB package. The CCM program will provide estimates of net coverage error and components of... (PI) and CCM PFU will be conducted. The purpose of the respondent debriefings is to obtain a...

Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia

PubMed Central

Berney, Michael; Cook, Gregory M.

2010-01-01

Mycobacteria are a group of obligate aerobes that require oxygen for growth, but paradoxically have the ability to survive and metabolize under hypoxia. The mechanisms responsible for this metabolic plasticity are unknown. Here, we report on the adaptation of Mycobacterium smegmatis to slow growth rate and hypoxia using carbon-limited continuous culture. When M. smegmatis is switched from a 4.6 h to a 69 h doubling time at a constant oxygen saturation of 50%, the cells respond through the down regulation of respiratory chain components and the F1Fo-ATP synthase, consistent with the cells lower demand for energy at a reduced growth rate. This was paralleled by an up regulation of molecular machinery that allowed more efficient energy generation (i.e. Complex I) and the use of alternative electron donors (e.g. hydrogenases and primary dehydrogenases) to maintain the flow of reducing equivalents to the electron transport chain during conditions of severe energy limitation. A hydrogenase mutant showed a 40% reduction in growth yield highlighting the importance of this enzyme in adaptation to low energy supply. Slow growing cells at 50% oxygen saturation subjected to hypoxia (0.6% oxygen saturation) responded by switching on oxygen scavenging cytochrome bd, proton-translocating cytochrome bc1-aa3 supercomplex, another putative hydrogenase, and by substituting NAD+-dependent enzymes with ferredoxin-dependent enzymes thus highlighting a new pattern of mycobacterial adaptation to hypoxia. The expression of ferredoxins and a hydrogenase provides a potential conduit for disposing of and transferring electrons in the absence of exogenous electron acceptors. The use of ferredoxin-dependent enzymes would allow the cell to maintain a high carbon flux through its central carbon metabolism independent of the NAD+/NADH ratio. These data demonstrate the remarkable metabolic plasticity of the mycobacterial cell and provide a new framework for understanding their ability to survive under low energy conditions and hypoxia. PMID:20062806

Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke.

PubMed

Gore, Aniket V; Lampugnani, Maria Grazia; Dye, Louis; Dejana, Elisabetta; Weinstein, Brant M

2008-01-01

Intracranial hemorrhage (ICH) is a particularly severe form of stroke whose etiology remains poorly understood, with a highly variable appearance and onset of the disease (Felbor et al., 2006; Frizzell, 2005; Lucas et al., 2003). In humans, mutations in any one of three CCM genes causes an autosomal dominant genetic ICH disorder characterized by cerebral cavernous malformations (CCM). Recent evidence highlighting multiple interactions between the three CCM gene products and other proteins regulating endothelial junctional integrity suggests that minor deficits in these other proteins could potentially predispose to, or help to initiate, CCM, and that combinations of otherwise silent genetic deficits in both the CCM and interacting proteins might explain some of the variability in penetrance and expressivity of human ICH disorders. Here, we test this idea by combined knockdown of CCM pathway genes in zebrafish. Reducing the function of rap1b, which encodes a Ras GTPase effector protein for CCM1/Krit1, disrupts endothelial junctions in vivo and in vitro, showing it is a crucial player in the CCM pathway. Importantly, a minor reduction of Rap1b in combination with similar reductions in the products of other CCM pathway genes results in a high incidence of ICH. These findings support the idea that minor polygenic deficits in the CCM pathway can strongly synergize to initiate ICH.

Red-pink pigmented Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov., isolated from rocks in Antarctica.

PubMed

Sedláček, Ivo; Králová, Stanislava; Kýrová, Kamila; Mašlaňová, Ivana; Busse, Hans-Jürgen; Staňková, Eva; Vrbovská, Veronika; Němec, Miroslav; Barták, Miloš; Holochová, Pavla; Švec, Pavel; Pantůček, Roman

2017-06-01

Four rod-shaped and Gram-stain-negative bacterial strains, CCM 8647, CCM 8649T, CCM 8643T and CCM 8648T, were isolated from rock samples collected on James Ross Island, Antarctica. Extensive biotyping, fatty acid profiling, chemotaxonomy, 16S rRNA gene sequencing and whole-genome sequencing was applied to isolates to clarify their taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all four isolates belonged to the genus Hymenobacter. Strains CCM 8649T and CCM 8647 were most closely related to Hymenobacter arizonensis OR362-8T (94.4 % 16S rRNA gene sequence similarity), strain CCM 8643T to Hymenobacter terrae DG7AT (96.3 %) and strain CCM 8648T to Hymenobacter glaciei VUG-A130T (96.3 %). The predominant fatty acids of CCM 8649T and CCM 8647 were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 1ω5c and iso-C15 : 0, whereas those of CCM 8643T and CCM 8648T were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 1ω5c. The quinone systems contained exclusively menaquinone MK-7. The major polyamine was sym-homospermidine. All four strains contained the major polar lipid phosphatidylethanolamine. The G+C content of genomic DNA ranged from 60-63 mol%. Whole-genome sequencing data supported the finding that isolates represented distinct species of the genus Hymenobacter. On the basis of the results obtained, three novel species are proposed for which the names Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov. are suggested, with the type strains CCM 8649T (=LMG 29441T=P5239T), CCM 8643T (=LMG 29435T=P3150T) and CCM 8648T (=LMG 29440T=P5086T), respectively.

Training Internists to Meet Critical Care Needs in the United States: A Consensus Statement from the Critical Care Societies Collaborative (CCSC)

PubMed Central

Pastores, Stephen M.; Martin, Greg S.; Baumann, Michael H.; Curtis, J. Randall; Farmer, J. Christopher; Fessler, Henry E.; Gupta, Rakesh; Hill, Nicholas S.; Hyzy, Robert C.; Kvetan, Vladimir; MacGregor, Drew A.; O’Grady, Naomi P.; Ognibene, Frederick P.; Rubenfeld, Gordon D.; Sessler, Curtis N.; Siegal, Eric; Simpson, Steven Q.; Spevetz, Antoinette; Ward, Nicholas S.; Zimmerman, Janice L.

2014-01-01

Objectives Multiple training pathways are recognized by the Accreditation Council for Graduate Medical Education (ACGME) for internal medicine (IM) physicians to certify in critical care medicine (CCM) via the American Board of Internal Medicine. While each involves 1 year of clinical fellowship training in CCM, substantive differences in training requirements exist among the various pathways. The Critical Care Societies Collaborative convened a task force to review these CCM pathways and to provide recommendations for unified and coordinated training requirements for IM-based physicians. Participants A group of CCM professionals certified in pulmonary-CCM and/or IM-CCM from ACGME-accredited training programs who have expertise in education, administration, research, and clinical practice. Data Sources and Synthesis Relevant published literature was accessed through a MEDLINE search and references provided by all task force members. Material published by the ACGME, American Board of Internal Medicine, and other specialty organizations was also reviewed. Collaboratively and iteratively, the task force reached consensus using a roundtable meeting, electronic mail, and conference calls. Main Results Internal medicine-CCM–based fellowships have disparate program requirements compared to other internal medicine subspecialties and adult CCM fellowships. Differences between IM-CCM and pulmonary-CCM programs include the ratio of key clinical faculty to fellows and a requirement to perform 50 therapeutic bronchoscopies. Competency-based training was considered uniformly desirable for all CCM training pathways. Conclusions The task force concluded that requesting competency-based training and minimizing variations in the requirements for IM-based CCM fellowship programs will facilitate effective CCM training for both programs and trainees. PMID:24637881

Sensitivity of temporal heart rate variability in Poincaré plot to changes in parasympathetic nervous system activity.

PubMed

Karmakar, Chandan K; Khandoker, Ahsan H; Voss, Andreas; Palaniswami, Marimuthu

2011-03-03

A novel descriptor (Complex Correlation Measure (CCM)) for measuring the variability in the temporal structure of Poincaré plot has been developed to characterize or distinguish between Poincaré plots with similar shapes. This study was designed to assess the changes in temporal structure of the Poincaré plot using CCM during atropine infusion, 70° head-up tilt and scopolamine administration in healthy human subjects. CCM quantifies the point-to-point variation of the signal rather than gross description of the Poincaré plot. The physiological relevance of CCM was demonstrated by comparing the changes in CCM values with autonomic perturbation during all phases of the experiment. The sensitivities of short term variability (SD1), long term variability (SD2) and variability in temporal structure (CCM) were analyzed by changing the temporal structure by shuffling the sequences of points of the Poincaré plot. Surrogate analysis was used to show CCM as a measure of changes in temporal structure rather than random noise and sensitivity of CCM with changes in parasympathetic activity. CCM was found to be most sensitive to changes in temporal structure of the Poincaré plot as compared to SD1 and SD2. The values of all descriptors decreased with decrease in parasympathetic activity during atropine infusion and 70° head-up tilt phase. In contrast, values of all descriptors increased with increase in parasympathetic activity during scopolamine administration. The concordant reduction and enhancement in CCM values with parasympathetic activity indicates that the temporal variability of Poincaré plot is modulated by the parasympathetic activity which correlates with changes in CCM values. CCM is more sensitive than SD1 and SD2 to changes of parasympathetic activity.

CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.

PubMed

Lant, Benjamin; Yu, Bin; Goudreault, Marilyn; Holmyard, Doug; Knight, James D R; Xu, Peter; Zhao, Linda; Chin, Kelly; Wallace, Evan; Zhen, Mei; Gingras, Anne-Claude; Derry, W Brent

2015-03-06

The mechanisms governing apical membrane assembly during biological tube development are poorly understood. Here, we show that extension of the C. elegans excretory canal requires cerebral cavernous malformation 3 (CCM-3), independent of the CCM1 orthologue KRI-1. Loss of ccm-3 causes canal truncations and aggregations of canaliculular vesicles, which form ectopic lumen (cysts). We show that CCM-3 localizes to the apical membrane, and in cooperation with GCK-1 and STRIPAK, promotes CDC-42 signalling, Golgi stability and endocytic recycling. We propose that endocytic recycling is mediated through the CDC-42-binding kinase MRCK-1, which interacts physically with CCM-3-STRIPAK. We further show canal membrane integrity to be dependent on the exocyst complex and the actin cytoskeleton. This work reveals novel in vivo roles of CCM-3·STRIPAK in regulating tube extension and membrane integrity through small GTPase signalling and vesicle dynamics, which may help explain the severity of CCM3 mutations in patients.

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

PubMed Central

Crowe, Adam M.; Casabon, Israël; Brown, Kirstin L.; Liu, Jie; Lian, Jennifer; Rogalski, Jason C.; Hurst, Timothy E.; Snieckus, Victor; Foster, Leonard J.

2017-01-01

ABSTRACT Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. PMID:28377529

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin.

PubMed

Retta, Saverio Francesco; Glading, Angela J

2016-12-01

Cerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity. This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State▿ †

PubMed Central

Zuber, Benoît; Chami, Mohamed; Houssin, Christine; Dubochet, Jacques; Griffiths, Gareth; Daffé, Mamadou

2008-01-01

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function. PMID:18567661

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

PubMed

Zuber, Benoît; Chami, Mohamed; Houssin, Christine; Dubochet, Jacques; Griffiths, Gareth; Daffé, Mamadou

2008-08-01

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic-susceptible and antibiotic-resistant bacterial pathogens

PubMed Central

HAYDEL, SHELLEY E.; REMENIH, CHRISTINE M.; WILLIAMS, LYNDA B.

2008-01-01

SYNOPSIS Objectives The capacity to properly address the worldwide incidence of infectious diseases lies in the ability to detect, prevent, and effectively treat these infections. Therefore, identifying and analyzing inhibitory agents are worthwhile endeavors in an era when few new classes of effective antimicrobials have been developed. The use of geological nanomaterials to heal skin infections has been evident since the earliest recorded history, and specific clay minerals may prove valuable in the treatment of bacterial diseases, including infections for which there are no effective antibiotics, such as Buruli ulcer and multi-drug resistant infections. Methods We have subjected two iron-rich clay minerals, which have previously been used to treat Buruli ulcer patients, to broth culture testing of antibiotic-susceptible and -resistant pathogenic bacteria to assess the feasibility of using clay minerals as therapeutic agents. Results One specific mineral, CsAg02, demonstrated bactericidal activity against pathogenic Escherichia coli, extended-spectrum β-lactamase (ESBL) E. coli, S. enterica serovar Typhimurium, Pseudomonas aeruginosa, and Mycobacterium marinum and a combined bacteriostatic/bactericidal effect against Staphylococcus aureus, penicillin-resistant S. aureus (PRSA), methicillin-resistant S. aureus (MRSA), and Mycobacterium smegmatis, while another mineral with similar structure and bulk crystal chemistry, CsAr02, had no effect on or enhanced bacterial growth. The <0.2 μm fraction of CsAg02 and CsAg02 heated to 200°C or 550°C retained bactericidal activity, while cation-exchanged CsAg02 and CsAg02 heated to 900°C no longer killed E. coli. Conclusions Our results indicate that specific mineral products have intrinsic, heat-stable antibacterial properties, which could provide an inexpensive treatment against numerous human bacterial infections. PMID:18070832

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Synergistic Response of Rifampicin with Hydroperoxides on Mycobacterium: A Mechanistic Study

PubMed Central

Patel, Yesha S.; Mehra, Sarika

2017-01-01

Prolonged chemotherapy as well as rapid development of antimicrobial resistance are two of the major concerns for treatment of mycobacterial infections. To enhance the effectiveness of current drug regimens, search for compounds having synergistic interaction with anti-mycobacterial drugs has become indispensable. Here, we have investigated the intervention by oxidative stress, a major factor in mycobacterial pathogenesis, in combination with rifampicin (RIF), a first-line drug used against Mycobacterium tuberculosis. We have observed that a sub-inhibitory concentration of cumene hydroperoxide (CHP), a hydrophobic oxidant, synergistically reduced the minimum inhibitory concentration of RIF by fourfold, with a Fractional Inhibitory Concentration Index (FICI) of 0.45. Also, this interaction was found to be robust and synergistic against different strains of M. smegmatis as well as on M. bovis BCG, with FICI ranging from 0.3 to 0.6. Various physiological, biochemical and molecular parameters were explored to understand the mechanism of synergy. It was observed that increased membrane permeability owing to the presence of the oxidant, led to higher uptake of the drug. Moreover, downregulation of the hydroperoxide reductases by RIF, a transcriptional inhibitor, prevented quenching of the reactive oxygen species produced in the presence of CHP. The lipid soluble reactive species triggered autocatalytic lipid peroxidation (LPO), observed here as extensive membrane damage eventually leading to growth inhibition. Furthermore, it was seen that in combination with hydrogen peroxide (H2O2), the effect was only additive, establishing LPO as a key aspect leading toward synergism. To conclude, this work suggests that targeting the bacterial membrane by a radical species can have a significant impact on the treatment of tuberculosis. PMID:29163385

Anticancer drug-based multifunctional nanogels through self-assembly of dextran-curcumin conjugates toward cancer theranostics.

PubMed

Nagahama, Koji; Sano, Yoshinori; Kumano, Takayuki

2015-06-15

Curcumin (CCM) has been received much attention in cancer theranostics because CCM exhibits both anticancer activity and strong fluorescence available for bio-imaging. However, CCM has never been utilized in clinical mainly due to its extremely low water solubility and its low cellular uptake into cancer cells. We fabricated novel CCM-based biodegradable nanoparticles through self-assembly of amphiphilic dextran-CCM conjugates. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran-CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran-CCMs nanoparticles could be a promising for creation of novel CCM-based cancer theranostics with high efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Comparative Studies of Binding Activity of Curcumin and Didemethylated Curcumin with Selenite: Hydrogen Bonding vs Acid-Base Interactions

NASA Astrophysics Data System (ADS)

Liao, Jiahn-Haur; Wu, Tzu-Hua; Chen, Ming-Yi; Chen, Wei-Ting; Lu, Shou-Yun; Wang, Yi-Hsuan; Wang, Shao-Pin; Hsu, Yen-Min; Huang, Yi-Shiang; Huang, Zih-You; Lin, Yu-Ching; Chang, Ching-Ming; Huang, Fu-Yung; Wu, Shih-Hsiung

2015-12-01

In this report, the in vitro relative capabilities of curcumin (CCM) and didemethylated curcumin (DCCM) in preventing the selenite-induced crystallin aggregation were investigated by turbidity tests and isothermal titration calorimetry (ITC). DCCM showed better activity than CCM. The conformers of CCM/SeO32- and DCCM/SeO32- complexes were optimized by molecular orbital calculations. Results reveal that the selenite anion surrounded by CCM through the H-bonding between CCM and selenite, which is also observed via IR and NMR studied. For DCCM, the primary driving force is the formation of an acid-base adduct with selenite showing that the phenolic OH group of DCCM was responsible for forming major conformer of DCCM. The formation mechanisms of selenite complexes with CCM or DCCM explain why DCCM has greater activity than CCM in extenuating the toxicity of selenite as to prevent selenite-induced lens protein aggregation.

Contemporary commercial music (CCM) survey: who's teaching what in nonclassical music.

PubMed

LoVetri, Jeannette L; Weekly, Edrie Means

2003-06-01

Currently, there is an increasing interest in and demand for training in a wide variety of nonclassical music--contemporary commercial music (CCM)--and most particularly for music theater. A survey of singing teachers was completed to elucidate their training, education, and experience with and methods of teaching CCM. Teachers were at colleges, universities, and conservatories as well as in private studios, both nationally and in several foreign countries. A substantial percentage of those teaching CCM had neither formal education in teaching it nor professional experience. Many of the respondents indicated conflict between classical and CCM styles. Respondents were generally familiar with voice science and voice medicine as well as certain CCM terminology. Teachers expressed an interest in obtaining more information, with an emphasis on healthy vocal production. These results are discussed, as well as implications for the singing teacher who desires specific training to teach CCM.

Independent Evaluation of the integrated Community Case Management of Childhood Illness Strategy in Malawi Using a National Evaluation Platform Design.

PubMed

Amouzou, Agbessi; Kanyuka, Mercy; Hazel, Elizabeth; Heidkamp, Rebecca; Marsh, Andrew; Mleme, Tiope; Munthali, Spy; Park, Lois; Banda, Benjamin; Moulton, Lawrence H; Black, Robert E; Hill, Kenneth; Perin, Jamie; Victora, Cesar G; Bryce, Jennifer

2016-03-01

We evaluated the impact of integrated community case management of childhood illness (iCCM) on careseeking for childhood illness and child mortality in Malawi, using a National Evaluation Platform dose-response design with 27 districts as units of analysis. "Dose" variables included density of iCCM providers, drug availability, and supervision, measured through a cross-sectional cellular telephone survey of all iCCM-trained providers. "Response" variables were changes between 2010 and 2014 in careseeking and mortality in children aged 2-59 months, measured through household surveys. iCCM implementation strength was not associated with changes in careseeking or mortality. There were fewer than one iCCM-ready provider per 1,000 under-five children per district. About 70% of sick children were taken outside the home for care in both 2010 and 2014. Careseeking from iCCM providers increased over time from about 2% to 10%; careseeking from other providers fell by a similar amount. Likely contributors to the failure to find impact include low density of iCCM providers, geographic targeting of iCCM to "hard-to-reach" areas although women did not identify distance from a provider as a barrier to health care, and displacement of facility careseeking by iCCM careseeking. This suggests that targeting iCCM solely based on geographic barriers may need to be reconsidered. © The American Society of Tropical Medicine and Hygiene.

Familial Cerebral Cavernous Malformations Are Associated with Adrenal Calcifications on CT Scans: An Imaging Biomarker for a Hereditary Cerebrovascular Condition.

PubMed

Strickland, Corinne D; Eberhardt, Steven C; Bartlett, Mary R; Nelson, Jeffrey; Kim, Helen; Morrison, Leslie A; Hart, Blaine L

2017-08-01

Purpose To determine if adrenal calcifications seen at computed tomography (CT) are associated with familial cerebral cavernous malformations (fCCMs) in carriers of the CCM1 Common Hispanic Mutation. Materials and Methods This study was approved by the institutional review board. The authors retrospectively reviewed abdominal CT scans in 38 patients with fCCM, 38 unaffected age- and sex-matched control subjects, and 13 patients with sporadic, nonfamilial cerebral cavernous malformation (CCM). The size, number, and laterality of calcifications and the morphologic characteristics of the adrenal gland were recorded. Brain lesion count was recorded from brain magnetic resonance (MR) imaging in patients with fCCM. The prevalence of adrenal calcifications in patients with fCCM was compared with that in unaffected control subjects and those with sporadic CCM by using the Fisher exact test. Additional analyses were performed to determine whether age and brain lesion count were associated with adrenal findings in patients with fCCM. Results Small focal calcifications (SFCs) (≤5 mm) were seen in one or both adrenal glands in 19 of the 38 patients with fCCM (50%), compared with 0 of the 38 unaffected control subjects (P < .001) and 0 of the 13 subjects with sporadic CCM (P = .001). Adrenal calcifications in patients with fCCM were more frequently left sided, with 17 of 19 patients having more SFCs in the left adrenal gland than the right adrenal gland and 50 of the 61 observed SFCs (82%) found in the left adrenal gland. No subjects had SFCs on the right side only. In patients with fCCM, the presence of SFCs showed a positive correlation with age (P < .001) and number of brain lesions (P < .001). Conclusion Adrenal calcifications identified on CT scans are common in patients with fCCM and may be a clinically silent manifestation of disease. © RSNA, 2017.

Terminalia laxiflora and Terminalia brownii contain a broad spectrum of antimycobacterial compounds including ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols.

PubMed

Salih, Enass Y A; Julkunen-Tiitto, Riitta; Lampi, Anna-Maija; Kanninen, Markku; Luukkanen, Olavi; Sipi, Marketta; Lehtonen, Mari; Vuorela, Heikki; Fyhrquist, Pia

2018-05-04

Terminalia laxiflora Engl. & Diels, (Sudanese Arabic name: Darout الدروت) and Terminalia brownii Fresen (Sudanese Arabic name: Alshaf ألشاف) (Combretaceae) are used in Sudanese traditional folk medicine and in other African countries for treatment of infectious diseases, TB and its symptoms, such as cough, bronchitis and chest pain. Because of the frequent use of T. laxiflora and T. brownii in African traditional medicine and due to the absence of studies regarding their antimycobacterial potential there was a need to screen extracts of T. laxiflora and T. brownii for their growth inhibitory potential and to study the chemical composition and compounds in growth inhibitory extracts. The plant species were collected in Sudan (Blue Nile Forest, Ed Damazin Forestry areas) and selected according to their uses in traditional medicine for the treatment of bacterial infections, including TB. Eighty extracts and fractions of the stem bark, stem wood, roots, leaves and fruits of T. laxiflora and T. brownii and nine pure compounds present in the active extracts were screened against Mycobacterium smegmatis ATCC 14468 using agar diffusion and microplate dilution methods. Inhibition zones and MIC values were estimated and compared to rifampicin. HPLC-UV/DAD, GC/MS and UHPLC/Q-TOF MS were employed to identify the compounds in the growth inhibitory extracts. The roots of T. laxiflora and T. brownii gave the best antimycobacterial effects (IZ 22-27mm) against Mycobacterium smegmatis. The lowest MIC of 625µg/ml was observed for an acetone extract of the root of T. laxiflora followed by methanol and ethyl acetate extracts, both giving MIC values of 1250µg/ml. Sephadex LH-20 column chromatography purification of T. brownii roots resulted in low MIC values of 62.5µg/ml and 125µg/ml for acetone and ethanol fractions, respectively, compared to 5000µg/ml for the crude methanol extract. Methyl (S)-flavogallonate is suggested to be the main active compound in the Sephadex LH- 20 acetone fraction, while ellagic acid xyloside and methyl ellagic acid xyloside are suggested to give good antimycobacterial activity in the Sephadex LH-20 ethanol fraction. RP-18 TLC purifications of an ethyl acetate extract of T. laxiflora roots resulted in the enrichment of punicalagin in one of the fractions (Fr5). This fraction gave a five times smaller MIC (500µg/ml) than the crude ethyl acetate extract (2500µg/ml) and this improved activity is suggested to be mostly due to punicalagin. 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone and β-sitosterol were found in antimycobacterial hexane extracts of the stem bark of both studied species. Of these compounds, 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone have not been previously identified in T. brownii and T. laxiflora. Moreover, both plant species contained friedelin, betulinic acid, β-amyrine and two unknown oleanane-type triterpenoids. Of the listed compounds, friedelin, triacontanol and sitostenone gave a MIC of 250µg/ml against M. smegmatis, whereas stigmasterol and β-sitosterol gave MIC values of 500µg/ml. Our results show that T. laxiflora and T. brownii contain antimycobacterial compounds of diverse polarities and support the traditional uses of various parts of T. laxiflora and T.brownii as decoctions for treatment of tuberculosis. Further investigations are warranted to explore additional (new) antimycobacterial compounds in the active extracts of T. laxiflora and T. brownii. Copyright © 2018. Published by Elsevier B.V.

Curcumin-incorporated albumin nanoparticles and its tumor image

NASA Astrophysics Data System (ADS)

Gong, Guangming; Pan, Qinqin; Wang, Kaikai; Wu, Rongchun; Sun, Yong; Lu, Ying

2015-01-01

Albumin is an ideal carrier for hydrophobic drugs. This paper reports a facile route to develop human serum albumin (HSA)-curcumin (CCM) nanoparticles, in which β-mercaptoethanol (β-ME) acted as an inducer and CCM acted as a bridge. Fluorescence quenching and conformational changes in HSA-CCM nanoparticles occurred during assembly. Disulfide bonds and hydrophobic interactions may play a key role in assembly. HSA-CCM nanoparticles were about 130 nm in size, and the solubility of CCM increased by more than 500 times. The HSA-CCM nanoparticles could accumulate at the cytoplasm of tumor cells and target the tumor tissues. Therefore, HSA nanoparticles fabricated by β-ME denaturation are promising nanocarriers for hydrophobic substances from chemotherapy drugs to imaging probes.

Curcumin-incorporated albumin nanoparticles and its tumor image.

PubMed

Gong, Guangming; Pan, Qinqin; Wang, Kaikai; Wu, Rongchun; Sun, Yong; Lu, Ying

2015-01-30

Albumin is an ideal carrier for hydrophobic drugs. This paper reports a facile route to develop human serum albumin (HSA)-curcumin (CCM) nanoparticles, in which β-mercaptoethanol (β-ME) acted as an inducer and CCM acted as a bridge. Fluorescence quenching and conformational changes in HSA-CCM nanoparticles occurred during assembly. Disulfide bonds and hydrophobic interactions may play a key role in assembly. HSA-CCM nanoparticles were about 130 nm in size, and the solubility of CCM increased by more than 500 times. The HSA-CCM nanoparticles could accumulate at the cytoplasm of tumor cells and target the tumor tissues. Therefore, HSA nanoparticles fabricated by β-ME denaturation are promising nanocarriers for hydrophobic substances from chemotherapy drugs to imaging probes.

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens

PubMed Central

Naz, Sadia; Ngo, Tony; Farooq, Umar

2017-01-01

Background The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis. The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Methods Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli, two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. Results High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis. Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Discussion Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner. PMID:28948099

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens.

PubMed

Naz, Sadia; Ngo, Tony; Farooq, Umar; Abagyan, Ruben

2017-01-01

The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis . The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli , two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis . Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner.

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

PubMed

Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

1995-10-01

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Strategy for Identifying Repurposed Drugs for the Treatment of Cerebral Cavernous Malformation

PubMed Central

Gibson, Christopher C.; Zhu, Weiquan; Davis, Chadwick T.; Bowman-Kirigin, Jay A.; Chan, Aubrey C.; Ling, Jing; Walker, Ashley E.; Goitre, Luca; Monache, Simona Delle; Retta, Saverio Francesco; Shiu, Yan-Ting E.; Grossmann, Allie H.; Thomas, Kirk R.; Donato, Anthony J.; Lesniewski, Lisa A.; Whitehead, Kevin J.; Li, Dean Y.

2014-01-01

Background Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans with no approved non-surgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results We developed an unbiased screening platform based on both cellular and animal models of loss-of-function of CCM2. Our discovery strategy consisted of four steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2; a secondary screen of functional changes in endothelial stability in these same cells; a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2; and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2,100 known drugs and bioactive compounds, and identified two candidates for further study, cholecalciferol (Vitamin D3) and tempol (a scavenger of superoxide). Each drug decreased lesion burden in a mouse model of CCM vascular disease by approximately 50%. Conclusions By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease. PMID:25486933

Attitudes of anesthesiology residents toward critical care medicine training.

PubMed

Durbin, C G; McLafferty, C L

1993-09-01

The number of anesthesiology residents pursuing critical care medicine (CCM) fellowship training has been decreasing in recent years. A significant number of training positions remain unfilled each year. Possible causes of this decline were evaluated by surveying residents regarding their attitudes toward practice and training in CCM. All 38 anesthesiology programs having accredited CCM fellowships were surveyed. Four of these and one program without CCM fellowships were used to develop the survey instrument. Four programs without CCM fellowships and 34 programs with CCM fellowships make up the survey group. Returned were 640 surveys from 37 (97%) programs accounting for over 30% of the possible residents. Resident interest in pursuing CCM training decreased as year of residency increased (P < 0.0001). Residents in programs with little patient care responsibility during intensive care unit (ICU) rotations expressed less interest in CCM training (P < 0.012). The administrative role of the anesthesiology department in the ICU also influenced resident interest (P < 0.014). Written responses to open-ended questions suggested resident concerns with the following: stress of chronic care, financial consequences of additional year of training, ICU call frequency and load, ICU role ambiguity, and shared decision-making in the ICU. A recurring question was, "Are there jobs (outside of academics) for anesthesiologist intensivists?" Most residents knew a CCM anesthesiologist they admired and knew that there were unfilled fellowship positions available. Defining the job market, improving curriculum and teaching, supporting deferment of student loans, and introducing residents and medical students to the ICU earlier may increase the interest in CCM practice among anesthesiology residents.

General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria

PubMed Central

2015-01-01

The chemical features that impact small-molecule permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibiotics. Antibacterials are known to have vastly different structural and physicochemical properties compared to nonantiinfective drugs, as illustrated herein by principal component analysis (PCA). To understand how these properties influence bacterial permeability, we have developed a systematic approach to evaluate the penetration of diverse compounds into bacteria with distinct cellular envelopes. Intracellular compound accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations are used to identify structural and physicochemical parameters that correlate with accumulation. An initial study using 10 sulfonyladenosines in Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis has identified nonobvious correlations between chemical structure and permeability that differ among the various bacteria. Effects of cotreatment with efflux pump inhibitors were also investigated. This sets the stage for use of this platform in larger prospective analyses of diverse chemotypes to identify global relationships between chemical structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery. PMID:25198656

Microbicidal Effects of Stored Aqueous Ozone Solution Generated by Nano-bubble Technology.

PubMed

Seki, Mineaki; Ishikawa, Tatsuya; Terada, Hiroshi; Nashimoto, Masayuki

2017-01-01

Clinically used disinfectants are often irritating and cause skin problems. Ozone water is unique among disinfectants. It does not damage skin cells and readily decomposes to oxygen without generating harmful residues. On the other hand, it rapidly loses its sanitizing activity. Recently developed nano-bubble ozone water (NBOW) can keep its sanitizing activity much longer. This study aimed to examine the microbicidal effects of NBOW after long-term storage. The concentration of ozone in NBOW was examined by measuring the NBOW redox potential. Microbicidal activity was evaluated by colony formation assays, after incubating bacteria with NBOW for set time periods. NBOW lost its microbicidal activity after 1 year of storage at 4°C. Stocked frozen, NBOW retained appreciable microbicidal activity after 1 year of storage. Mycobacterium smegmatis, one of the most disinfectant-resistant bacteria, was killed within 15 min. NBOW was resistant to freeze-thawing. NBOW that had been stored frozen possessed sufficient microbicidal activity to kill bacteria even after 1 year of storage. Moreover, it was shown that NBOW is freeze-thaw resistant. NBOW possesses desirable features rendering it an attractive alternative disinfectant. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Decoding the similarities and differences among mycobacterial species

PubMed Central

Vedithi, Sundeep Chaitanya; Blundell, Tom L.

2017-01-01

Mycobacteriaceae comprises pathogenic species such as Mycobacterium tuberculosis, M. leprae and M. abscessus, as well as non-pathogenic species, for example, M. smegmatis and M. thermoresistibile. Genome comparison and annotation studies provide insights into genome evolutionary relatedness, identify unique and pathogenicity-related genes in each species, and explore new targets that could be used for developing new diagnostics and therapeutics. Here, we present a comparative analysis of ten-mycobacterial genomes with the objective of identifying similarities and differences between pathogenic and non-pathogenic species. We identified 1080 core orthologous clusters that were enriched in proteins involved in amino acid and purine/pyrimidine biosynthetic pathways, DNA-related processes (replication, transcription, recombination and repair), RNA-methylation and modification, and cell-wall polysaccharide biosynthetic pathways. For their pathogenicity and survival in the host cell, pathogenic species have gained specific sets of genes involved in repair and protection of their genomic DNA. M. leprae is of special interest owing to its smallest genome (1600 genes and ~1300 psuedogenes), yet poor genome annotation. More than 75% of the pseudogenes were found to have a functional ortholog in the other mycobacterial genomes and belong to protein families such as transferases, oxidoreductases and hydrolases. PMID:28854187

The assessment of changes to the nontuberculous mycobacterial metabolome in response to anti-TB drugs.

PubMed

Drapal, Margit; Wheeler, Paul R; Fraser, Paul D

2018-06-26

Mycobacterium species can cause a range of nontuberculous infections of healthy and immunocompromised people as well as infect people during and after surgical procedures. The similarity of nontuberculous mycobacteria (NTM) to the tuberculosis bacilli (TB) could ultimately enable the use of anti-TB drugs for the genus. Hence, three NTM (M. smegmatis, M. phlei and M. avium) were cultured under different lab conditions, causing two mycobacterial phenotypes (active and dormant), and treated with isoniazid (INH) and ethambutol (EMB) independently or in combination. Metabolite profiling was applied to facilitate the investigation and characterisation of intracellular targets affected by the antibiotics. Aliquots of the cell culture were taken over the treatment period and the metabolite profile of the cells analysed by GC/MS. Comparative analysis of the metabolite levels to untreated mycobacteria confirmed the successful action of the antibiotics on the metabolism of all three species. Furthermore, single metabolites and metabolite pathways affected by the antibiotics could be identified and included, besides the known target sites for INH and EMB on mycobacterial cells, changes in e.g. nucleotide and saccharide levels. The combined treatment highlighted the property of EMB to enhance the effects of INH even under hypoxic culture conditions.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

PubMed

Syal, Kirtimaan; Maiti, Krishnagopal; Naresh, Kottari; Avaji, Prakash Gouda; Chatterji, Dipankar; Jayaraman, Narayanaswamy

2016-10-01

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100 μg mL(-1). Especially the biofilm coat is ruptured by ~80-85 % in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-05-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.

Synthesis, in vitro anticancer and antimycobacterial evaluation of new 5-(2,5-dimethoxyphenyl)-1,3,4-thiadiazole-2-amino derivatives.

PubMed

Polkam, Naveen; Rayam, Parsharamulu; Anireddy, Jaya Shree; Yennam, Satyanarayana; Anantaraju, Hasitha Shilpa; Dharmarajan, Sriram; Perumal, Yogeeswari; Kotapalli, Sudha Sravanti; Ummanni, Ramesh; Balasubramanian, Sridhar

2015-04-01

A series of 2,5-disubstituted-1,3,4-thiadiazole derivatives 5a-5l, 7a-7e and 9 have been synthesised and screened for in vitro antimycobacterial activity against Mycobacterium smegmatis MC-155. In addition these compounds have also been screened for cytotoxic activity against cancer cell lines HT-29, MDA-MB-231 by MTT colorimetric assay. The compounds are well characterized by spectral analysis viz. (1)H NMR, (13)C NMR, FT-IR, mass and HRMS. Screening results indicate that compounds 5g, 7a possess good antitubercular activity with MIC value 65.74 and 40.86, respectively, compounds 5g, 7a, 7b, 7d, 7e and 9 displayed promising cytotoxic activity against the cell lines tested. 5g and 7a stand out to be potent antimycobacterial and anticancer agents among the tested series. Further the title compounds were also tested on human normal cells HEK293T and are found to be safer with lesser cytotoxicity. It is interesting to observe that compound 5g has come out to be safer, potent anticancer and antimycobacterial agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Comparative Studies of Binding Activity of Curcumin and Didemethylated Curcumin with Selenite: Hydrogen Bonding vs Acid-Base Interactions.

PubMed

Liao, Jiahn-Haur; Wu, Tzu-Hua; Chen, Ming-Yi; Chen, Wei-Ting; Lu, Shou-Yun; Wang, Yi-Hsuan; Wang, Shao-Pin; Hsu, Yen-Min; Huang, Yi-Shiang; Huang, Zih-You; Lin, Yu-Ching; Chang, Ching-Ming; Huang, Fu-Yung; Wu, Shih-Hsiung

2015-12-04

In this report, the in vitro relative capabilities of curcumin (CCM) and didemethylated curcumin (DCCM) in preventing the selenite-induced crystallin aggregation were investigated by turbidity tests and isothermal titration calorimetry (ITC). DCCM showed better activity than CCM. The conformers of CCM/SeO3(2-) and DCCM/SeO3(2-) complexes were optimized by molecular orbital calculations. Results reveal that the selenite anion surrounded by CCM through the H-bonding between CCM and selenite, which is also observed via IR and NMR studied. For DCCM, the primary driving force is the formation of an acid-base adduct with selenite showing that the phenolic OH group of DCCM was responsible for forming major conformer of DCCM. The formation mechanisms of selenite complexes with CCM or DCCM explain why DCCM has greater activity than CCM in extenuating the toxicity of selenite as to prevent selenite-induced lens protein aggregation.

The eHealth Enhanced Chronic Care Model: a theory derivation approach.

PubMed

Gee, Perry M; Greenwood, Deborah A; Paterniti, Debora A; Ward, Deborah; Miller, Lisa M Soederberg

2015-04-01

Chronic illnesses are significant to individuals and costly to society. When systematically implemented, the well-established and tested Chronic Care Model (CCM) is shown to improve health outcomes for people with chronic conditions. Since the development of the original CCM, tremendous information management, communication, and technology advancements have been established. An opportunity exists to improve the time-honored CCM with clinically efficacious eHealth tools. The first goal of this paper was to review research on eHealth tools that support self-management of chronic disease using the CCM. The second goal was to present a revised model, the eHealth Enhanced Chronic Care Model (eCCM), to show how eHealth tools can be used to increase efficiency of how patients manage their own chronic illnesses. Using Theory Derivation processes, we identified a "parent theory", the Chronic Care Model, and conducted a thorough review of the literature using CINAHL, Medline, OVID, EMBASE PsychINFO, Science Direct, as well as government reports, industry reports, legislation using search terms "CCM or Chronic Care Model" AND "eHealth" or the specific identified components of eHealth. Additionally, "Chronic Illness Self-management support" AND "Technology" AND several identified eHealth tools were also used as search terms. We then used a review of the literature and specific components of the CCM to create the eCCM. We identified 260 papers at the intersection of technology, chronic disease self-management support, the CCM, and eHealth and organized a high-quality subset (n=95) using the components of CCM, self-management support, delivery system design, clinical decision support, and clinical information systems. In general, results showed that eHealth tools make important contributions to chronic care and the CCM but that the model requires modification in several key areas. Specifically, (1) eHealth education is critical for self-care, (2) eHealth support needs to be placed within the context of community and enhanced with the benefits of the eCommunity or virtual communities, and (3) a complete feedback loop is needed to assure productive technology-based interactions between the patient and provider. The revised model, eCCM, offers insight into the role of eHealth tools in self-management support for people with chronic conditions. Additional research and testing of the eCCM are the logical next steps.

Integrated community case management for childhood illnesses: explaining policy resistance in Kenya.

PubMed

Juma, Pamela A; Owuor, Karen; Bennett, Sara

2015-12-01

There has been a re-emphasis recently on community health workers to provide child health care services including integrated community case management for childhood illness (iCCM). This research analysed iCCM policy development in Kenya and in particular the types of decision-making criteria used by Kenyan policy-makers in considering whether to advance iCCM policy. Data were collected through document reviews (n = 41) and semi-structured interviews (n = 19) with key stakeholders in iCCM policy including government officials, development partners, bilateral donors, and civil society organizations. Initial analysis was guided by the policy triangle with further analysis of factors affecting policy decision-making drawing upon a simple framework developed by Grindle and Thomas (Policy makers, policy choices and policy outcomes: the political economy of reform in developing countries. 1989; Policy Sci 22: :213-48.). Policy development for iCCM has been slow in Kenya, compared with other Sub-Saharan African countries. At the time of the study, the Government had just completed the Community Health Training Manual which incorporated iCCM as a module, but this was the only formal expression of iCCM in Kenya. We found technical considerations, notably concerns about community health workers dispensing antibiotics to be a key factor slowing iCCM policy development, but this also overlapped with bureaucratic considerations, such as how the development of community health worker cadres may affect clinicians, as well as initial concerns about how an integrated approach might affect vertically oriented programs. International actors through agreements such as the Millennium Development Goals helped to get child survival onto the national policy agenda and such actors were active promoters of iCCM policy change. However international funders had not committed funding to scale-up iCCM policy, and this probably constrained their influence over iCCM policy debate. Kenyan actors' concerns about iCCM underline the importance of adapting global policies to local conditions, and also generating local evidence to inform decision-making. © The Author 2015. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Time to Remission for Depression with Collaborative Care Management (CCM) in Primary Care.

PubMed

Garrison, Gregory M; Angstman, Kurt B; O'Connor, Stephen S; Williams, Mark D; Lineberry, Timothy W

2016-01-01

Collaborative care management (CCM) has been shown to have superior outcomes to usual care (UC) for depressed patients with a fixed end point. This study was a survival analysis over time comparing CCM with UC using remission (9-item Patient Health Questionnaire [PHQ-9] score <5) and persistent depressive symptoms (PDSs; PHQ-9 score ≥10) as end points. A retrospective cohort study of 7340 patients with depression cared for at 4 outpatient primary care clinics was conducted from March 2008 through June 2013. All adult patients diagnosed with depression (International Classification of Diseases, 9th Revision [ICD-9], codes 296.2-3) or dysthymia (ICD-9 code 300.4) with an initial PHQ-9 score ≥10 were included. CCM was implemented at all clinics between 2008 and 2010. Kaplan-Meyer survival curves for time to remission and PDSs were plotted. A Cox proportional hazards model was used to adjust for expected differences between patients choosing CCM versus UC. Median time to remission was 86 days (95% confidence interval [CI], 81-91 days) for the CCM group versus 614 days (95% CI, 565-692 days) for the UC group. Likewise, median duration of PDSs was 31 days (95% CI, 30-33 days) for the CCM group versus 154 days (95% CI, 138-182 days) for the UC group. In the Cox proportional hazards model, which controlled for covariates such as age, sex, race, diagnosis, and initial PHQ-9 score, CCM was associated with faster remission (hazard ratio of the CCM group [HRCCM], 2.48; 95% CI, 2.31-2.65). This study demonstrated that patients enrolled in CCM have a faster rate of remission and a shorter duration of PDSs than patients choosing UC. © Copyright 2016 by the American Board of Family Medicine.

Policy challenges facing integrated community case management in Sub-Saharan Africa.

PubMed

Bennett, Sara; George, Asha; Rodriguez, Daniela; Shearer, Jessica; Diallo, Brahima; Konate, Mamadou; Dalglish, Sarah; Juma, Pamela; Namakhoma, Ireen; Banda, Hastings; Chilundo, Baltazar; Mariano, Alda; Cliff, Julie

2014-07-01

To report an in-depth analysis of policy change for integrated community case management of childhood illness (iCCM) in six sub-Saharan African countries. We analysed how iCCM policies developed and the barriers and facilitators to policy change. Qualitative retrospective case studies drawing from document reviews, semi-structured interviews and in-country validation workshops were conducted in Burkina Faso, Kenya, Malawi, Mali, Mozambique and Niger. These countries were selected to maximise variation in iCCM policy status, community health worker (CHW) models and different African regions. Country iCCM policies evolved in an ad hoc fashion, but were substantially influenced by the history of primary health care and the nature of CHW programmes. Technical officers within Ministries of Health led iCCM policy change with support from international donors, but neither communities nor political leadership was mobilised. Concerns about achieving the Millennium Development Goals, together with recognition of the shortcomings of existing child health programmes, led to the adoption of iCCM policies. Availability of external financing played a critical role in facilitating policy change. iCCM policy change has been promoted by international agencies, but national governments have struggled to align iCCM with country health systems. Greater investment is needed in tailoring global policy initiatives to match country needs. High-level, political ownership of iCCM policies could facilitate policy change, as could clearer strategies for ensuring the long-term sustainability of such policies. © 2014 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

Policy challenges facing integrated community case management in Sub-Saharan Africa

PubMed Central

Bennett, Sara; George, Asha; Rodriguez, Daniela; Shearer, Jessica; Diallo, Brahima; Konate, Mamadou; Dalglish, Sarah; Juma, Pamela; Namakhoma, Ireen; Banda, Hastings; Chilundo, Baltazar; Mariano, Alda; Cliff, Julie

2014-01-01

Objective To report an in-depth analysis of policy change for integrated community case management of childhood illness (iCCM) in six sub-Saharan African countries. We analysed how iCCM policies developed and the barriers and facilitators to policy change. Methods Qualitative retrospective case studies drawing from document reviews, semi-structured interviews and in-country validation workshops were conducted in Burkina Faso, Kenya, Malawi, Mali, Mozambique and Niger. These countries were selected to maximise variation in iCCM policy status, community health worker (CHW) models and different African regions. Results Country iCCM policies evolved in an ad hoc fashion, but were substantially influenced by the history of primary health care and the nature of CHW programmes. Technical officers within Ministries of Health led iCCM policy change with support from international donors, but neither communities nor political leadership was mobilised. Concerns about achieving the Millennium Development Goals, together with recognition of the shortcomings of existing child health programmes, led to the adoption of iCCM policies. Availability of external financing played a critical role in facilitating policy change. Conclusions iCCM policy change has been promoted by international agencies, but national governments have struggled to align iCCM with country health systems. Greater investment is needed in tailoring global policy initiatives to match country needs. High-level, political ownership of iCCM policies could facilitate policy change, as could clearer strategies for ensuring the long-term sustainability of such policies. PMID:24750516

Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisher, Oriana S.; Deng, Hanqiang; Liu, Dou

Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystalmore » structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho–ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.« less

Curcumin-loaded chitosan-cholesterol micelles: evaluation in monolayers and 3D cancer spheroid model.

PubMed

Muddineti, Omkara Swami; Kumari, Preeti; Ray, Eupa; Ghosh, Balaram; Biswas, Swati

2017-06-02

To improve the bioavailability and anticancer potential of curcumin by using a cholesterol-conjugated chitosan micelle. Methods & methods: Cholesterol was conjugated to chitosan (15 kDa) to form self-assembled micelles, which loaded curcumin. Physicochemical characterization and formulation optimization of the drug-loaded micelles (curcumin-loaded chitosan-cholesterol micelles [C-CCM]) were performed. In vitro cellular uptake and viability of C-CCM were investigated in melanoma and breast cancer cell lines. The antitumor efficacy was evaluated in 3D lung cancer spheroid model. The optimized C-CCM had size of approximately 162 nm with loading efficiency of approximately 36%. C-CCM was taken up efficiently by the cells, and it reduced cancer cell viability significantly compared with free curcumin. C-CCM enhanced the antitumor efficacy in spheroids, suggesting that C-CCM could be used as an effective chemotherapy in cancer.

Reducing agent-free synthesis of curcumin-loaded albumin nanoparticles by self-assembly at room temperature.

PubMed

Safavi, Maryam Sadat; Shojaosadati, Seyed Abbas; Yang, Hye Gyeong; Kim, Yejin; Park, Eun Ji; Lee, Kang Choon; Na, Dong Hee

2017-08-30

The purpose of this study was to prepare curcumin-loaded bovine serum albumin nanoparticles (CCM-BSA-NPs) by reducing agent-free self-assembly at room temperature. A 2 4 factorial design approach was used to investigate the CCM-BSA-NP preparation process at different pH values, temperatures, dithiothreitol amounts, and CCM/BSA mass ratios. Increasing the ionic strength enabled preparation of CCM-BSA-NPs at 25°C without reducing agent. CCM-BSA-NPs prepared under the optimized conditions at 25°C showed a particle size of 110±6nm, yield of 88.5%, and drug loading of 7.1%. The CCM-BSA-NPs showed strong antioxidant activity and neuroprotective effects in glutamate-induced mouse hippocampal neuronal HT22 cells. This study suggests that ionic strength can be a key parameter affecting the preparation of albumin-based NPs. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA Editing Responses to Oxidative Stress between a Wild Abortive Type Male-Sterile Line and Its Maintainer Line

PubMed Central

Xiong, Jie; Tao, Tao; Luo, Zhi; Yan, Shuaigang; Liu, Yi; Yu, Xinqiao; Liu, Guolan; Xia, Hui; Luo, Lijun

2017-01-01

RNA editing of mitochondrial gene transcripts plays a central role during plant development and evolutionary adaptation. RNA editing has previously been reported to differ between the rice cytoplasmic male sterile (CMS) line and its maintainer line, which has been suggested as a cause for their different performances under environmental stress. To specifically test this hypothesis, a wild abortive (WA) CMS line (Huhan-1A) and its maintainer line (Huhan-1B) were utilized to investigate performances in response to oxidative stress, as well as RNA editing efficiencies on transcripts of six selected mitochondrial genes. Compared to the maintainer line, Huhan-1A represented both lower plant height and total antioxidant capacity, possessed higher total soluble protein and chlorophyll contents, accumulated less H2O2 content on the 3rd day after treatment (DAT), and exhibited higher survival ratio after re-watering. Furthermore, a total of 90 editing sites were detected on transcripts of six mitochondrial genes (atp9, nad2, nad7, nad9, ccmB, and ccmC) in both Huhan-1A and Huhan-1B on the 0, 1st, and 3rd DAT. Forty-eight sites were furthermore determined as stress-responsive sites (SRS). Generally, in response to oxidative stress, SRS in Huhan-1A increased the resulting editing efficiencies, while SRS in Huhan-1B decreased the resulting editing efficiencies. In addition, 33 and 22 sites at ccmB and ccmC were differentially edited between Huhan-1A and Huhan-1B, respectively, on the 0, 1st, and 3rd DAT. Editing efficiencies of ccmB and ccmC were generally lower in Huhan-1A (ccmB, 37.3–47.8%; ccmC, 41.2–52.3%) than those in Huhan-1B (ccmB, 82.6–86.5%; ccmC, 81.0–82.9%). Deficiencies of RNA editing in Huhan-1A at ccmB and ccmC could lead to the loss of transmembrane domains in their protein structures. Consequently, differences in RNA editing at ccmB and ccmC between the WA-CMS line and its maintainer line partially explained their different performances under stress. Moreover, we detected differences in expressions of pentatricopeptide repeat (PPR) genes between both lines, as well as significant correlations with RNA editing. Our study indicated potential associations of RNA editing and PPR genes in rice tolerance to abiotic stresses. However, the underlying molecular mechanisms of stress-adaptation, which are attributed to RNA editing on transcripts of mitochondrial genes, require further investigation. PMID:29234339

The eHealth Enhanced Chronic Care Model: A Theory Derivation Approach

PubMed Central

Greenwood, Deborah A; Paterniti, Debora A; Ward, Deborah; Miller, Lisa M Soederberg

2015-01-01

Background Chronic illnesses are significant to individuals and costly to society. When systematically implemented, the well-established and tested Chronic Care Model (CCM) is shown to improve health outcomes for people with chronic conditions. Since the development of the original CCM, tremendous information management, communication, and technology advancements have been established. An opportunity exists to improve the time-honored CCM with clinically efficacious eHealth tools. Objective The first goal of this paper was to review research on eHealth tools that support self-management of chronic disease using the CCM. The second goal was to present a revised model, the eHealth Enhanced Chronic Care Model (eCCM), to show how eHealth tools can be used to increase efficiency of how patients manage their own chronic illnesses. Methods Using Theory Derivation processes, we identified a “parent theory”, the Chronic Care Model, and conducted a thorough review of the literature using CINAHL, Medline, OVID, EMBASE PsychINFO, Science Direct, as well as government reports, industry reports, legislation using search terms “CCM or Chronic Care Model” AND “eHealth” or the specific identified components of eHealth. Additionally, “Chronic Illness Self-management support” AND “Technology” AND several identified eHealth tools were also used as search terms. We then used a review of the literature and specific components of the CCM to create the eCCM. Results We identified 260 papers at the intersection of technology, chronic disease self-management support, the CCM, and eHealth and organized a high-quality subset (n=95) using the components of CCM, self-management support, delivery system design, clinical decision support, and clinical information systems. In general, results showed that eHealth tools make important contributions to chronic care and the CCM but that the model requires modification in several key areas. Specifically, (1) eHealth education is critical for self-care, (2) eHealth support needs to be placed within the context of community and enhanced with the benefits of the eCommunity or virtual communities, and (3) a complete feedback loop is needed to assure productive technology-based interactions between the patient and provider. Conclusions The revised model, eCCM, offers insight into the role of eHealth tools in self-management support for people with chronic conditions. Additional research and testing of the eCCM are the logical next steps. PMID:25842005

Implementing collaborative care for depression treatment in primary care: A cluster randomized evaluation of a quality improvement practice redesign

PubMed Central

2011-01-01

Background Meta-analyses show collaborative care models (CCMs) with nurse care management are effective for improving primary care for depression. This study aimed to develop CCM approaches that could be sustained and spread within Veterans Affairs (VA). Evidence-based quality improvement (EBQI) uses QI approaches within a research/clinical partnership to redesign care. The study used EBQI methods for CCM redesign, tested the effectiveness of the locally adapted model as implemented, and assessed the contextual factors shaping intervention effectiveness. Methods The study intervention is EBQI as applied to CCM implementation. The study uses a cluster randomized design as a formative evaluation tool to test and improve the effectiveness of the redesign process, with seven intervention and three non-intervention VA primary care practices in five different states. The primary study outcome is patient antidepressant use. The context evaluation is descriptive and uses subgroup analysis. The primary context evaluation measure is naturalistic primary care clinician (PCC) predilection to adopt CCM. For the randomized evaluation, trained telephone research interviewers enrolled consecutive primary care patients with major depression in the evaluation, referred enrolled patients in intervention practices to the implemented CCM, and re-surveyed at seven months. Results Interviewers enrolled 288 CCM site and 258 non-CCM site patients. Enrolled intervention site patients were more likely to receive appropriate antidepressant care (66% versus 43%, p = 0.01), but showed no significant difference in symptom improvement compared to usual care. In terms of context, only 40% of enrolled patients received complete care management per protocol. PCC predilection to adopt CCM had substantial effects on patient participation, with patients belonging to early adopter clinicians completing adequate care manager follow-up significantly more often than patients of clinicians with low predilection to adopt CCM (74% versus 48%%, p = 0.003). Conclusions Depression CCM designed and implemented by primary care practices using EBQI improved antidepressant initiation. Combining QI methods with a randomized evaluation proved challenging, but enabled new insights into the process of translating research-based CCM into practice. Future research on the effects of PCC attitudes and skills on CCM results, as well as on enhancing the link between improved antidepressant use and symptom outcomes, is needed. Trial Registration ClinicalTrials.gov: NCT00105820 PMID:22032247

Development of clinical contents model markup language for electronic health records.

PubMed

Yun, Ji-Hyun; Ahn, Sun-Ju; Kim, Yoon

2012-09-01

To develop dedicated markup language for clinical contents models (CCM) to facilitate the active use of CCM in electronic health record systems. Based on analysis of the structure and characteristics of CCM in the clinical domain, we designed extensible markup language (XML) based CCM markup language (CCML) schema manually. CCML faithfully reflects CCM in both the syntactic and semantic aspects. As this language is based on XML, it can be expressed and processed in computer systems and can be used in a technology-neutral way. CCML HAS THE FOLLOWING STRENGTHS: it is machine-readable and highly human-readable, it does not require a dedicated parser, and it can be applied for existing electronic health record systems.

Transformation of Strontium during formation of biogenic calcium carbonate

NASA Astrophysics Data System (ADS)

Ohnuki, T.; Kozai, N.; Sakamoto, F.; Yamashita, M.; Horiieke, T.; Utsunomiya, S.

2016-12-01

Some amounts of radionuclides contaminated water containing 90Sr generated in the Fukushima Daiichi Nuclear Power Plant were leaked to sea water in the port. One of the possible method to eliminate 90Sr is co-precipitated with biogenic carbonates minerals (CCM). Specific bacteria are known to form biogenic CCM in groundwater. In the present study, we have screened specific bacterium to form CCM in saline water, and studied transformation of Sr during biogenic CCM. A marine microbe of strain TK2d, which is screened from Tokyo bay to form CCM in saline solution, was grown in the medium solution contained urea and Sr. The concentratuion of Sr2+ in the solution was monitored by ICP-OES (ICP-OES; 720 Agilent Technologies, Inc., USA) during the formation of biogenic CCM. The precipitates were analyzed by SEM, TEM, and XAFS. When 1.0 mM Sr was dissolved in the medium solution, the concentration of Sr decreased up to 0.02 mM within 10 days, indicating that most of Sr in the solution was eliminated within 10 days. SEM and TEM analyses showed that needle shaped CCM containing Ca and Sr were formed. The CCM was not single crystalline, but poly-crystalline of calcite and aragonite. The elemental mapping showed that Sr was present at the same position of Ca, indicating that Sr was coprecipitated with Ca. The XANES analysis of Sr in the precipitates showed that the XANES spectrum was not the same as that of Sr coprecipitated with an abiotic Ca carbonates. Linear combination fitting of XANES spectra by those of SrCl2 and SrCO3 showed that both Sr2+ and SrCO3 were present in CCM. Longer contact time resulted in higher content of SrCO3, indicating that Sr was incorporated gradually with time into CCM structure. Thus, Sr was changed its chemical species from adsorbed one to the incorporated one in biogenic CCM in saline solution. This work was partially supported by a research grant from the Japan Science and Technology Agency, Japan (research grant No. 260502).

Medicare Chronic Care Management Payments and Financial Returns to Primary Care Practices: A Modeling Study.

PubMed

Basu, Sanjay; Phillips, Russell S; Bitton, Asaf; Song, Zirui; Landon, Bruce E

2015-10-20

Physicians have traditionally been reimbursed for face-to-face visits. A new non-visit-based payment for chronic care management (CCM) of Medicare patients took effect in January 2015. To estimate financial implications of CCM payment for primary care practices. Microsimulation model incorporating national data on primary care use, staffing, expenditures, and reimbursements. National Ambulatory Medical Care Survey and other published sources. Medicare patients. 10 years. Practice-level. Comparison of CCM delivery approaches by staff and physicians. Net revenue per full-time equivalent (FTE) physician; time spent delivering CCM services. If nonphysician staff were to deliver CCM services, net revenue to practices would increase despite opportunity and staffing costs. Practices could expect approximately $332 per enrolled patient per year (95% CI, $234 to $429) if CCM services were delivered by registered nurses (RNs), approximately $372 (CI, $276 to $468) if services were delivered by licensed practical nurses, and approximately $385 (CI, $286 to $485) if services were delivered by medical assistants. For a typical practice, this equates to more than $75 ,00 of net annual revenue per FTE physician and 12 hours of nursing service time per week if 50% of eligible patients enroll. At a minimum, 131 Medicare patients (CI, 115 to 140 patients) must enroll for practices to recoup the salary and overhead costs of hiring a full-time RN to provide CCM services. If physicians were to deliver all CCM services, approximately 25% of practices nationwide could expect net revenue losses due to opportunity costs of face-to-face visit time. The CCM program may alter long-term primary care use, which is difficult to predict. Practices that rely on nonphysician team members to deliver CCM services will probably experience substantial net revenue gains but must enroll a sufficient number of eligible patients to recoup costs. None.

Assessment of the monitoring and evaluation system for integrated community case management (ICCM) in Ethiopia: a comparison against global benchmark indicators.

PubMed

Mamo, Dereje; Hazel, Elizabeth; Lemma, Israel; Guenther, Tanya; Bekele, Abeba; Demeke, Berhanu

2014-10-01

Program managers require feasible, timely, reliable, and valid measures of iCCM implementation to identify problems and assess progress. The global iCCM Task Force developed benchmark indicators to guide implementers to develop or improve monitoring and evaluation (M&E) systems. To assesses Ethiopia's iCCM M&E system by determining the availability and feasibility of the iCCM benchmark indicators. We conducted a desk review of iCCM policy documents, monitoring tools, survey reports, and other rele- vant documents; and key informant interviews with government and implementing partners involved in iCCM scale-up and M&E. Currently, Ethiopia collects data to inform most (70% [33/47]) iCCM benchmark indicators, and modest extra effort could boost this to 83% (39/47). Eight (17%) are not available given the current system. Most benchmark indicators that track coordination and policy, human resources, service delivery and referral, supervision, and quality assurance are available through the routine monitoring systems or periodic surveys. Indicators for supply chain management are less available due to limited consumption data and a weak link with treatment data. Little information is available on iCCM costs. Benchmark indicators can detail the status of iCCM implementation; however, some indicators may not fit country priorities, and others may be difficult to collect. The government of Ethiopia and partners should review and prioritize the benchmark indicators to determine which should be included in the routine M&E system, especially since iCCMdata are being reviewed for addition to the HMIS. Moreover, the Health Extension Worker's reporting burden can be minimized by an integrated reporting approach.

Mandatory labeling requirements and over-the-counter cough and cold medication use in early childhood.

PubMed

DeGroot, Julie; Anderson, Laura N; Chen, Yang; Birken, Catherine S; Parkin, Patricia C; Carsley, Sarah; Khovratovich, Marina; Mamdani, Muhammad; Maguire, Jonathon L

2016-03-16

Due to rare but severe adverse events, Health Canada in October 2009 required manufacturers to relabel over-the-counter (OTC) cough and cold medication (CCM) to state that the products should not be used in children <6 years of age. The main objective of this study was to determine whether this labeling standard decreased OTC CCM use among young children with a recent cough, cold or flu. An interrupted time series study was conducted using data from the TARGet Kids! practice-based research network. A total of 3,515 healthy children 1-5 years of age were recruited from 2008-2011; of these, 1,072 had a cough, cold or flu in the previous month. Parents completed a standardized survey instrument. For the primary analysis, use of OTC CCMs prior to and after October 1, 2009 was compared using time series analyses. For the secondary analysis, multivariable logistic regression was used to identify predictors of recent OTC CCM use. OTC CCM use was reported in 222 of 1,072 (20.7%) children with a cough, cold or flu within the previous month. OTC CCM use declined from 22.2% to 17.8% following the October 2009 Health Canada labeling standard (p = 0.014). Maternal age <35 years (OR 1.49; 95% CI: 1.05-2.13) and having older siblings (OR 1.65; 95% CI: 1.16-2.35) were independently associated with OTC CCM use. Labeling legislation against OTC CCM use for children <6 years resulted in a small decrease in OTC CCM use. Stronger measures may be needed to curtail OTC CCM use, particularly for younger parents and those with multiple children.

Collaborative Chronic Care Models for Mental Health Conditions: Cumulative Meta-Analysis and Meta-Regression to Guide Future Research and Implementation

PubMed Central

Grogan-Kaylor, Andrew; Perron, Brian E.; Kilbourne, Amy M.; Woltmann, Emily; Bauer, Mark S.

2013-01-01

Objective Prior meta-analysis indicates that collaborative chronic care models (CCMs) improve mental and physical health outcomes for individuals with mental disorders. This study aimed to investigate the stability of evidence over time and identify patient and intervention factors associated with CCM effects in order to facilitate implementation and sustainability of CCMs in clinical practice. Method We reviewed 53 CCM trials that analyzed depression, mental quality of life (QOL), or physical QOL outcomes. Cumulative meta-analysis and meta-regression were supplemented by descriptive investigations across and within trials. Results Most trials targeted depression in the primary care setting, and cumulative meta-analysis indicated that effect sizes favoring CCM quickly achieved significance for depression outcomes, and more recently achieved significance for mental and physical QOL. Four of six CCM elements (patient self-management support, clinical information systems, system redesign, and provider decision support) were common among reviewed trials, while two elements (healthcare organization support and linkages to community resources) were rare. No single CCM element was statistically associated with the success of the model. Similarly, meta-regression did not identify specific factors associated with CCM effectiveness. Nonetheless, results within individual trials suggest that increased illness severity predicts CCM outcomes. Conclusions Significant CCM trials have been derived primarily from four original CCM elements. Nonetheless, implementing and sustaining this established model will require healthcare organization support. While CCMs have typically been tested as population-based interventions, evidence supports stepped care application to more severely ill individuals. Future priorities include developing implementation strategies to support adoption and sustainability of the model in clinical settings while maximizing fit of this multi-component framework to local contextual factors. PMID:23938600

Development of Clinical Contents Model Markup Language for Electronic Health Records

PubMed Central

Yun, Ji-Hyun; Kim, Yoon

2012-01-01

Objectives To develop dedicated markup language for clinical contents models (CCM) to facilitate the active use of CCM in electronic health record systems. Methods Based on analysis of the structure and characteristics of CCM in the clinical domain, we designed extensible markup language (XML) based CCM markup language (CCML) schema manually. Results CCML faithfully reflects CCM in both the syntactic and semantic aspects. As this language is based on XML, it can be expressed and processed in computer systems and can be used in a technology-neutral way. Conclusions CCML has the following strengths: it is machine-readable and highly human-readable, it does not require a dedicated parser, and it can be applied for existing electronic health record systems. PMID:23115739

Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets.

PubMed

Prusa, Jerome; Jensen, Drake; Santiago-Collazo, Gustavo; Pope, Steven S; Garner, Ashley L; Miller, Justin J; Ruiz Manzano, Ana; Galburt, Eric A; Stallings, Christina L

2018-07-01

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP o ) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β' and σ subunits, a core domain (CD) that contacts the RNAP β' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP o stabilization in vitro , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general. Copyright © 2018 American Society for Microbiology.

Clinical course of untreated cerebral cavernous malformations: a meta-analysis of individual patient data.

PubMed

Horne, Margaret A; Flemming, Kelly D; Su, I-Chang; Stapf, Christian; Jeon, Jin Pyeong; Li, Da; Maxwell, Susanne S; White, Philip; Christianson, Teresa J; Agid, Ronit; Cho, Won-Sang; Oh, Chang Wan; Wu, Zhen; Zhang, Jun-Ting; Kim, Jeong Eun; Ter Brugge, Karel; Willinsky, Robert; Brown, Robert D; Murray, Gordon D; Al-Shahi Salman, Rustam

2016-02-01

Cerebral cavernous malformations (CCMs) can cause symptomatic intracranial haemorrhage (ICH), but the estimated risks are imprecise and predictors remain uncertain. We aimed to obtain precise estimates and predictors of the risk of ICH during untreated follow-up in an individual patient data meta-analysis. We invited investigators of published cohorts of people aged at least 16 years, identified by a systematic review of Ovid MEDLINE and Embase from inception to April 30, 2015, to provide individual patient data on clinical course from CCM diagnosis until first CCM treatment or last available follow-up. We used survival analysis to estimate the 5-year risk of symptomatic ICH due to CCMs (primary outcome), multivariable Cox regression to identify baseline predictors of outcome, and random-effects models to pool estimates in a meta-analysis. Among 1620 people in seven cohorts from six studies, 204 experienced ICH during 5197 person-years of follow-up (Kaplan-Meier estimated 5-year risk 15·8%, 95% CI 13·7-17·9). The primary outcome of ICH within 5 years of CCM diagnosis was associated with clinical presentation with ICH or new focal neurological deficit (FND) without brain imaging evidence of recent haemorrhage versus other modes of presentation (hazard ratio 5·6, 95% CI 3·2-9·7) and with brainstem CCM location versus other locations (4·4, 2·3-8·6), but age, sex, and CCM multiplicity did not add independent prognostic information. The 5-year estimated risk of ICH during untreated follow-up was 3·8% (95% CI 2·1-5·5) for 718 people with non-brainstem CCM presenting without ICH or FND, 8·0% (0·1-15·9) for 80 people with brainstem CCM presenting without ICH or FND, 18·4% (13·3-23·5) for 327 people with non-brainstem CCM presenting with ICH or FND, and 30·8% (26·3-35·2) for 495 people with brainstem CCM presenting with ICH or FND. Mode of clinical presentation and CCM location are independently associated with ICH within 5 years of CCM diagnosis. These findings can inform decisions about CCM treatment. UK Medical Research Council, Chief Scientist Office of the Scottish Government, and UK Stroke Association. Copyright © 2016 Horne et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved.

CCM-AMI, a Polyethylene Glycol Micelle with Amifostine, as an Acute Radiation Syndrome Protectant in C57BL/6 Mice.

PubMed

Chen, Chia-Hung; Kuo, Min-Liang; Wang, Jen-Ling; Liao, Wei-Chuan; Chang, Li-Ching; Chan, Leong-Perng; Lin, Johnson

2015-09-01

Acute radiation syndrome results from radiation exposure, such as in accidental nuclear disasters. Safe and effective radioprotectants, mitigators, and treatment drugs must be developed as medical countermeasures against radiation exposure. Here, the authors evaluated CCM-Ami, a novel polyethylene glycol micelle encapsulated with amifostine, for its radioprotective properties after total-body irradiation from a 60Co source. Male C57BL/6 mice (6-8 wk old) were intravenously injected with 45 mg kg(-1) of CCM-Ami 90 min before exposure to 7.2 and 8.5 Gy irradiation at a dose rate of 0.04 Gy min(-1). Both survival benefit and hematopoietic protection were observed after prophylactic CCM-Ami administration when compared with the effects measured in excipient control and amifostine groups. Pharmacokinetic results showed that after the intravenous injection, the plasma concentration of WR-1065, the active form of amifostine, was higher in CCM-Ami-treated mice than in amifostine-treated mice. These findings suggest that CCM-Ami-mediated hematopoietic protection plays a key role in enhancing survival of mice exposed to radiation toxicity and thus indicate that CCM-Ami is a radioprotectant that can be used safely and effectively in nuclear disasters.

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.

PubMed

de Araujo, Charlotte; Arefeen, Dewan; Tadesse, Yohannes; Long, Benedict M; Price, G Dean; Rowlett, Roger S; Kimber, Matthew S; Espie, George S

2014-09-01

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.

Chronic care management for dependence on alcohol and other drugs: the AHEAD randomized trial.

PubMed

Saitz, Richard; Cheng, Debbie M; Winter, Michael; Kim, Theresa W; Meli, Seville M; Allensworth-Davies, Don; Lloyd-Travaglini, Christine A; Samet, Jeffrey H

2013-09-18

People with substance dependence have health consequences, high health care utilization, and frequent comorbidity but often receive poor-quality care. Chronic care management (CCM) has been proposed as an approach to improve care and outcomes. To determine whether CCM for alcohol and other drug dependence improves substance use outcomes compared with usual primary care. The AHEAD study, a randomized trial conducted among 563 people with alcohol and other drug dependence at a Boston, Massachusetts, hospital-based primary care practice. Participants were recruited from September 2006 to September 2008 from a freestanding residential detoxification unit and referrals from an urban teaching hospital and advertisements; 95% completed 12-month follow-up. Participants were randomized to receive CCM (n=282) or no CCM (n=281). Chronic care management included longitudinal care coordinated with a primary care clinician; motivational enhancement therapy; relapse prevention counseling; and on-site medical, addiction, and psychiatric treatment, social work assistance, and referrals (including mutual help). The no CCM (control) group received a primary care appointment and a list of treatment resources including a telephone number to arrange counseling. The primary outcome was self-reported abstinence from opioids, stimulants, or heavy drinking. Biomarkers were secondary outcomes. There was no significant difference in abstinence from opioids, stimulants, or heavy drinking between the CCM (44%) and control (42%) groups (adjusted odds ratio, 0.84; 95% CI, 0.65-1.10; P=.21). No significant differences were found for secondary outcomes of addiction severity, health-related quality of life, or drug problems. No subgroup effects were found except among those with alcohol dependence, in whom CCM was associated with fewer alcohol problems (mean score, 10 vs 13; incidence rate ratio, 0.85; 95% CI, 0.72-1.00; P=.048). Among persons with alcohol and other drug dependence, CCM compared with a primary care appointment but no CCM did not increase self-reported abstinence over 12 months. Whether more intensive or longer-duration CCM is effective requires further investigation. clinicaltrials.gov Identifier: NCT00278447.

An ethical justification for the Chronic Care Model (CCM)

PubMed Central

Oprea, Liviu; Braunack‐Mayer, Annette; Rogers, Wendy A.; Stocks, Nigel

2009-01-01

Abstract Background:  Chronic diseases are major causes of morbidity and mortality in developed countries. Their effects can be mitigated by high quality evidence‐based care, but this is not the norm in most systems. The Chronic Care Model (CCM) is an evidence‐based policy response to this practice gap, which uses multiple strategies to promote the quality of chronic care. Objective:  To review CCM with an ethical lens. Methods:  We reviewed the published empirical and non‐empirical articles of CCM to analyse the ethical underpinnings of this model. Results and conclusions:  We argue that its principal ethical value lies in the institutional cooperation it builds between the stakeholders involved in health care services. First, we briefly describe CCM and argue that the pathways through which it aims to improve patients’ health outcomes are not made explicit. Second, we argue that the potential of CCM to be more beneficent, compared with traditional health care systems, depends on its capacity to promote mutual trust between health care providers and patients. There is no evidence to date that the implementation of CCM enhances mutual trust between health care professionals and patients. Third, we argue that CCM seeks to enhance human agency, allowing increased expression of individual autonomy and increased respect for individuals thereby expanding human freedom and avoiding social discrimination. However, we review the communication patterns that characterize the model of doctor–patient relationship promoted by this model and argue that these communication patterns raise ethical concerns that may prevent the model from reaching its expected outcomes. PMID:19906213

Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lau, E Y; Felton, J S; Lightstone, F C

2006-06-06

A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclicmore » amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.« less

Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

PubMed

Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

2017-12-01

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

Understanding delayed T-cell priming, lung recruitment, and airway luminal T-cell responses in host defense against pulmonary tuberculosis.

PubMed

Shaler, Christopher R; Horvath, Carly; Lai, Rocky; Xing, Zhou

2012-01-01

Mycobacterium tuberculosis (M.tb), the causative bacterium of pulmonary tuberculosis (TB), is a serious global health concern. Central to M.tb effective immune avoidance is its ability to modulate the early innate inflammatory response and prevent the establishment of adaptive T-cell immunity for nearly three weeks. When compared with other intracellular bacterial lung pathogens, such as Legionella pneumophila, or even closely related mycobacterial species such as M. smegmatis, this delay is astonishing. Customarily, the alveolar macrophage (AM) acts as a sentinel, detecting and alerting surrounding cells to the presence of an invader. However, in the case of M.tb, this may be impaired, thus delaying the recruitment of antigen-presenting cells (APCs) to the lung. Upon uptake by APC populations, M.tb is able to subvert and delay the processing of antigen, MHC class II loading, and the priming of effector T cell populations. This delay ultimately results in the deferred recruitment of effector T cells to not only the lung interstitium but also the airway lumen. Therefore, it is of upmost importance to dissect the mechanisms that contribute to the delayed onset of immune responses following M.tb infection. Such knowledge will help design the most effective vaccination strategies against pulmonary TB.

One pot green fabrication of metallic silver nanoscale materials using Crescentia cujete L. and assessment of their bactericidal activity.

PubMed

Prabukumar, Seetharaman; Rajkuberan, Chandrasekaran; Sathishkumar, Gnanasekar; Illaiyaraja, Mani; Sivaramakrishnan, Sivaperumal

2018-06-01

In this study, the leaf extract of an important medicinal plant Crescentia cujete L. (CC) was employed as a green reducing agent to synthesise highly-stable C. cujete silver nanoparticles (CCAgNPs). The reduction of Ag + to Ag 0 nanoparticles was initially observed by a colour change which generates an intense surface plasmon resonance peak at 417 nm using a UV-Vis spectrophotometer. Various optimisation factors such as temperature, pH, time and the stoichiometric proportion of the reaction mixture were performed, which influence the size, dispersity and synthesis rate of CCAgNPs. In addition, surface chemistry of synthesised CCAgNPs through Fourier transform infrared spectroscopy reveals the reducing/stabilising agent present in the aqueous extract of C. cujete and synthesised CCAgNPs. Transmission electron microscopy analysis features the spherical shape of CCAgNPs with an average size of 39.74 nm. Furthermore, an X-ray diffraction study confirms that the synthesised CCAgNPs were face-centred cubic crystalline in nature. The CCAgNPs display tremendous bactericidal activity against human pathogens Bacillus subtilis , Staphylococcus epidermidis , Rhodococcus rhodochrous , Salmonella typhi , Mycobacterium smegmatis , Shigella flexneri and Vibrio cholerae via penetrating into the bacterial cell membrane and causing failure of an internal chain reaction.

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases

PubMed Central

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2014-01-01

N-hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and mycobacteria. NMOs catalyze the hydroxylation of lysine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of l-kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington’s and Alzheimer’s diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin monooxygenases. Fluorescently-labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a Kd value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with Kd values of 2.1 ± 0.2 μM and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we showed that this assay can be used to identify inhibitors of NMOs. A Z’-factor of 0.77 was calculated and we show that the assay exhibits good tolerance to temperature, incubation time, and DMSO concentration. PMID:22410281

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships

PubMed Central

Pope, Welkin H.; Mavrich, Travis N.; Garlena, Rebecca A.; Guerrero-Bustamante, Carlos A.; Jacobs-Sera, Deborah; Montgomery, Matthew T.; Russell, Daniel A.; Warner, Marcie H.

2017-01-01

ABSTRACT The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are “singletons” with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. PMID:28811342

Biocompatible high performance hyperbranched epoxy/clay nanocomposite as an implantable material.

PubMed

Barua, Shaswat; Dutta, Nipu; Karmakar, Sanjeev; Chattopadhyay, Pronobesh; Aidew, Lipika; Buragohain, Alak K; Karak, Niranjan

2014-04-01

Polymeric biomaterials are in extensive use in the domain of tissue engineering and regenerative medicine. High performance hyperbranched epoxy is projected here as a potential biomaterial for tissue regeneration. Thermosetting hyperbranched epoxy nanocomposites were prepared with Homalomena aromatica rhizome oil-modified bentonite as well as organically modified montmorillonite clay. Fourier transformed infrared spectroscopy, x-ray diffraction and scanning and transmission electron microscopic techniques confirmed the strong interfacial interaction of clay layers with the epoxy matrix. The poly(amido amine)-cured thermosetting nanocomposites exhibited high mechanical properties like impact resistance (>100 cm), scratch hardness (>10 kg), tensile strength (48-58 MPa) and elongation at break (11.9-16.6%). Cytocompatibility of the thermosets was found to be excellent as evident by MTT and red blood cell hemolytic assays. The nanocomposites exhibited antimicrobial activity against Staphylococcus aureus (ATCC 11632), Escherichia coli (ATCC 10536), Mycobacterium smegmatis (ATCC14468) and Candida albicans (ATCC 10231) strains. In vivo biocompatibility of the best performing nanocomposite was ascertained by histopathological study of the brain, heart, liver and skin after subcutaneous implantation in Wistar rats. The material supported the proliferation of dermatocytes without induction of any sign of toxicity to the above organs. The adherence and proliferation of cells endorse the nanocomposite as a non-toxic biomaterial for tissue regeneration.

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

PubMed

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD

PubMed Central

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-01-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosyn­thesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-l-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit. PMID:24817736

Single-molecule DNA detection with an engineered MspA protein nanopore

PubMed Central

Butler, Tom Z.; Pavlenok, Mikhail; Derrington, Ian M.; Niederweis, Michael; Gundlach, Jens H.

2008-01-01

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ≈20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ≈100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications. PMID:19098105

The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

PubMed

Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

2014-01-01

Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

Photobactericidal porphyrin-cellulose nanocrystals: synthesis, characterization, and antimicrobial properties.

PubMed

Feese, Elke; Sadeghifar, Hasan; Gracz, Hanna S; Argyropoulos, Dimitris S; Ghiladi, Reza A

2011-10-10

Adherence and survival of pathogenic bacteria on surfaces leading to concomitant transmission to new hosts significantly contributes to the proliferation of pathogens, which in turn considerably increases the threat to human health, particularly by antibiotic-resistant bacteria. Consequently, more research into effective surface disinfection and alternative materials (fabrics, plastics, or coatings) with antimicrobial and other bioactive characteristics is desirable. This report describes the synthesis and characterization of cellulose nanocrystals that were surface-modified with a cationic porphyrin. The porphyrin was appended onto the cellulose surface via the Cu(I)-catalyzed Huisgen-Meldal-Sharpless 1,3-dipolar cycloaddition having occurred between azide groups on the cellulosic surface and porphyrinic alkynes. The resulting, generally insoluble, crystalline material, CNC-Por (5), was characterized by infrared and diffusion (1)H NMR spectroscopies, gel permeation chromatography, and thermogravimetric analysis. Although only suspended, and not dissolved, in an aqueous system, CNC-Por (5) showed excellent efficacy toward the photodynamic inactivation of Mycobacterium smegmatis and Staphylococcus aureus , albeit only slight activity against Escherichia coli . The synthesis, properties, and activity of CNC-Por (5) described herein serve as a benchmark toward our overall objectives of developing novel, potent, bioactive, photobactericidal materials that are effective against a range of bacteria, with potential utilization in the health care and food preparation industries.

Convergent cross-mapping and pairwise asymmetric inference.

PubMed

McCracken, James M; Weigel, Robert S

2014-12-01

Convergent cross-mapping (CCM) is a technique for computing specific kinds of correlations between sets of times series. It was introduced by Sugihara et al. [Science 338, 496 (2012).] and is reported to be "a necessary condition for causation" capable of distinguishing causality from standard correlation. We show that the relationships between CCM correlations proposed by Sugihara et al. do not, in general, agree with intuitive concepts of "driving" and as such should not be considered indicative of causality. It is shown that the fact that the CCM algorithm implies causality is a function of system parameters for simple linear and nonlinear systems. For example, in a circuit containing a single resistor and inductor, both voltage and current can be identified as the driver depending on the frequency of the source voltage. It is shown that the CCM algorithm, however, can be modified to identify relationships between pairs of time series that are consistent with intuition for the considered example systems for which CCM causality analysis provided nonintuitive driver identifications. This modification of the CCM algorithm is introduced as "pairwise asymmetric inference" (PAI) and examples of its use are presented.

Demand generation and social mobilisation for integrated community case management (iCCM) and child health: Lessons learned from successful programmes in Niger and Mozambique.

PubMed

Sharkey, Alyssa B; Martin, Sandrine; Cerveau, Teresa; Wetzler, Erica; Berzal, Rocio

2014-12-01

We present the approaches used in and outcomes resulting from integrated community case management (iCCM) programmes in Niger and Mozambique with a strong focus on demand generation and social mobilisation. We use a case study approach to describe the programme and contextual elements of the Niger and Mozambique programmes. Awareness and utilisation of iCCM services and key family practices increased following the implementation of the Niger and Mozambique iCCM and child survival programmes, as did care-seeking within 24 hours and care-seeking from appropriate, trained providers in Mozambique. These approaches incorporated interpersonal communication activities and community empowerment/participation for collective change, partnerships and networks among key stakeholder groups within communities, media campaigns and advocacy efforts with local and national leaders. iCCM programmes that train and equip community health workers and successfully engage and empower community members to adopt new behaviours, have appropriate expectations and to trust community health workers' ability to assess and treat illnesses can lead to improved care-seeking and utilisation, and community ownership for iCCM.

Novel Naja atra cardiotoxin 1 (CTX-1) derived antimicrobial peptides with broad spectrum activity

PubMed Central

Santospirito, Davide; Polverini, Eugenia; Flisi, Sara; Cavirani, Sandro; Taddei, Simone

2018-01-01

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent β-strand of the first “finger” of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50–6.3 μg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families. PMID:29364903

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin-binding protein PonA1 reveal potential mechanisms of antibiotic resistance.

PubMed

Filippova, Ekaterina V; Kieser, Karen J; Luan, Chi-Hao; Wawrzak, Zdzislaw; Kiryukhina, Olga; Rubin, Eric J; Anderson, Wayne F

2016-06-01

Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic-resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin-binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor-free form and in complex with penicillin V are reported. We used site-directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β-lactams. Structural comparison of the PonA1 apo-form and the antibiotic-bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4'-α3 surrounding the penicillin-binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β-lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall-targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis.

PubMed

Eckelt, Elke; Jarek, Michael; Frömke, Cornelia; Meens, Jochen; Goethe, Ralph

2014-12-06

Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis. In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria. Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

PubMed

McBee, Megan E; Chionh, Yok H; Sharaf, Mariam L; Ho, Peiying; Cai, Maggie W L; Dedon, Peter C

2017-01-01

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis , and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.

Synergy, Salary, and Satisfaction: Benefits of Training in Critical Care Medicine and Infectious Diseases Gleaned From a National Pilot Survey of Dually Trained Physicians.

PubMed

Kadri, Sameer S; Rhee, Chanu; Magda, Gabriela; Strich, Jeffrey R; Cai, Rongman; Sun, Junfeng; Decker, Brooke K; O'Grady, Naomi P

2016-10-01

An increasing number of physicians are seeking dual training in critical care medicine (CCM) and infectious diseases (ID). Understanding experiences and perceptions of CCM-ID physicians could inform career choices and programmatic innovation. All physicians trained and/or certified in both CCM and ID to date in the United States were sent a Web-based questionnaire in 2015. Responses enabled a cross-sectional analysis of physician demographics and training and practice characteristics and satisfaction. Of 202 CCM-ID physicians, 196 were alive and reachable. The response rate was 79%. Forty-six percent trained and 34% practice in the northeastern United States. Only 40% received dual training at the same institution. Eighty-three percent identified as either an intensivist with ID expertise (44%) or as equally an intensivist and ID physician (38%). Median salary was $265 000 (interquartile range [IQR], $215 000-$350 000). Practice settings were split between academic (45%) and community settings (42%). Two-thirds are clinicians but 62% conduct some research and 26% practice outpatient ID. Top reasons to dually specialize included clinical synergy (70%), procedural activity (50%), and less interest in pulmonology (49%). Although 38% cited less proficiency with bronchoscopy as a disadvantage, 87% seldom need pulmonary consultation in the intensive care unit. Median career satisfaction was 4 (IQR, 4-5) out of 5, and 76% would dually train again. CCM-ID graduates prefer the acute care setting, predominantly CCM or a combination of CCM and ID. They find combination training and practice to be synergistic and satisfying, but most have had to seek CCM and ID training independently at separate institutions. Given these findings, avenues for combined training in CCM-ID should be considered. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Monitoring iCCM: a feasibility study of the indicator guide for monitoring and evaluating integrated community case management.

PubMed

Roberton, Timothy; Kasungami, Dyness; Guenther, Tanya; Hazel, Elizabeth

2016-07-01

Most countries in sub-Saharan Africa have now adopted integrated community case management (iCCM) of common childhood illnesses as a strategy to improve child health. In March 2014, the iCCM Task Force published an Indicator Guide for Monitoring and Evaluating iCCM: a 'menu' of recommended indicators with globally agreed definitions and methodology, to guide countries in developing robust iCCM monitoring systems. The Indicator Guide was conceived as an evolving document that would incorporate collective experience and learning as iCCM programmes themselves evolve. This article presents findings from two studies that examined the feasibility of collecting the Indicator Guide's 18 routine monitoring indicators with the iCCM monitoring systems that countries currently have in place. We reviewed iCCM monitoring tools, protocols and reports from a purposive sample of 10 countries in sub-Saharan Africa. We developed a scorecard system to assess which of the Indicator Guide's 18 routine monitoring indicators could be calculated with the given monitoring tools, and at which level of the health system the relevant information would be available. We found that the data needed to calculate many of the Indicator Guide's routine monitoring indicators are already being collected through existing monitoring systems, although much of these data are only available at health facility level and not aggregated to district or national levels. Our results highlight challenge of using supervision checklists as a data source, and the need for countries to maintain accurate deployment data for CHWs and CHW supervisors. We suggest that some of the recommended indicators need revising. Routine monitoring will be more feasible, effective and efficient if iCCM programmes focus on a smaller set of high-value indicators that are easy to measure, reliably interpreted and useful both for global and national stakeholders and for frontline health workers themselves. © The Author 2016. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine.

A randomized comparison of 5 versus 12 hours per day of cardiac contractility modulation treatment for heart failure patients: A preliminary report.

PubMed

Kloppe, Axel; Mijic, Dejan; Schiedat, Fabian; Bogossian, Harilaos; Mügge, Andreas; Rousso, Benny; Lemke, Bernd

2016-01-01

Cardiac contractility modulation (CCM) signals are non-excitatory electrical signals delivered during the absolute refractory period intended to improve contraction and cardiac function. Clinical trials have shown that CCM treatment significantly improves exercise tolerance and quality of life in symptomatic heart failure patients. Studies with CCM therapy typically include CCM delivery for 3, 5 or 7 h per day, although other configurations are also commonly used. Each has been associated with improved outcomes in heart failure, but it is not clear whether different application durations are associated with the various degrees of benefit. The purpose of the current pilot evaluation study was to evaluate the quality of life, exercise tolerance, and cardiac function, over a 6-month period when CCM was delivered for 5 h/day vs. 12 h/day. Increasing the daily CCM therapy duration is safe and as good as the standard CCM periods of application per day. This single center pilot evaluation study involved 19 medically refractory symptomatic patients with heart failure and reduced left ventricular function who underwent implantation of an Optimizer™ system (Impulse Dynamics, Orangeburg, NY, USA). Patients were randomized into one of two treatment groups; 5 h/day CCM treatment or 12 h/day CCM treatment. Subjects and evaluating physicians were blinded to the study group. Subjects returned to the hospital after 12 and 24 weeks. Efficacy evaluations included changes from baseline to 24 weeks in Minnesota Living With Heart Failure Questionnaire score (MLWHFQ), maximal oxygen consumption in the cardio-pulmonary stress test (peak VO2), New York Heart Association classification (NYHA), 6-min walk distance (6MWD), and ejection fraction (EF). At the end of 24 weeks, clinical improvement was observed in the entire cohort in all efficacy measures (mean change from baseline of -17.1 in MLWHFQ, -0.86 in NYHA, and improvement trend of 1.48 mL O2/kg/min in peak VO2, 31.3 m in 6MWD, and 2.25% in EF). There were no significant differences, either clinically or statistically, between the groups receiving CCM for 5 h/day vs. 12 h/day. Three subjects were voluntarily withdrawn before completing the study. One subject died from pneumonia after 125 days, and 6 serious adverse events were reported, none of which was classified as related to either the device or the procedure. Together with previously reported experience with CCM, delivery of CCM therapy is equally safe and appears similarly effective over the range of shorter (5 h) to longer (12 h) daily periods of application. Given the small sample size, further studies are warranted.

Dynamics of Carbon-Concentrating Mechanism Induction and Protein Relocalization during the Dark-to-Light Transition in Synchronized Chlamydomonas reinhardtii1[W][OPEN

PubMed Central

Mitchell, Madeline C.; Meyer, Moritz T.; Griffiths, Howard

2014-01-01

In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction. PMID:25106822

Results and evaluation of key comparison CCM.P-K12.1 for very low helium flow rates (leak rates)

NASA Astrophysics Data System (ADS)

Šetina, Janez; Vičar, Martin; Pražák, Dominik

2015-01-01

The Czech Metrology Institute (CMI) took part and failed to prove equivalence in the Key Comparison CCM.P-K12 of helium leak artefacts (leak rates) into vacuum. After identifying and eliminating the cause of the failure, the Institute of Metals and Technology (IMT) of Slovenia, a successful participant in CCM.P-K12, volunteered to serve as pilot and link in a following bilateral comparison of IMT and CMI that obtained designation CCM.P-K12.1 in June 2012. It was decided to perform the comparison with a glass permeation helium leak artefact at nominally 3.10-11 mol/s (7.4.10-7 mbar.L.s-1) at 23 °C. Both the laboratory standards took part in CCM.P-K12 and were considered as primary. The comparison measurements were performed in October 2012. CMI proved equivalence both with IMT and with the reference value of the key comparison CCM.P-K12. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Expression Profiling-Based Identification of CO2-Responsive Genes Regulated by CCM1 Controlling a Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii1

PubMed Central

Miura, Kenji; Yamano, Takashi; Yoshioka, Satoshi; Kohinata, Tsutomu; Inoue, Yoshihiro; Taniguchi, Fumiya; Asamizu, Erika; Nakamura, Yasukazu; Tabata, Satoshi; Yamato, Katsuyuki T.; Ohyama, Kanji; Fukuzawa, Hideya

2004-01-01

Photosynthetic acclimation to CO2-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO2-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO2-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO2-rich conditions with those of cells grown under CO2-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO2 inducible genes and 32 genes repressed by low CO2 whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. Among low-CO2 inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO2 inducible and/or CCM1-regulated genes are discussed in relation to the CCM. PMID:15235119

Running STAR-CCM+ Software on the Peregrine System | High-Performance

Science.gov Websites

/bin/lmutil lmstat -c 1999@wind-lms.nrel.gov -a module load star-ccm export TMPDIR="/scratch/$USER + -power -rsh "ssh -oStrictHostKeyChecking=no" -machinefile nodelist -np $(($nodes*$cores , type the commands from the SLURM script and make sure the job runs: module load star-ccm export TMPDIR

Cytochrome P450 and matrix metalloproteinase genetic modifiers of disease severity in Cerebral Cavernous Malformation type 1

PubMed Central

Choquet, Hélène; Trapani, Eliana; Goitre, Luca; Trabalzini, Lorenza; Akers, Amy; Fontanella, Marco; Hart, Blaine L.; Morrison, Leslie A.; Pawlikowska, Ludmila; Kim, Helen; Retta, Saverio Francesco

2016-01-01

Background Familial Cerebral Cavernous Malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions. CCM lesions manifest across a range of different phenotypes, including wide differences in lesion number, size and susceptibility to intracerebral hemorrhage (ICH). Oxidative stress plays an important role in cerebrovascular disease pathogenesis, raising the possibility that inter-individual variability in genes related to oxidative stress may contribute to the phenotypic differences observed in CCM1 disease. Here, we investigated whether candidate oxidative stress-related cytochrome P450 (CYP) and matrix metalloproteinase (MMP) genetic markers grouped by superfamilies, families or genes, or analyzed individually influence the severity of CCM1 disease. Methods Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging (SWI) were performed to determine total and large (≥5 mm in diameter) lesion counts as well as ICH in 188 Hispanic CCM1 patients harboring the founder KRIT1/CCM1 ‘common Hispanic mutation’ (CCM1–CHM). Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 1,122 genetic markers (both single nucleotide polymorphisms (SNPs) and insertion/deletions) grouped by CYP and MMP superfamily, family or gene for association with total or large lesion count and ICH adjusted for age at enrollment and gender. Genetic markers bearing the associations were then analyzed individually. Results The CYP superfamily showed a trend toward association with total lesion count (P=0.057) and large lesion count (P=0.088) in contrast to the MMP superfamily. The CYP4 and CYP8 families were associated with either large lesion count or total lesion count (P=0.014), and two other families (CYP46 and the MMP Stromelysins) were associated with ICH (P=0.011 and 0.007, respectively). CYP4F12 rs11085971, CYP8A1 rs5628, CYP46A1 rs10151332, and MMP3 rs117153070 single SNPs, mainly bearing the above-mentioned associations, were also individually associated with CCM1 disease severity. Conclusions Overall, our candidate oxidative stress-related genetic markers set approach outlined CYP and MMP families and identified suggestive SNPs that may impact the severity of CCM1 disease, including the development of numerous and large CCM lesions and ICH. These novel genetic risk factors of prognostic value could serve as early objective predictors of disease outcome and might ultimately provide better options for disease prevention and treatment. PMID:26795600

Cytochrome P450 and matrix metalloproteinase genetic modifiers of disease severity in Cerebral Cavernous Malformation type 1.

PubMed

Choquet, Hélène; Trapani, Eliana; Goitre, Luca; Trabalzini, Lorenza; Akers, Amy; Fontanella, Marco; Hart, Blaine L; Morrison, Leslie A; Pawlikowska, Ludmila; Kim, Helen; Retta, Saverio Francesco

2016-03-01

Familial Cerebral Cavernous Malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions. CCM lesions manifest across a range of different phenotypes, including wide differences in lesion number, size and susceptibility to intracerebral hemorrhage (ICH). Oxidative stress plays an important role in cerebrovascular disease pathogenesis, raising the possibility that inter-individual variability in genes related to oxidative stress may contribute to the phenotypic differences observed in CCM1 disease. Here, we investigated whether candidate oxidative stress-related cytochrome P450 (CYP) and matrix metalloproteinase (MMP) genetic markers grouped by superfamilies, families or genes, or analyzed individually influence the severity of CCM1 disease. Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging (SWI) were performed to determine total and large (≥5mm in diameter) lesion counts as well as ICH in 188 Hispanic CCM1 patients harboring the founder KRIT1/CCM1 'common Hispanic mutation' (CCM1-CHM). Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 1,122 genetic markers (both single nucleotide polymorphisms (SNPs) and insertion/deletions) grouped by CYP and MMP superfamily, family or gene for association with total or large lesion count and ICH adjusted for age at enrollment and gender. Genetic markers bearing the associations were then analyzed individually. The CYP superfamily showed a trend toward association with total lesion count (P=0.057) and large lesion count (P=0.088) in contrast to the MMP superfamily. The CYP4 and CYP8 families were associated with either large lesion count or total lesion count (P=0.014), and two other families (CYP46 and the MMP Stromelysins) were associated with ICH (P=0.011 and 0.007, respectively). CYP4F12 rs11085971, CYP8A1 rs5628, CYP46A1 rs10151332, and MMP3 rs117153070 single SNPs, mainly bearing the above-mentioned associations, were also individually associated with CCM1 disease severity. Overall, our candidate oxidative stress-related genetic markers set approach outlined CYP and MMP families and identified suggestive SNPs that may impact the severity of CCM1 disease, including the development of numerous and large CCM lesions and ICH. These novel genetic risk factors of prognostic value could serve as early objective predictors of disease outcome and might ultimately provide better options for disease prevention and treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Absorption mechanism of whey-protein-delivered curcumin using Caco-2 cell monolayers.

PubMed

Li, Ming; Cui, Jie; Ngadi, Michael O; Ma, Ying

2015-08-01

Curcumin (CCM) is a bioactive polyphenolic compound that suffers a low bioavailability because of its low water solubility. In this work β-lactoglobulin (β-Lg) and nanoemulsion were used as carriers to deliver curcumin. The pH stability of β-Lg-CCM was investigated. The digestion of β-Lg-CCM and the nanoemulsion was studied using an in vitro gastrointestinal model. The effect of different carriers on the permeability of curcumin was assessed using the Caco-2 cell monolayer model. The results revealed that the water solubility and the pH stability of curcumin significantly increased by binding with β-Lg. In SDS-PAGE experiments the β-Lg-CCM complex and nanoemulsion were found to be resistant to pepsin digestion but sensitive to trypsin. In the permeability experiment it was shown that the digested nanoemulsion and β-Lg-CCM improved significantly the permeation rate of curcumin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bovine vaginal strain Kocuria kristinae and its characterization.

PubMed

Styková, Eva; Nemcová, Radomíra; Gancarčíková, Soňa; Valocký, Igor; Lauková, Andrea

2016-05-01

Kocuria spp. are widely distributed in nature. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria. In general, limited knowledge exists concerning the properties associated with the representants of the genus Kocuria, Kocuria kristinae as well. Following our previous results, K. kristinae Kk2014 Biocenol(™) (CCM 8628) was isolated from vagina of a healthy cow. Its taxonomical allottation was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system and phenotypic characteristics. Kk2014 strain showed strong adherence capability to the vaginal mucus, produced organic acids which can play a role in prevention of unsuitable contamination, and showed in vitro antagonistic/antimicrobial activity against strains Arcanobacterium pyogenes CCM 5753, Fusobacterium necrophorum CCM 5982, Streptococcus equi subsp. zooepidemicus CCM 7316, and Gardnerella vaginalis CCM 6221. Antimicrobial activity ranged from 100 to 200 AU/mL, up to 32 mm in size, respectively.

CCM in the FUV

NASA Astrophysics Data System (ADS)

Cartledge, S. I. B.; Clayton, G. C.; Gordon, K. D.

2005-12-01

Over 90% of Milky Way extinction curves sampled from IR through UV wavelengths (UV coverage by IUE) conform to the CCM family of curves based on R(V), a single parameter linked to grain size. The far-ultraviolet (FUV) portion of this formulation, however, was based only on a few sight lines observed by Copernicus, and it has recently been shown that in certain cases CCM does not predict extinction in the FUV as accurately as it does at longer wavelengths. In this poster paper, we present preliminary results from our examination of the agreement between all publicly-available FUSE FUV spectra of reddened O and B stars and their corresponding extinction curves inferred from longer wavelength fluxes. The eventual goal of our project is to reformulate CCM, particularly in the FUV where we have compiled more than 90 sight lines observed by FUSE. We are also exploring correlations between bulk sight line properties and the form of the reconstituted CCM curves.

Effect of Curcumin Supplementation on Physiological Fatigue and Physical Performance in Mice

PubMed Central

Huang, Wen-Ching; Chiu, Wan-Chun; Chuang, Hsiao-Li; Tang, Deh-Wei; Lee, Zon-Min; Wei, Li; Chen, Fu-An; Huang, Chi-Chang

2015-01-01

Curcumin (CCM) is a well-known phytocompound and food component found in the spice turmeric and has multifunctional bioactivities. However, few studies have examined its effects on exercise performance and physical fatigue. We aimed to evaluate the potential beneficial effects of CCM supplementation on fatigue and ergogenic function following physical challenge in mice. Male ICR mice were divided into four groups to receive vehicle or CCM (180 μg/mL) by oral gavage at 0, 12.3, 24.6, or 61.5 mL/kg/day for four weeks. Exercise performance and anti-fatigue function were evaluated after physical challenge by forelimb grip strength, exhaustive swimming time, and levels of physical fatigue-associated biomarkers serum lactate, ammonia, blood urea nitrogen (BUN), and glucose and tissue damage markers such as aspartate transaminase (AST), alanine transaminase (ALT), and creatine kinase (CK). CCM supplementation dose-dependently increased grip strength and endurance performance and significantly decreased lactate, ammonia, BUN, AST, ALT, and CK levels after physical challenge. Muscular glycogen content, an important energy source for exercise, was significantly increased. CCM supplementation had few subchronic toxic effects. CCM supplementation may have a wide spectrum of bioactivities for promoting health, improving exercise performance and preventing fatigue. PMID:25647661

Demand generation and social mobilisation for integrated community case management (iCCM) and child health: Lessons learned from successful programmes in Niger and Mozambique

PubMed Central

Sharkey, Alyssa B; Martin, Sandrine; Cerveau, Teresa; Wetzler, Erica; Berzal, Rocio

2014-01-01

Aim We present the approaches used in and outcomes resulting from integrated community case management (iCCM) programmes in Niger and Mozambique with a strong focus on demand generation and social mobilisation. Methods We use a case study approach to describe the programme and contextual elements of the Niger and Mozambique programmes. Results Awareness and utilisation of iCCM services and key family practices increased following the implementation of the Niger and Mozambique iCCM and child survival programmes, as did care–seeking within 24 hours and care–seeking from appropriate, trained providers in Mozambique. These approaches incorporated interpersonal communication activities and community empowerment/participation for collective change, partnerships and networks among key stakeholder groups within communities, media campaigns and advocacy efforts with local and national leaders. Conclusions iCCM programmes that train and equip community health workers and successfully engage and empower community members to adopt new behaviours, have appropriate expectations and to trust community health workers’ ability to assess and treat illnesses can lead to improved care–seeking and utilisation, and community ownership for iCCM. PMID:25520800

Overcoming roadblocks: current and emerging reimbursement strategies for integrated mental health services in primary care.

PubMed

O'Donnell, Allison N; Williams, Mark; Kilbourne, Amy M

2013-12-01

The Chronic Care Model (CCM) has been shown to improve medical and psychiatric outcomes for persons with mental disorders in primary care settings, and has been proposed as a model to integrate mental health care in the patient-centered medical home under healthcare reform. However, the CCM has not been widely implemented in primary care settings, primarily because of a lack of a comprehensive reimbursement strategy to compensate providers for day-to-day provision of its core components, including care management and provider decision support. Drawing upon the existing literature and regulatory guidelines, we provide a critical analysis of challenges and opportunities in reimbursing CCM components under the current fee-for-service system, and describe an emerging financial model involving bundled payments to support core CCM components to integrate mental health treatment into primary care settings. Ultimately, for the CCM to be used and sustained over time to integrate physical and mental health care, effective reimbursement models will need to be negotiated across payers and providers. Such payments should provide sufficient support for primary care providers to implement practice redesigns around core CCM components, including care management, measurement-based care, and mental health specialist consultation.

Evaluation of stability of interface between CCM (Co-Cr-Mo) UCLA abutment and external hex implant.

PubMed

Yoon, Ki-Joon; Park, Young-Bum; Choi, Hyunmin; Cho, Youngsung; Lee, Jae-Hoon; Lee, Keun-Woo

2016-12-01

The purpose of this study is to evaluate the stability of interface between Co-Cr-Mo (CCM) UCLA abutment and external hex implant. Sixteen external hex implant fixtures were assigned to two groups (CCM and Gold group) and were embedded in molds using clear acrylic resin. Screw-retained prostheses were constructed using CCM UCLA abutment and Gold UCLA abutment. The external implant fixture and screw-retained prostheses were connected using abutment screws. After the abutments were tightened to 30 Ncm torque, 5 kg thermocyclic functional loading was applied by chewing simulator. A target of 1.0 × 10 6 cycles was applied. After cyclic loading, removal torque values were recorded using a driving torque tester, and the interface between implant fixture and abutment was evaluated by scanning electronic microscope (SEM). The means and standard deviations (SD) between the CCM and Gold groups were analyzed with independent t-test at the significance level of 0.05. Fractures of crowns, abutments, abutment screws, and fixtures and loosening of abutment screws were not observed after thermocyclic loading. There were no statistically significant differences at the recorded removal torque values between CCM and Gold groups ( P >.05). SEM analysis revealed that remarkable wear patterns were observed at the abutment interface only for Gold UCLA abutments. Those patterns were not observed for other specimens. Within the limit of this study, CCM UCLA abutment has no statistically significant difference in the stability of interface with external hex implant, compared with Gold UCLA abutment.

PUPTH Prehospital Air Medical Plasma (PAMP) Trial

DTIC Science & Technology

2014-07-01

collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for...projects. Scott Gunn, MD, is an Associate Professor of CCM and Emergency Medicine and Director , Combined Emergency Medicine/Internal Medicine/CCM...Residency Program. Dr. Gunn is also Director of the CCM Clinical Trials Program. As such, he is experienced in the design and monitoring of clinical trials

Strengthening the primary care workforce to deliver community case management for child health in rural Indonesia.

PubMed

Setiawan, Agus; Dawson, Angela

2017-10-02

Objectives The aim of the present study was to report on the implementation of community case management (CCM) to reduce infant mortality in a rural district, namely Kutai Timur, Kalimantan Indonesia. Methods An interpretive qualitative methodology was used. In-depth interviews were conducted with 18 primary healthcare workers (PHCWs), and PHCWs were observed during a consultation with mothers to gain insight into the delivery of the new protocol and workforce issues. The field notes and interview transcripts were analysed thematically. Results PHCWs reported that their performance had improved as a result of increased knowledge and confidence. The implementation of CCM had also reportedly enhanced the PHCWs' clinical reasoning. However, the participants noted confusion surrounding their role in prescribing medication. Conclusions CCM is viewed as a useful model of care in terms of enhancing the capacity of rural PHCWs to provide child health care and improve the uptake of life-saving interventions. However, work is needed to strengthen the workforce and to fully integrate CCM into maternal and child health service delivery across Indonesia. What is known about the topic? Indonesia has successfully reduced infant mortality in the past 10 years. However, concerns remain regarding issues related to disparities between districts. The number of infant deaths in rural areas tends to be staggeringly high compared with that in the cities. One of the causes is inadequate access to child health care. What does this paper add? CCM is a model of care that is designed to address childhood illnesses in limited-resource settings. In CCM, PHCWs are trained to deliver life-saving interventions to sick children in rural communities. In the present study, CCM improved the capacity of PHCWs to treat childhood illnesses. What are the implications for practitioners? CCM can be considered to strengthen PHCWs' competence in addressing infant mortality in areas where access to child health care is challenging. Policy regarding task shifting needs to be examined further so that CCM can be integrated into current health service delivery in Indonesia.

Critical Care Medicine and Infectious Diseases: An Emerging Combined Subspecialty in the United States

PubMed Central

Kadri, Sameer S.; Rhee, Chanu; Fortna, Gregory S.; O'Grady, Naomi P.

2015-01-01

The recent rise in unfilled training positions among infectious diseases (ID) fellowship programs nationwide indicates that ID is declining as a career choice among internal medicine residency graduates. Supplementing ID training with training in critical care medicine (CCM) might be a way to regenerate interest in the specialty. Hands-on patient care and higher salaries are obvious attractions. High infection prevalence and antibiotic resistance in intensive care units, expanding immunosuppressed host populations, and public health crises such as the recent Ebola outbreak underscore the potential synergy of CCM-ID training. Most intensivists receive training in pulmonary medicine and only 1% of current board-certified intensivists are trained in ID. While still small, this cohort of CCM-ID certified physicians has continued to rise over the last 2 decades. ID and CCM program leadership nationwide must recognize these trends and the merits of the CCM-ID combination to facilitate creation of formal dual-training opportunities. PMID:25944345

Rapid, automated mosaicking of the human corneal subbasal nerve plexus.

PubMed

Vaishnav, Yash J; Rucker, Stuart A; Saharia, Keshav; McNamara, Nancy A

2017-11-27

Corneal confocal microscopy (CCM) is an in vivo technique used to study corneal nerve morphology. The largest proportion of nerves innervating the cornea lie within the subbasal nerve plexus, where their morphology is altered by refractive surgery, diabetes and dry eye. The main limitations to clinical use of CCM as a diagnostic tool are the small field of view of CCM images and the lengthy time needed to quantify nerves in collected images. Here, we present a novel, rapid, fully automated technique to mosaic individual CCM images into wide-field maps of corneal nerves. We implemented an OpenCV image stitcher that accounts for corneal deformation and uses feature detection to stitch CCM images into a montage. The method takes 3-5 min to process and stitch 40-100 frames on an Amazon EC2 Micro instance. The speed, automation and ease of use conferred by this technique is the first step toward point of care evaluation of wide-field subbasal plexus (SBP) maps in a clinical setting.

Examination of tracer transport in the NCAR CCM2 by comparison of CFCl3 simulations with ALE/GAGE observations

NASA Technical Reports Server (NTRS)

Hartley, Dana E.; Williamson, David L.; Rasch, Philip J.; Prinn, Ronald G.

1994-01-01

The latest version of the National Center for Atmospheric Research (NCAR) community climate model (CCM2) contains a semi-Lagrangian tracer transport scheme for the purpose of advecting water vapor and for including chemistry in the climate model. One way to diagnose the CCM2 transport is to simulate CFCl3 in the CCM2 since it has a well-known industry-based source distribution and a photochemical sink and to compare the model results to Atmospheric Lifetime Experiment/Global Atmospheric Gases Experiment ALE/GAGE observations around the globe. In this paper we focus on this comparison and discuss the synoptic scale issues of tracer transport where appropriate. We compare the model and observations on both 12-hour and monthly timescales. The higher-frequency events allow us to diagnose the synoptic scale transport in the CCM2 associated with the observational sites and to determine uncertainties in our high-resolution source distribution. We find that the CCM2 does simulate many of the key features such as pollution events and some seasonal transports, but there are still some dynamical features of tracer transport such as the storm track dynamics and cross-equatorial flow that merit further study in both the model and the real atmosphere.

Evaluation of stability of interface between CCM (Co-Cr-Mo) UCLA abutment and external hex implant

PubMed Central

Yoon, Ki-Joon; Park, Young-Bum; Choi, Hyunmin; Cho, Youngsung; Lee, Jae-Hoon

2016-01-01

PURPOSE The purpose of this study is to evaluate the stability of interface between Co-Cr-Mo (CCM) UCLA abutment and external hex implant. MATERIALS AND METHODS Sixteen external hex implant fixtures were assigned to two groups (CCM and Gold group) and were embedded in molds using clear acrylic resin. Screw-retained prostheses were constructed using CCM UCLA abutment and Gold UCLA abutment. The external implant fixture and screw-retained prostheses were connected using abutment screws. After the abutments were tightened to 30 Ncm torque, 5 kg thermocyclic functional loading was applied by chewing simulator. A target of 1.0 × 106 cycles was applied. After cyclic loading, removal torque values were recorded using a driving torque tester, and the interface between implant fixture and abutment was evaluated by scanning electronic microscope (SEM). The means and standard deviations (SD) between the CCM and Gold groups were analyzed with independent t-test at the significance level of 0.05. RESULTS Fractures of crowns, abutments, abutment screws, and fixtures and loosening of abutment screws were not observed after thermocyclic loading. There were no statistically significant differences at the recorded removal torque values between CCM and Gold groups (P>.05). SEM analysis revealed that remarkable wear patterns were observed at the abutment interface only for Gold UCLA abutments. Those patterns were not observed for other specimens. CONCLUSION Within the limit of this study, CCM UCLA abutment has no statistically significant difference in the stability of interface with external hex implant, compared with Gold UCLA abutment. PMID:28018564

A New Water-Soluble Nanomicelle Formed through Self-Assembly of Pectin-Curcumin Conjugates: Preparation, Characterization, and Anticancer Activity Evaluation.

PubMed

Bai, Feng; Diao, Jiajing; Wang, Ying; Sun, Shixin; Zhang, Hongmei; Liu, Yunyun; Wang, Yanqing; Cao, Jian

2017-08-16

Curcumin is a dominating active component of Curcuma longa and has been studied widely because of its prominent biological activities. The extremely low aqueous solubility, stability, and bioavailability of curcumin limit its application in the field of medicine. In this study, we developed pectin-curcumin (PEC-CCM) conjugates that could self-assemble water-soluble nanomicelles in aqueous solution. The structure of PEC-CCM conjugates was characterized by ultraviolet-visible spectra, fluorescence spectra, Fourier transform infrared spectroscopy, and 1 H nuclear magnetic resonance spectroscopy. The thermal property of PEC-CCM conjugates was investigated by thermogravimetric analysis. It was found that PEC-CCM conjugates had formed nanomicelles in aqueous medium via self-assembly. These nanomicelles were observed as small spheres or ellipsoids and aggregated with a size range of 70-190 nm by transmission electron microscopy analysis. In a solution of nanomicelles, the stability of curcumin was improved, and its antioxidant property was preserved. The anticancer activity of PEC-CCM conjugates was quantified by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay using a hepatic cancer cell line (HepG2), a breast cancer cell line (MCF-7), a cervical cancer cell line (HeLa), and a human normal kidney cell line (293A). It was found that the curcumin of PEC-CCM conjugates had a more significant inhibitory effect on cancer cells and was less cytotoxic to normal cells than free curcumin was. PEC-CCM conjugates have great potential for some food and pharmaceutical applications.

Under-Five Child Mortality and Morbidity Associated with Consanguineous Child Marriage in Pakistan: Retrospective Analysis using Pakistan Demographic and Health Surveys, 1990-91, 2006-07, 2012-13.

PubMed

Mustafa, Mudasir; Zakar, Rubeena; Zakar, Muhammad Zakria; Chaudhry, Ashraf; Nasrullah, Muazzam

2017-05-01

Objective To assess the combined effect of consanguineous and child marriages (CCM) on children health, which has not previously been explored, either globally or locally. Methods We analyzed secondary data from a series of cross-sectional, nationally representative Pakistan Demographic and Health Surveys 1990-91, 2006-07, and 2012-13. A total of 5406 mothers with 10,164 children were included in the analysis. Child health was assessed by variables such as history of diarrhea, acute respiratory infection (ARI), ARI with fever, Under-5 child mortality (U5CM) and small-size birth (SSB). Associations among variables were assessed by calculating unadjusted Odd Ratios (OR) and adjusted OR (AOR). Results A majority (n = 6,247, 61%) of the births were to mothers having CCM as compare to non-CCM (3917, 39%). There was a significant association between CCM and U5CM during 1990-91 (AOR 1.24, 95% CI 1.03-1.49) and 2006-07 (AOR 1.25, 95% CI 1.05-1.51), and infant mortality in 1990-91 (AOR 1.39, 95% CI 1.05-1.85) and 2006-07 (AOR 1.61, 95% CI 1.17-2.21). A significant association was also found between CCM and SSB infants in the period 2006-07 (AOR 1.19, 95% CI 1.01-1.42) and 2012-13 (AOR 1.22, 95% CI 1.02-1.46). We noted no effect of CCM on diarrhea, ARI, and ARI with fever. Conclusion CCM increases the likelihood of U5CM, infant mortality and SSB infants. Further quantitative and qualitative research should be conducted to assess the effects of environmental, congenital and genetic factors on the health of children born to mothers in CCM.

Folic acid-targeted disulfide-based cross-linking micelle for enhanced drug encapsulation stability and site-specific drug delivery against tumors

PubMed Central

Zhang, Yumin; Zhou, Junhui; Yang, Cuihong; Wang, Weiwei; Chu, Liping; Huang, Fan; Liu, Qiang; Deng, Liandong; Kong, Deling; Liu, Jianfeng; Liu, Jinjian

2016-01-01

Although the shortcomings of small molecular antitumor drugs were efficiently improved by being entrapped into nanosized vehicles, premature drug release and insufficient tumor targeting demand innovative approaches that boost the stability and tumor responsiveness of drug-loaded nanocarriers. Here, we show the use of the core cross-linking method to generate a micelle with enhanced drug encapsulation ability and sensitivity of drug release in tumor. This kind of micelle could increase curcumin (Cur) delivery to HeLa cells in vitro and improve tumor accumulation in vivo. We designed and synthesized the core cross-linked micelle (CCM) with polyethylene glycol and folic acid-polyethylene glycol as the hydrophilic units, pyridyldisulfide as the cross-linkable and hydrophobic unit, and disulfide bond as the cross-linker. CCM showed spherical shape with a diameter of 91.2 nm by the characterization of dynamic light scattering and transmission electron microscope. Attributed to the core cross-linking, drug-loaded CCM displayed higher Nile Red or Cur-encapsulated stability and better sensitivity to glutathione than noncross-linked micelle (NCM). Cellular uptake and in vitro antitumor studies proved the enhanced endocytosis and better cytotoxicity of CCM-Cur against HeLa cells, which had a high level of glutathione. Meanwhile, the folate receptor-mediated drug delivery (FA-CCM-Cur) further enhanced the endocytosis and cytotoxicity. Ex vivo imaging studies showed that CCM-Cur and FA-CCM-Cur possessed higher tumor accumulation until 24 hours after injection. Concretely, FA-CCM-Cur exhibited the highest tumor accumulation with 1.7-fold of noncross-linked micelle Cur and 2.8-fold of free Cur. By combining cross-linking of the core with active tumor targeting of FA, we demonstrated a new and effective way to design nanocarriers for enhanced drug encapsulation, smart tumor responsiveness, and elevated tumor accumulation. PMID:27051287

Profiles of cardiovascular biomarkers according to severity stages of Chagas cardiomyopathy.

PubMed

Echeverría, Luis E; Rojas, Lyda Z; Calvo, Lauren S; Roa, Zayne M; Rueda-Ochoa, Oscar L; Morillo, Carlos A; Muka, Taulant; Franco, Oscar H

2017-01-15

Up 30 to 40% of Chagas patients exhibit cardiomyopathy with different degrees of cardiac involvement. Biomarkers may help in differentiation of the severity of Chagas cardiomyopathy (CCM). This study sought to examine the diagnostic value of a panel of biomarkers to distinguish the severity of (CCM). 100 patients with CCM were included in this cross-sectional study. Based on electrocardiogram and echocardiogram, CCM patients were classified in three stages according to disease's severity. Levels of high-sensitivity cardiac troponin T (Hs-cTnT), N-terminal pro B-type natriuretic peptide (NT-proBNP), galectin-3 (Gal-3), neutrophil gelatinase-associated lipocalin (NGAL), soluble ST2 (sST2) and cystatin-c (Cys-c) were measured. Logistic regression models were used to assess the association between levels of natural log-transformed values of biomarkers and stages C/D versus B. We also calculated the area under curve (AUC) for each of the models. In models adjusted for age, sex, body mass index, kidney function and medication use, increased levels of NT-proBNP (per 1 unit natural log-transformed values, odds ratio (OR)=5.55; 95CI%:1.65-18.72) and Hs-cTnT (per 1 unit natural log-transformed values, OR=7.11; 95CI%:1.41-35.90) showed significant association with the severity of CCM per 1 unit increase of biomarkers. The accuracy of NT-proBNP and Hs-cTnT for diagnosis of the severity of CCM was high: AUC of 0.968 and 0.956 respectively. No significant difference was found in the AUC between NT-proBNP and Hs-cTnT. No association was found between Gal-3, NGAL, sST2 and Cys-C and severity of CCM. NT-proBNP and Hs-cTnT have both same diagnostic value in distinguishing severity of CCM. Copyright © 2016. Published by Elsevier Ireland Ltd.

The Chronic Care Model and Technological Research and Innovation: A Scoping Review at the Crossroads

PubMed Central

Berntsen, Gro Karine Rosvold; Koricho, Absera Teshome; Sygna, Karin; Ruland, Cornelia

2015-01-01

Background Information and communication technologies (ICT) are key to optimizing the outcomes of the Chronic Care Model (CCM), currently acknowledged as the best synthesis of available evidence for chronic illness prevention and management. At the same time, CCM can offer a needed framework for increasing the relevance and feasibility of ICT innovation and research in health care. Little is known about how and to what extent CCM and ICT research inform each other to leverage mutual strengths. The current study examines: What characterizes work being done at the crossroads of CCM and ICT research and innovation? Objective Our aim is identify the gaps and potential that lie between the research domains CCM and ICT, thus enabling more substantive questions and opportunities for accelerating improvements in ICT-supported chronic care. Methods Using a scoping study approach, we developed a search strategy applied to medical and technical databases resulting in 1054 titles and abstracts that address CCM and ICT. After iteratively adapting our inclusion/exclusion criteria to balance between breadth and feasibility, 26 publications from 20 studies were found to fulfill our criteria. Following initial coding of each article according to predefined categories (eg, type of article, CCM component, ICT, health issue), a 1st level analysis was conducted resulting in a broad range of categories. These were gradually reduced by constantly comparing them for underlying commonalities and discrepancies. Results None of the studies included were from technical databases and interventions relied mostly on “old-fashioned” technologies. Technologies supporting “productive interactions” were often one-way (provider to patient), and it was sometimes difficult to decipher how CCM was guiding intervention design. In particular, the major focus on ICT to support providers did not appear unique to the challenges of chronic care. Challenges in facilitating CCM components through ICT included poorly designed user interfaces, digital divide issues, and lack of integration with existing infrastructure. Conclusions The CCM is a highly influential guide for health care development, which recognizes the need for alignment of system tools such as ICT. Yet, there seem to be alarmingly few touch points between the subject fields of “health service development” and “ICT-innovation”. Bridging these gaps needs explicit and urgent attention as the synergies between these domains have enormous potential. Policy makers and funding agencies need to facilitate the joining of forces between high-tech innovative expertise and experts in the chronic care system redesign that is required for tackling the current epidemic of long-term multiple conditions. PMID:25677200

Costing commodity and human resource needs for integrated community case management in thie differing community health strategies of Ethiopia, Kenya and Zambia.

PubMed

Nefdt, Rory; Ribaira, Eric; Diallo, Khassoum

2014-10-01

To ensure correct and appropriate funding is available, there is a need to estimate resource needs for improved planning and implementation of integrated Community Case Management (iCCM). To compare and estimate costs for commodity and human resource needs for iCCM, based on treatment coverage rates, bottlenecks and national targets in Ethiopia, Kenya and Zambia from 2014 to 2016. Resource needs were estimated using Ministry of Health (MoH) targets fronm 2014 to 2016 for implementation of case management of pneumonia, diarrhea and malaria through iCCM based on epidemiological, demographic, economic, intervention coverage and other health system parameters. Bottleneck analysis adjusted cost estimates against system barriers. Ethiopia, Kenya and Zambia were chosen to compare differences in iCCM costs in different programmatic implementation landscapes. Coverage treatment rates through iCCM are lowest in Ethiopia, followed by Kenya and Zambia, but Ethiopia had the greatest increases between 2009 and 2012. Deployment of health extension workers (HEWs) in Ethiopia is more advanced compared to Kenya and Zambia, which have fewer equivalent cadres (called commu- nity health workers (CHWs)) covering a smaller proportion of the population. Between 2014 and 2016, the propor- tion of treatments through iCCM compared to health centres are set to increase from 30% to 81% in Ethiopia, 1% to 18% in Kenya and 3% to 22% in Zambia. The total estimated cost of iCCM for these three years are USD 75,531,376 for Ethiopia, USD 19,839,780 for Kenya and USD 33,667,742 for Zambia. Projected per capita expen- diture for 2016 is USD 0.28 for Ethiopia, USD 0.20 in Kenya and USD 0.98 in Zambia. Commodity costs for pneumonia and diarrhea were a small fraction of the total iCCM budget for all three countries (less than 3%), while around 80% of the costs related to human resources. Analysis of coverage, demography and epidemiology data improves estimates of fimding requirements for iCCM. Bottleneck analysis adjusts cost estimates by including system barriers, thus reflecting a more accurate estimate of potential resource utilization. Adding pneumonia and diarrhea interventions to existing large scale community-based malaria case management programs is likely to require relatively small and nationally affordable investments. iCCM can be implemented for USD 0.09 to 0.98 per capita per annum, depending on the stage of scale-up and targets set by the MoH.

The chronic care model and technological research and innovation: a scoping review at the crossroads.

PubMed

Gammon, Deede; Berntsen, Gro Karine Rosvold; Koricho, Absera Teshome; Sygna, Karin; Ruland, Cornelia

2015-02-06

Information and communication technologies (ICT) are key to optimizing the outcomes of the Chronic Care Model (CCM), currently acknowledged as the best synthesis of available evidence for chronic illness prevention and management. At the same time, CCM can offer a needed framework for increasing the relevance and feasibility of ICT innovation and research in health care. Little is known about how and to what extent CCM and ICT research inform each other to leverage mutual strengths. The current study examines: What characterizes work being done at the crossroads of CCM and ICT research and innovation? Our aim is identify the gaps and potential that lie between the research domains CCM and ICT, thus enabling more substantive questions and opportunities for accelerating improvements in ICT-supported chronic care. Using a scoping study approach, we developed a search strategy applied to medical and technical databases resulting in 1054 titles and abstracts that address CCM and ICT. After iteratively adapting our inclusion/exclusion criteria to balance between breadth and feasibility, 26 publications from 20 studies were found to fulfill our criteria. Following initial coding of each article according to predefined categories (eg, type of article, CCM component, ICT, health issue), a 1st level analysis was conducted resulting in a broad range of categories. These were gradually reduced by constantly comparing them for underlying commonalities and discrepancies. None of the studies included were from technical databases and interventions relied mostly on "old-fashioned" technologies. Technologies supporting "productive interactions" were often one-way (provider to patient), and it was sometimes difficult to decipher how CCM was guiding intervention design. In particular, the major focus on ICT to support providers did not appear unique to the challenges of chronic care. Challenges in facilitating CCM components through ICT included poorly designed user interfaces, digital divide issues, and lack of integration with existing infrastructure. The CCM is a highly influential guide for health care development, which recognizes the need for alignment of system tools such as ICT. Yet, there seem to be alarmingly few touch points between the subject fields of "health service development" and "ICT-innovation". Bridging these gaps needs explicit and urgent attention as the synergies between these domains have enormous potential. Policy makers and funding agencies need to facilitate the joining of forces between high-tech innovative expertise and experts in the chronic care system redesign that is required for tackling the current epidemic of long-term multiple conditions.

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria.

PubMed

Crowe, Adam M; Casabon, Israël; Brown, Kirstin L; Liu, Jie; Lian, Jennifer; Rogalski, Jason C; Hurst, Timothy E; Snieckus, Victor; Foster, Leonard J; Eltis, Lindsay D

2017-04-04

Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis , this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene ( ipdAB ) and an acyl-CoA reductase gene ( ipdC ). In the presence of cholesterol, Δ ipdC and Δ ipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα- H -4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and ( R )-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A Δ fadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by Δ ipdF and Δ echA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a Δ ipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. IMPORTANCE Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis , this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics. Copyright © 2017 Crowe et al.

Hydrogenetic Ferromanganese Crusts of the California Continental Margin

NASA Astrophysics Data System (ADS)

Conrad, Tracey A.

Hydrogenetic Ferromanganese (Fe-Mn) crusts grow from seawater and in doing so sequester elements of economic interest and serve as archives of past seawater chemistry. Ferromanganese crusts have been extensively studied in open-ocean environments. However, few studies have examined continent-proximal Fe-Mn crusts especially from the northeast Pacific. This thesis addresses Fe-Mn crusts within the northeast Pacific California continental margin (CCM), which is a dynamic geological and oceanographic environment. In the first of three studies, I analyzed the chemical and mineralogical composition of Fe-Mn crusts and show that continental-proximal processes greatly influence the chemistry and mineralogy of CCM Fe-Mn crusts. When compared to global open-ocean Fe-Mn crusts, CCM crusts have higher concentrations of iron, silica, and thorium with lower concentrations of many elements of economic interest including manganese, cobalt, and tellurium, among other elements. The mineralogy of CCM Fe-Mn crusts is also unique with more birnessite and todorokite present than found in open-ocean samples. Unlike open-ocean Fe-Mn crusts, carbonate-fluorapatite is not present in CCM crusts. This lack of phosphatization makes CCM Fe-Mn crusts excellent candidates for robust paleoceanography records. The second and third studies in this thesis use isotope geochemistry on select CCM Fe-Mn crusts from four seamounts in the CCM to study past terrestrial inputs into the CCM and sources and behavior of Pb and Nd isotopes over the past 7 million years along the northeast Pacific margin. The second study focuses on riverine inputs into the Monterey Submarine Canyon System and sources of the continental material. Osmium isotopes in the crusts are compared to the Cenozoic Os seawater curve to develop an age model for the samples that show the crusts range in age of initiation of crust growth from approximately 20 to 6 Myr. Lead and neodymium isotopes measured in select Fe-Mn crusts show that large amounts of terrestrial material entered the CCM via the Monterey Canyon from prior to 6.8+/-0.5 until 4.5 +/-0.5 Myr ago. These data combined with reconstructions of the paleo-coastline indicate that incision of the modern Monterey Canyon started around 7 Myr ago. Isotope plots of potential source regions indicate that the source of the material is the border of the southern Sierra Nevada and western Basin and Range. This answers a long-standing and fundamental question about the timing and formation of the Monterey Canyon, the dominant feature of the Monterey Bay. The third study presented here uses the differences in lead and neodymium isotopic values in CCM Fe-Mn crusts over time compared to open-ocean Pacific, North Pacific, and Arctic Ocean Fe-Mn crusts to identify regional time-series trends and sources for these important oceanographic tracers. I found that sediment fluxes and inputs of terrestrial material from North American rivers effects the lead and neodymium isotope composition of regional seawater.

High-Order Coupled Cluster Method (CCM) Calculations for Quantum Magnets with Valence-Bond Ground States

NASA Astrophysics Data System (ADS)

Farnell, D. J. J.; Richter, J.; Zinke, R.; Bishop, R. F.

2009-04-01

In this article, we prove that exact representations of dimer and plaquette valence-bond ket ground states for quantum Heisenberg antiferromagnets may be formed via the usual coupled cluster method (CCM) from independent-spin product (e.g. Néel) model states. We show that we are able to provide good results for both the ground-state energy and the sublattice magnetization for dimer and plaquette valence-bond phases within the CCM. As a first example, we investigate the spin-half J 1- J 2 model for the linear chain, and we show that we are able to reproduce exactly the dimerized ground (ket) state at J 2/ J 1=0.5. The dimerized phase is stable over a range of values for J 2/ J 1 around 0.5, and results for the ground-state energies are in good agreement with the results of exact diagonalizations of finite-length chains in this regime. We present evidence of symmetry breaking by considering the ket- and bra-state correlation coefficients as a function of J 2/ J 1. A radical change is also observed in the behavior of the CCM sublattice magnetization as we enter the dimerized phase. We then consider the Shastry-Sutherland model and demonstrate that the CCM can span the correct ground states in both the Néel and the dimerized phases. Once again, very good results for the ground-state energies are obtained. We find CCM critical points of the bra-state equations that are in agreement with the known phase transition point for this model. The results for the sublattice magnetization remain near to the "true" value of zero over much of the dimerized regime, although they diverge exactly at the critical point. Finally, we consider a spin-half system with nearest-neighbor bonds for an underlying lattice corresponding to the magnetic material CaV4O9 (CAVO). We show that we are able to provide excellent results for the ground-state energy in each of the plaquette-ordered, Néel-ordered, and dimerized regimes of this model. The exact plaquette and dimer ground states are reproduced by the CCM ket state in their relevant limits. Furthermore, we estimate the range over which the Néel order is stable, and we find the CCM result is in reasonable agreement with the results obtained by other methods. Our new approach has the dual advantages that it is simple to implement and that existing CCM codes for independent-spin product model states may be used from the outset. Furthermore, it also greatly extends the range of applicability to which the CCM may be applied. We believe that the CCM now provides an excellent choice of method for the study of systems with valence-bond quantum ground states.

Outcome after surgical or conservative management of cerebral cavernous malformations.

PubMed

Moultrie, Fiona; Horne, Margaret A; Josephson, Colin B; Hall, Julie M; Counsell, Carl E; Bhattacharya, Jo J; Papanastassiou, Vakis; Sellar, Robin J; Warlow, Charles P; Murray, Gordon D; Al-Shahi Salman, Rustam

2014-08-12

There have been few comparative studies of microsurgical excision vs conservative management of cerebral cavernous malformations (CCM) and none of them has reliably demonstrated a statistically and clinically significant difference. We conducted a prospective, population-based study to identify and independently validate definite CCM diagnoses first made in 1999-2003 in Scottish adult residents. We used multiple sources of prospective follow-up to assess adults' dependence and to identify and independently validate outcome events. We used univariate and multivariable survival analyses to test the influence of CCM excision on outcome, adjusted for prognostic factors and baseline imbalances. Of 134 adults, 25 underwent CCM excision; these adults were younger (34 vs 43 years at diagnosis, p = 0.004) and more likely to present with symptomatic intracranial hemorrhage or focal neurologic deficit than adults managed conservatively (48% vs 26%; odds ratio 2.7, 95% confidence interval [CI] 1.1-6.5). During 5 years of follow-up, CCM excision was associated with a deterioration to an Oxford Handicap Scale score 2-6 sustained over at least 2 successive years (adjusted hazard ratio [HR] 2.2, 95% CI 1.1-4.3) and the occurrence of symptomatic intracranial hemorrhage or new focal neurologic deficit (adjusted HR 3.6, 95% CI 1.3-10.0). CCM excision was associated with worse outcomes over 5 years compared to conservative management. Long-term follow-up will determine whether this difference is sustained over patients' lifetimes. Meanwhile, a randomized controlled trial appears justified. This study provides Class III evidence that CCM excision worsens short-term disability scores and increases the risk of symptomatic intracranial hemorrhage and new focal neurologic deficits. © 2014 American Academy of Neurology.

Critical care medicine training and certification for emergency physicians.

PubMed

Huang, David T; Osborn, Tiffany M; Gunnerson, Kyle J; Gunn, Scott R; Trzeciak, Stephen; Kimball, Edward; Fink, Mitchell P; Angus, Derek C; Dellinger, R Phillip; Rivers, Emanuel P

2005-09-01

Demand for critical care services is increasing. Unless the supply of intensivists increases, critically ill patients will not have access to intensivists. Recent critical care society recommendations include increased graduate medical education support and expansion of the J-1 visa waiver program for foreign medical graduates. This article proposes additional recommendations, based on strengthening the relationship between emergency medicine and critical care medicine. Demand for critical care services is increasing. Unless the supply of intensivists increases, critically ill patients will not have access to intensivists. Recent critical care society recommendations include increased graduate medical education support and expansion of the J-1 visa waiver program for foreign medical graduates. This article proposes additional recommendations, based on strengthening the relationship between emergency medicine (EM) and critical care medicine (CCM). Critical care is a continuum that includes prehospital, emergency department (ED), and intensive care unit (ICU) care teams. Both EM and CCM require expertise in treating life-threatening acute illness, with many critically ill patients often presenting first to the ED. Increased patient volumes and acuity have resulted in longer ED lengths of stay and more critical care delivery in the ED. However, the majority of CCM fellowships do not accept EM residents, and those who successfully complete a fellowship do not have access to a U.S. certification exam in CCM. Despite these barriers, interest in CCM training among EM physicians is increasing. Dual EM/CCM-trained physicians not only will help alleviate the intensivist shortage but also will strengthen critical care delivery in the ED and facilitate coordination at the ED-ICU interface. We therefore propose that all accreditation bodies work cooperatively to create a route to CCM certification for emergency physicians who complete a critical care fellowship.

The Chronic Care Model and Diabetes Management in US Primary Care Settings: A Systematic Review

PubMed Central

Stellefson, Michael; Stopka, Christine

2013-01-01

Introduction The Chronic Care Model (CCM) uses a systematic approach to restructuring medical care to create partnerships between health systems and communities. The objective of this study was to describe how researchers have applied CCM in US primary care settings to provide care for people who have diabetes and to describe outcomes of CCM implementation. Methods We conducted a literature review by using the Cochrane database of systematic reviews, CINAHL, and Health Source: Nursing/Academic Edition and the following search terms: “chronic care model” (and) “diabet*.” We included articles published between January 1999 and October 2011. We summarized details on CCM application and health outcomes for 16 studies. Results The 16 studies included various study designs, including 9 randomized controlled trials, and settings, including academic-affiliated primary care practices and private practices. We found evidence that CCM approaches have been effective in managing diabetes in US primary care settings. Organizational leaders in health care systems initiated system-level reorganizations that improved the coordination of diabetes care. Disease registries and electronic medical records were used to establish patient-centered goals, monitor patient progress, and identify lapses in care. Primary care physicians (PCPs) were trained to deliver evidence-based care, and PCP office–based diabetes self-management education improved patient outcomes. Only 7 studies described strategies for addressing community resources and policies. Conclusion CCM is being used for diabetes care in US primary care settings, and positive outcomes have been reported. Future research on integration of CCM into primary care settings for diabetes management should measure diabetes process indicators, such as self-efficacy for disease management and clinical decision making. PMID:23428085

Performance of Versions 1,2 and 3 of the Goddard Earth Observing System (GEOS) Chemistry-Climate Model (CCM)

NASA Technical Reports Server (NTRS)

Pawson, Steven; Stolarski, Richard S.; Nielsen, J. Eric; Duncan, Bryan N.

2008-01-01

Version 1 of the Goddard Earth Observing System Chemistry-Climate Model (GEOS CCM) was used in the first CCMVa1 model evaluation and forms the basis for several studies of links between ozone and the circulation. That version of the CCM was based on the GEOS-4 GCM. Versions 2 and 3 of the GEOS CCM are based on the GEOS-5 GCM, which retains the "Lin-Rood" dynamical core but has a totally different set of physical parameterizatiOns to GEOS-4. In Version 2 of the GEOS CCM the Goddard stratospheric chemistry module is retained. Difference between Versions 1 and 2 thus reflect the physics changes of the underlying GCMs. Several comparisons between these two models are made, several of which reveal improvements in Version 2 (including a more realistic representation of the interannual variability of the Antarctic vortex). In Version 3 of the GEOS CCM, the stratospheric chemistry mechanism is replaced by the "GMI COMBO" code that includes tropospheric chemistry and different computational approaches. An advantage of this model version. is the reduction of high ozone biases that prevail at low chlorine loadings in Versions 1 and 2. This poster will compare and contrast various aspects of the three model versions that are relevant for understanding interactions between ozone and climate.

Chemical Modification and Detoxification of the Pseudomonas aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-Oxide by Environmental and Pathogenic Bacteria.

PubMed

Thierbach, Sven; Birmes, Franziska S; Letzel, Matthias C; Hennecke, Ulrich; Fetzner, Susanne

2017-09-15

2-Heptyl-4-hydroxyquinoline N-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus introduced a hydroxyl group at C-3 of HQNO, whereas Mycobacterium abscessus, M. fortuitum, and M. smegmatis performed an O-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. Bacillus spp. produced the glycosylated derivative 2-heptyl-1-(β-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC 50 values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against P. aeruginosa. Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising P. aeruginosa.

Penicyrones A and B, an epimeric pair of α-pyrone-type polyketides produced by the marine-derived Penicillium sp.

PubMed

Bu, Ying-Yue; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

2016-01-01

Two polyketides containing an α-pyrone unit, named penicyrones A (1) and B (2), were isolated from a culture broth of the marine-derived Penicillium sp. TPU1271 together with nine known compounds: verrucosidin (3), fructigenine A (4), verrucofortine (5), cyclo-(L-Trp-L-Phe) (6), cyclopenol (7), cyclopenin (8), penipratynolene (9), aspterric acid (10) and viridicatol (11). The structures of 1 and 2 were elucidated by analyzing the spectroscopic data of 1, 2 and their O-acetyl derivatives (1a and 2a). Compounds 1 and 2 were epimers of each other at the C-9 position. The absolute configurations of 1 and 2 were assigned on the basis of NOESY data for 1, 2, 1a and 2a, a conformational analysis and the identity of the biogenetic pathway with verrucosidin (3). The planar structure of penicyrones was found in the SciFinder as a compound in the commercial chemical libraries; however, the stereostructure and spectroscopic data were not available. Therefore, this is the first study on the isolation and structure elucidation, including the absolute configurations, of penicyrones A (1) and B (2) as fungal metabolites. Compound 3 exhibited growth inhibitory activity against Mycobacterium smegmatis at 40 μg per disc (inhibition zone of 11 mm). This is the first study to demonstrate that verrucosidin (3) exhibited anti-mycobacterial activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watrous, Jeramie D.; Roach, Patrick J.; Alexandrov, Theodore

Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a "holy grail" in microbiology. This work describes a highly sensitive, broadly applicable, and costeffective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes withinmore » bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097–1100]. The antifungal effect of strain SHC52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is amonochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sathyamoorthy, N.; Qureshi, N.; Takayama, K.

When a dialyzed, cell-free extract of Mycobacterium smegmatis was incubated with (/sup 14/C)trehalose and unlabeled trehalose 6-monomycolate (TM), radiolabeled TM was formed. This appears to be an enzymatic mycolic acid exchange reaction. The TM was purified by DEAE cellulose and silicic acid column chromatography, followed by reverse-phase HPLC using a C/sub 18/-bonded silica column with a linear gradient of 0-60% hexane-isopropanol (2:1, v/v) in isopropanol-water (9:1, v/v). The donor lipid, the /sup 14/C-labeled product, and authentic TM all comigrated on HPLC. Three peak fractions were obtained from HPLC and analyzed by laser desorption mass spectrometry (LDMS) and the structural seriesmore » of mycolic acids were identified. The major TM components gave molecular ions (M+K)/sup +/ at m/z 1486, 1500, and 1528. This corresponded to the presence of dienyl mycolic acids with M/sub r/ of 1106, 1120, and 1148, respectively. Using organically synthesized TM, the authors confirmed that the donor lipid as well as the labeled product of this reaction are indeed TM. This enzyme has now been partially purified by ammonium sulfate precipitation and QAE-Sephadex A-50 column chromatography. This newly discovered mycolic acid exchange reaction might be an integral part of the last step in the biosynthesis of mycolic acid as well as the mycolic acid utilization pathway in Mycobacteria.« less

Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase

PubMed Central

Yano, Takahiro; Kassovska-Bratinova, Sacha; Teh, J. Shin; Winkler, Jeffrey; Sullivan, Kevin; Isaacs, Andre; Schechter, Norman M.; Rubin, Harvey

2011-01-01

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species. PMID:21193400

Microfluidic Screening of Electric Fields for Electroporation

PubMed Central

Garcia, Paulo A.; Ge, Zhifei; Moran, Jeffrey L.; Buie, Cullen R.

2016-01-01

Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX®, which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX® after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes. PMID:26893024

One-pot facile green synthesis of biocidal silver nanoparticles

NASA Astrophysics Data System (ADS)

Nudrat Hazarika, Shabiha; Gupta, Kuldeep; Shamin, Khan Naseem Ahmed Mohammed; Bhardwaj, Pushpender; Boruah, Ratan; Yadav, Kamlesh K.; Naglot, Ashok; Deb, P.; Mandal, M.; Doley, Robin; Veer, Vijay; Baruah, Indra; Namsa, Nima D.

2016-07-01

The plant root extract mediated green synthesis method produces monodispersed spherical shape silver nanoparticles (AgNPs) with a size range of 15-30 nm as analyzed by atomic force and transmission electron microscopy. The material showed potent antibacterial and antifungal properties. Synthesized AgNPs display a characteristic surface plasmon resonance peak at 420 nm in UV-Vis spectroscopy. X-ray diffractometer analysis revealed the crystalline and face-centered cubic geometry of in situ prepared AgNPs. Agar well diffusion and a colony forming unit assay demonstrated the potent biocidal activity of AgNPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas diminuta and Mycobacterium smegmatis. Intriguingly, the phytosynthesized AgNPs exhibited activity against pathogenic fungi, namely Trichophyton rubrum, Aspergillus versicolor and Candida albicans. Scanning electron microscopy observations indicated morphological changes in the bacterial cells incubated with silver nanoparticles. The genomic DNA isolated from the bacteria was incubated with an increasing concentration of AgNPs and the replication fidelity of 16S rDNA was observed by performing 18 and 35 cycles PCR. The replication efficiency of small (600 bp) and large (1500 bp) DNA fragments in the presence of AgNPs were compromised in a dose-dependent manner. The results suggest that the Thalictrum foliolosum root extract mediated synthesis of AgNPs could be used as a promising antimicrobial agent against clinical pathogens.

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

PubMed

Steiger, Stefanie; Kuhn, Sabine; Ronchese, Franca; Harper, Jacquie L

2015-12-01

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1β production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Positive and negative feedback regulatory loops of thiol-oxidative stress response mediated by an unstable isoform of sigmaR in actinomycetes.

PubMed

Kim, Min-Sik; Hahn, Mi-Young; Cho, Yoobok; Cho, Sang-Nae; Roe, Jung-Hye

2009-09-01

Alternate sigma factors provide an effective way of diversifying bacterial gene expression in response to environmental changes. In Streptomyces coelicolor where more than 65 sigma factors are predicted, sigma(R) is the major regulator for response to thiol-oxidative stresses. sigma(R) becomes available when its bound anti-sigma factor RsrA is oxidized at sensitive cysteine thiols to form disulphide bonds. sigma(R) regulon includes genes for itself and multiple thiol-reducing systems, which constitute positive and negative feedback loops respectively. We found that the positive amplification loop involves an isoform of sigma(R) (sigma(R')) with an N-terminal extension of 55 amino acids, produced from an upstream start codon. A major difference between constitutive sigma(R) and inducible sigma(R') is that the latter is markedly unstable (t(1/2) approximately 10 min) compared with the former (> 70 min). The rapid turnover of sigma(R') is partly due to induced ClpP1/P2 proteases from the sigma(R) regulon. This represents a novel way of elaborating positive and negative feedback loops in a control circuit. Similar phenomenon may occur in other actinomycetes that harbour multiple start codons in the sigR homologous gene. We observed that sigH gene, the sigR orthologue in Mycobacterium smegmatis, produces an unstable larger isoform of sigma(H) upon induction by thiol-oxidative stress.

Commiphora leptophloeos Phytochemical and Antimicrobial Characterization

PubMed Central

de Souza Pereira, Jorge J.; Pereira, Aline de P. C.; Jandú, Jannyson J. B.; da Paz, Josinete A.; Crovella, Sergio; dos Santos Correia, Maria T.; de Azevêdo Silva, Jaqueline

2017-01-01

Commiphora leptophloeos is a plant specie usually known for its medicinal purposes in local communities in Northeast Brazil. In order to evaluate its therapeutic potential, we aimed to determine the phytochemical and antimicrobial properties of C. leptophloeos extracts. Thin Layer Chromatography (TLC) was able to detect the presence of phenolic compounds, flavonoids and reducing sugars. Three phenolic compounds were identified by HPLC and described as Gallic, Chlorogenic and Protocatechuic acids. On the other hand, H1NMR analysis revealed the presence of hinokinin, a bioactive lignan further characterized in the present work. The minimum inhibitory concentration (MIC) values for hinokinin ranged from 0.0485 to 3.125 mg/mL in different S. aureus clinical isolates and showed a bactericidal activity against MRSA isolated from blood (MMC 0.40 mg/mL) and postoperative secretion (MMC = 3.125 mg/mL). C. leptophloeos extracts also showed antimicrobial activity against Mycobacterium species such as M. smegmatis (MIC = 12.5 mg/mL) and M. tuberculosis (MIC = 52 mg/mL). Additionally, we determined the toxicity of C. leptophloeos by in vitro HC50 tests with hemolytic activity detected of 313 ± 0.5 μg/mL. Our results showed that C. leptophloeos possesses inhibitory properties against MRSA as well as several other clinically important microorganisms. Furthermore, the present work is the first report of the presence of hinokinin in Commiphora genus. PMID:28174564

In vitro α-glucosidase inhibition, antioxidant, anticancer, and antimycobacterial properties of ethyl acetate extract of Aegle tamilnadensis Abdul Kader (Rutaceae) leaf.

PubMed

R, Pratap Chandran; S, Nishanth Kumar; S, Manju; S, Abdul Kader; B S, Dileep Kumar

2015-01-01

The present study was aimed to investigate in vitro α-glucosidase inhibition, antioxidant, anticancer, and antimycobacterial activities of the ethyl acetate extract of A. tamilnadensis leaves. The extract recorded strong α-glucosidase inhibition with an IC50 value of 100 μg/ml. The antioxidant potential of the extract was evaluated by nitric oxide radical inhibition, lipid peroxidation inhibition, ferric thiocyanate, and ABTS radical scavenging assay, and the extract recorded significant antioxidant activity. The ferric thiocyanate activity of extract was superior to butylated hydroxyl anisol (BHA), the standard antioxidant agent. The anticancer activity of the extract was evaluated against (1) breast cancer cell lines (MDAM B-231), (2) cervical cancer cell lines (HeLa), and (3) lung cancer cell line (A 549) using MTT assay, and significant activity was recorded against A 549 with an IC50 value of 64 μg/ml. Further studies on the morphology, acridine orange/ethidium bromide staining, and cell cycle analysis by flow cytometry confirm the extract-induced apoptosis in A 549. This extract also recorded significant anti-tuberculosis activity against Mycobacterium smegmatis. The current study suggests that the ethyl acetate extract of A. tamilnadensis is a potential source of natural α-glucosidase inhibitor and antioxidant for protection as well as prevention of life-threatening diseases like cancer.

CASL VMA FY16 Milestone Report (L3:VMA.VUQ.P13.07) Westinghouse Mixing with COBRA-TF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gordon, Natalie

2016-09-30

COBRA-TF (CTF) is a low-resolution code currently maintained as CASL's subchannel analysis tool. CTF operates as a two-phase, compressible code over a mesh comprised of subchannels and axial discretized nodes. In part because CTF is a low-resolution code, simulation run time is not computationally expensive, only on the order of minutes. Hi-resolution codes such as STAR-CCM+ can be used to train lower-fidelity codes such as CTF. Unlike STAR-CCM+, CTF has no turbulence model, only a two-phase turbulent mixing coefficient, β. β can be set to a constant value or calculated in terms of Reynolds number using an empirical correlation. Resultsmore » from STAR-CCM+ can be used to inform the appropriate value of β. Once β is calibrated, CTF runs can be an inexpensive alternative to costly STAR-CCM+ runs for scoping analyses. Based on the results of CTF runs, STAR-CCM+ can be run for specific parameters of interest. CASL areas of application are CIPS for single phase analysis and DNB-CTF for two-phase analysis.« less

Curcumin inhibits vasculogenic mimicry through the downregulation of erythropoietin-producing hepatocellular carcinoma-A2, phosphoinositide 3-kinase and matrix metalloproteinase-2

PubMed Central

LIANG, YIMING; HUANG, MIN; LI, JIANWEN; SUN, XINLIN; JIANG, XIAODAN; LI, LIANGPING; KE, YIQUAN

2014-01-01

Glioblastomas (GBMs) are the most common and aggressive malignant primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry (VM) is often detected. VM is the formation of de novo vascular networks by highly invasive tumor cells, instead of endothelial cells. An understanding of the mechanisms of VM formation will contribute to the targeted therapy of GBMs. In the present study, the efficacy of curcumin (CCM) on VM formation and its mechanisms were investigated. It was found that CCM inhibits the VM formation, proliferation, migration and invasion of human glioma U251 cells in a dose-dependent manner. Furthermore, CCM downregulated the protein and mRNA expression of erythropoietin-producing hepatocellular carcinoma-A2, phosphoinositide 3-kinase and matrix metalloproteinase-2, indicating that CCM may function through these factors for the inhibition of VM formation. These data provide novel insights into the use of CCM to antagonize VM, and may contribute to the angiogenesis-targeted therapy of malignant glioma. PMID:25202424

Heg1 and Ccm1/2 proteins control endocardial mechanosensitivity during zebrafish valvulogenesis

PubMed Central

Otten, Cécile; Renz, Marc

2018-01-01

Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology. PMID:29364115

Quality of care in integrated community case management services in Bugoye, Uganda: a retrospective observational study.

PubMed

Miller, James S; English, Lacey; Matte, Michael; Mbusa, Rapheal; Ntaro, Moses; Bwambale, Shem; Kenney, Jessica; Siedner, Mark J; Reyes, Raquel; Lee, Patrick T; Mulogo, Edgar; Stone, Geren S

2018-02-27

Village health workers (VHWs) in five villages in Bugoye subcounty (Kasese District, Uganda) provide integrated community case management (iCCM) services, in which VHWs evaluate and treat malaria, pneumonia, and diarrhoea in children under 5 years of age. VHWs use a "Sick Child Job Aid" that guides them through the evaluation and treatment of these illnesses. A retrospective observational study was conducted to measure the quality of iCCM care provided by 23 VHWs in 5 villages in Bugoye subcounty over a 2-year period. Patient characteristics and clinical services were summarized using existing aggregate programme data. Lot quality assurance sampling of individual patient records was used to estimate adherence to the iCCM algorithm, VHW-level quality (based on adherence to the iCCM protocol), and change over time in quality of care (using generalized estimating equations regression modelling). For each of 23 VHWs, 25 patient visits were randomly selected from a 2-year period after iCCM care initiation. In these visits, 97% (150) of patients with diarrhoea were treated with oral rehydration and zinc, 95% (216) of patients with pneumonia were treated with amoxicillin, and 94% (240) of patients with malaria were treated with artemisinin-based combination therapy or rectal artesunate. However, only 44% (44) of patients with a negative rapid test for malaria were appropriately referred to a health facility. Overall, 75% (434) of patients received all the correct evaluation and management steps. Only 9 (39%) of the 23 VHWs met the pre-determined LQAS threshold for high-quality care over the 2-year observation period. Quality of care increased significantly in the first 6 months after initiation of iCCM services (p = 0.003), and then plateaued during months 7-24. Quality of care was high for uncomplicated malaria, pneumonia and diarrhoea. Overall quality of care was lower, in part because VHWs often did not follow the guidelines to refer patients with fever who tested negative for malaria. Quality of care appears to improve in the initial months after iCCM implementation, as VHWs gain initial experience in iCCM care.

The added value of a mobile application of Community Case Management on referral, re-consultation and hospitalization rates of children aged under 5 years in two districts in Northern Malawi: study protocol for a pragmatic, stepped-wedge cluster-randomized controlled trial.

PubMed

Hardy, Victoria; O'Connor, Yvonne; Heavin, Ciara; Mastellos, Nikolaos; Tran, Tammy; O'Donoghue, John; Fitzpatrick, Annette L; Ide, Nicole; Wu, Tsung-Shu Joseph; Chirambo, Griphin Baxter; Muula, Adamson S; Nyirenda, Moffat; Carlsson, Sven; Andersson, Bo; Thompson, Matthew

2017-10-11

There is evidence to suggest that frontline community health workers in Malawi are under-referring children to higher-level facilities. Integrating a digitized version of paper-based methods of Community Case Management (CCM) could strengthen delivery, increasing urgent referral rates and preventing unnecessary re-consultations and hospital admissions. This trial aims to evaluate the added value of the Supporting LIFE electronic Community Case Management Application (SL eCCM App) compared to paper-based CCM on urgent referral, re-consultation and hospitalization rates, in two districts in Northern Malawi. This is a pragmatic, stepped-wedge cluster-randomized trial assessing the added value of the SL eCCM App on urgent referral, re-consultation and hospitalization rates of children aged 2 months and older to up to 5 years, within 7 days of the index visit. One hundred and two health surveillance assistants (HSAs) were stratified into six clusters based on geographical location, and clusters randomized to the timing of crossover to the intervention using simple, computer-generated randomization. Training workshops were conducted prior to the control (paper-CCM) and intervention (paper-CCM + SL eCCM App) in assigned clusters. Neither participants nor study personnel were blinded to allocation. Outcome measures were determined by abstraction of clinical data from patient records 2 weeks after recruitment. A nested qualitative study explored perceptions of adherence to urgent referral recommendations and a cost evaluation determined the financial and time-related costs to caregivers of subsequent health care utilization. The trial was conducted between July 2016 and February 2017. This is the first large-scale trial evaluating the value of adding a mobile application of CCM to the assessment of children aged under 5 years. The trial will generate evidence on the potential use of mobile health for CCM in Malawi, and more widely in other low- and middle-income countries. ClinicalTrials.gov, ID: NCT02763345 . Registered on 3 May 2016.

Cerebral Cavernous Malformation

MedlinePlus

... and individuals frequently have multiple CCMs found via magnetic resonance imaging. Individuals with CCM are faced with a diagnosis ... and individuals frequently have multiple CCMs found via magnetic resonance imaging. Individuals with CCM are faced with a diagnosis ...

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease.

PubMed

Girard, Romuald; Zeineddine, Hussein A; Orsbon, Courtney; Tan, Huan; Moore, Thomas; Hobson, Nick; Shenkar, Robert; Lightle, Rhonda; Shi, Changbin; Fam, Maged D; Cao, Ying; Shen, Le; Neander, April I; Rorrer, Autumn; Gallione, Carol; Tang, Alan T; Kahn, Mark L; Marchuk, Douglas A; Luo, Zhe-Xi; Awad, Issam A

2016-09-15

Cerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. We hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. Twenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. Serial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r(2)=0.84, p<0.0001). Micro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel functions of CCM1 delimit the relationship of PTB/PH domains.

PubMed

Zhang, Jun; Dubey, Pallavi; Padarti, Akhil; Zhang, Aileen; Patel, Rinkal; Patel, Vipulkumar; Cistola, David; Badr, Ahmed

2017-10-01

Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. A new β1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and relationship of PTB, PH and FERM domains has been proposed, which extends the importance of the NPXY-PTB/PH interaction on the CSC signaling and/or other cell receptors with great potential pointing to new therapeutic strategies. The study provides new insight into the structural characteristics of PTB/PH domains, essential structural elements of PTB/PH domain required for NPXY motif-binding, and function and relationship among PTB, PH and FERM domains. Copyright © 2017 Elsevier B.V. All rights reserved.

The association between chronic care management and the quality of thrombosis care

PubMed Central

Drewes, H.W.; Baan, C.A.; Westert, G.P.; Meijboom, B.R.; Lambooij, M.

2010-01-01

Introduction The oral anticoagulant therapy (OAT), used to prevent thrombosis, is associated with substantial avoidable hospitalization. Aim Identify the associations between chronic care management and the quality of OAT as suggested by the chronic care model (CCM) of Wagner. Methods Regression analysis with data of 61 thrombosis clinics and inductive analysis with 63 interviews with health care professionals of 23 thrombosis clinics. Results Results show substantial differences between regions in the quality of thrombosis care and the CCM activities. However, the variation in quality of care was not associated with the differences in CCM activities. The inductive analysis indicates that there are problems in the cooperation between caregivers. Several preferred CCM activities (e.g., multidisciplinary protocol) as well as the barriers to implement these activities (e.g., conflicting interests) were put forward by the health care professionals. Conclusion It can be concluded that there is variation in quality of thrombosis care between regions. This variation could not be explained by the observed differences in CCM activities. However, fragmentation is a major source of inefficiency according to health care professionals. The paper concludes with suggestions to improve chronic care management for thrombosis.

Flyback CCM inverter for AC module applications: iterative learning control and convergence analysis

NASA Astrophysics Data System (ADS)

Lee, Sung-Ho; Kim, Minsung

2017-12-01

This paper presents an iterative learning controller (ILC) for an interleaved flyback inverter operating in continuous conduction mode (CCM). The flyback CCM inverter features small output ripple current, high efficiency, and low cost, and hence it is well suited for photovoltaic power applications. However, it exhibits the non-minimum phase behaviour, because its transfer function from control duty to output current has the right-half-plane (RHP) zero. Moreover, the flyback CCM inverter suffers from the time-varying grid voltage disturbance. Thus, conventional control scheme results in inaccurate output tracking. To overcome these problems, the ILC is first developed and applied to the flyback inverter operating in CCM. The ILC makes use of both predictive and current learning terms which help the system output to converge to the reference trajectory. We take into account the nonlinear averaged model and use it to construct the proposed controller. It is proven that the system output globally converges to the reference trajectory in the absence of state disturbances, output noises, or initial state errors. Numerical simulations are performed to validate the proposed control scheme, and experiments using 400-W AC module prototype are carried out to demonstrate its practical feasibility.

Critical Care Medicine and Infectious Diseases: An Emerging Combined Subspecialty in the United States.

PubMed

Kadri, Sameer S; Rhee, Chanu; Fortna, Gregory S; O'Grady, Naomi P

2015-08-15

The recent rise in unfilled training positions among infectious diseases (ID) fellowship programs nationwide indicates that ID is declining as a career choice among internal medicine residency graduates. Supplementing ID training with training in critical care medicine (CCM) might be a way to regenerate interest in the specialty. Hands-on patient care and higher salaries are obvious attractions. High infection prevalence and antibiotic resistance in intensive care units, expanding immunosuppressed host populations, and public health crises such as the recent Ebola outbreak underscore the potential synergy of CCM-ID training. Most intensivists receive training in pulmonary medicine and only 1% of current board-certified intensivists are trained in ID. While still small, this cohort of CCM-ID certified physicians has continued to rise over the last 2 decades. ID and CCM program leadership nationwide must recognize these trends and the merits of the CCM-ID combination to facilitate creation of formal dual-training opportunities. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Comparison of manually produced and automated cross country movement maps using digital image processing techniques

NASA Technical Reports Server (NTRS)

Wynn, L. K.

1985-01-01

The Image-Based Information System (IBIS) was used to automate the cross country movement (CCM) mapping model developed by the Defense Mapping Agency (DMA). Existing terrain factor overlays and a CCM map, produced by DMA for the Fort Lewis, Washington area, were digitized and reformatted into geometrically registered images. Terrain factor data from Slope, Soils, and Vegetation overlays were entered into IBIS, and were then combined utilizing IBIS-programmed equations to implement the DMA CCM model. The resulting IBIS-generated CCM map was then compared with the digitized manually produced map to test similarity. The numbers of pixels comprising each CCM region were compared between the two map images, and percent agreement between each two regional counts was computed. The mean percent agreement equalled 86.21%, with an areally weighted standard deviation of 11.11%. Calculation of Pearson's correlation coefficient yielded +9.997. In some cases, the IBIS-calculated map code differed from the DMA codes: analysis revealed that IBIS had calculated the codes correctly. These highly positive results demonstrate the power and accuracy of IBIS in automating models which synthesize a variety of thematic geographic data.

Antimycobacterial activity of medicinal plants used by the Mayo people of Sonora, Mexico.

PubMed

Coronado-Aceves, Enrique Wenceslao; Sánchez-Escalante, José Jesús; López-Cervantes, Jaime; Robles-Zepeda, Ramón Enrique; Velázquez, Carlos; Sánchez-Machado, Dalia Isabel; Garibay-Escobar, Adriana

2016-08-22

Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), which generates 9 million new cases worldwide each year. The Mayo ethnicity of southern Sonora, Mexico is more than 2000 years old, and the Mayos possess extensive knowledge of traditional medicine. To evaluate the antimycobacterial activity levels of extracts of medicinal plants used by the Mayos against Mtb and Mycobacterium smegmatis (Msm) in the treatment of TB, respiratory diseases and related symptoms. A total of 34 plant species were collected, and 191 extracts were created with n-hexane, dichloromethane, ethyl acetate (EtOAc), methanol and water. Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against Mtb H37Rv using the microplate alamar blue assay (MABA) and against Msm using the resazurin microplate assay (REMA) at 6 and 2 days of exposure, respectively, and at concentrations of 250-1.9µg/mL (n-hexane extracts) and 1000-7.81µg/mL (extracts obtained with dichloromethane, EtOAc, methanol and water). Rhynchosia precatoria (Willd.) DC. (n-hexane root extract), Euphorbia albomarginata Torr. and A. Gray. (EtOAc shoot extract) and Helianthus annuus L. (n-hexane stem extract) were the most active plants against Mtb H37Rv, with MICs of 15.6, 250, 250µg/mL and MBCs of 31.25, 250, 250µg/mL, respectively. R. precatoria (root) was the only active plant against Msm, with MIC and MBC values of ≥250µg/mL. None of the aqueous extracts were active. This study validates the medicinal use of certain plants used by the Mayo people in the treatment of TB and related symptoms. R. precatoria, E. albomarginata and H. annuus are promising plant sources of active compounds that act against Mtb H37Rv. To our knowledge, this is the first time that their antimycobacterial activity has been reported. Crude extracts obtained with n-hexane, EtOAc and dichloromethane were the most active against Mtb H37Rv. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Facilitators and barriers of implementing the chronic care model in primary care: a systematic review.

PubMed

Kadu, Mudathira K; Stolee, Paul

2015-02-06

The Chronic Care Model (CCM) is a framework developed to redesign care delivery for individuals living with chronic diseases in primary care. The CCM and its various components have been widely adopted and evaluated, however, little is known about different primary care experiences with its implementation, and the factors that influence its successful uptake. The purpose of this review is to synthesize findings of studies that implemented the CCM in primary care, in order to identify facilitators and barriers encountered during implementation. This study identified English-language, peer-reviewed research articles, describing the CCM in primary care settings. Searches were performed in three data bases: Web of Knowledge, Pubmed and Scopus. Article abstracts and titles were read based on whether they met the following inclusion criteria: 1) studies published after 2003 that described or evaluated the implementation of the CCM; 2) the care setting was primary care; 3) the target population of the study was adults over the age of 18 with chronic conditions. Studies were categorized by reference, study design and methods, participants and setting, study objective, CCM components used, and description of the intervention. The next stage of data abstraction involved qualitative analysis of cited barriers and facilitators using the Consolidating Framework for Research Implementation. This review identified barriers and facilitators of implementation across various primary care settings in 22 studies. The major emerging themes were those related to the inner setting of the organization, the process of implementation and characteristics of the individual healthcare providers. These included: organizational culture, its structural characteristics, networks and communication, implementation climate and readiness, presence of supportive leadership, and provider attitudes and beliefs. These findings highlight the importance of assessing organizational capacity and needs prior to and during the implementation of the CCM, as well as gaining a better understanding of health care providers' and organizational perspective.

Cardiac contractility modulation in heart failure patients: Randomized comparison of signal delivery through one vs. two ventricular leads.

PubMed

Röger, Susanne; Said, Samir; Kloppe, Axel; Lawo, Thomas; Emig, Ulf; Rousso, Benny; Gutterman, David; Borggrefe, Martin; Kuschyk, Jürgen

2017-01-01

Cardiac contractility modulation (CCM) is an electrical stimulation treatment for symptomatic heart failure (HF) patients. The procedure involves implantation of two ventricular leads for delivery of CCM impulses. The purpose of this study is to compare the efficacy and safety of CCM when the signal is delivered through one vs. two ventricular leads. This prospective blinded randomized trial enrolled 48 patients. Eligible subjects had symptoms despite optimal HF medications, left ventricular ejection fraction <40% and peakVO 2 ≥9ml O 2 /kg/min. All patients received a CCM system with two ventricular leads, and were randomized to CCM active through both or just one ventricular lead; 25 patients were randomized to receive signal delivery through two leads (Group A) and 23 patients to signal delivery through one lead (Group B). The study compared the mean changes from baseline to 6 months follow-up in peakVO 2 , New York Heart Association (NYHA) classification, and quality of life (by MLWHFQ). Following 6 months, similar and significant (p<0.05) improvements from baseline in NYHA (-0.7±0.5 vs. -0.9±0.7) and MLWHFQ (-14±20 vs. -16±22) were observed in Group A and in Group B. PeakVO 2 showed improvement trends in both groups (0.34±1.52 vs. 0.10±2.21ml/kg/min; p=ns). No patient died. Serious adverse event rates (20 events in 10 subjects) were not different between groups. No statistically significant difference was found in any of the study endpoints. The efficacy and safety of CCM in this study were similar when the signal was delivered through either one or two ventricular leads. These results support the potential use of a single ventricular lead for delivery of CCM. Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Nationwide implementation of integrated community case management of childhood illness in Rwanda

PubMed Central

Mugeni, Catherine; Levine, Adam C; Munyaneza, Richard M; Mulindahabi, Epiphanie; Cockrell, Hannah C; Glavis-Bloom, Justin; Nutt, Cameron T; Wagner, Claire M; Gaju, Erick; Rukundo, Alphonse; Habimana, Jean Pierre; Karema, Corine; Ngabo, Fidele; Binagwaho, Agnes

2014-01-01

ABSTRACT Background: Between 2008 and 2011, Rwanda introduced integrated community case management (iCCM) of childhood illness nationwide. Community health workers in each of Rwanda's nearly 15,000 villages were trained in iCCM and equipped for empirical diagnosis and treatment of pneumonia, diarrhea, and malaria; for malnutrition surveillance; and for comprehensive reporting and referral services. Methods: We used data from the Rwanda health management information system (HMIS) to calculate monthly all-cause under-5 mortality rates, health facility use rates, and community-based treatment rates for childhood illness in each district. We then compared a 3-month baseline period prior to iCCM implementation with a seasonally matched comparison period 1 year after iCCM implementation. Finally, we compared the actual changes in all-cause child mortality and health facility use over this time period with the changes that would have been expected based on baseline trends in Rwanda. Results: The number of children receiving community-based treatment for diarrhea and pneumonia increased significantly in the 1-year period after iCCM implementation, from 0.83 cases/1,000 child-months to 3.80 cases/1,000 child-months (P = .01) and 0.25 cases/1,000 child-months to 5.28 cases/1,000 child-months (P<.001), respectively. On average, total under-5 mortality rates declined significantly by 38% (P<.001), and health facility use declined significantly by 15% (P = .006). These decreases were significantly greater than would have been expected based on baseline trends. Conclusions: This is the first study to demonstrate decreases in both child mortality and health facility use after implementing iCCM of childhood illness at a national level. While our study design does not allow for direct attribution of these changes to implementation of iCCM, these results are in line with those of prior studies conducted at the sub-national level in other low-income countries. PMID:25276592

Access to integrated community case management of childhood illnesses services in rural Ethiopia: a qualitative study of the perspectives and experiences of caregivers

PubMed Central

Shaw, Bryan; Amouzou, Agbessi; Miller, Nathan P; Tafesse, Mengistu; Bryce, Jennifer; Surkan, Pamela J

2016-01-01

Background: In 2010, Ethiopia began scaling up the integrated community case management (iCCM) of childhood illness strategy throughout the country allowing health extension workers (HEWs) to treat children in rural health posts. After 2 years of iCCM scale up, utilization of HEWs remains low. Little is known about factors related to the use of health services in this setting. This research aimed to elicit perceptions and experiences of caregivers to better understand reasons for low utilization of iCCM services. Methods: A rapid ethnographic assessment was conducted in eight rural health post catchment areas in two zones: Jimma and West Hararghe. In total, 16 focus group discussions and 78 in-depth interviews were completed with mothers, fathers, HEWs and community health volunteers. Results: In spite of the HEW being a core component of iCCM, we found that the lack of availability of HEWs at the health post was one of the most common barriers to the utilization of iCCM services mentioned by caregivers. Financial and geographic challenges continue to influence caregiver decisions despite extension of free child health services in communities. Acceptability of HEWs was often low due to a perceived lack of sensitivity of HEWs and concerns about medicines given at the health post. Social networks acted both to facilitate and hinder use of HEWs. Many mothers stated a preference for using the health post, but some were unable to do so due to objections or alternative care-seeking preferences of gatekeepers, often mothers-in-law and husbands. Conclusion: Caregivers in Ethiopia face many challenges in using HEWs at the health post, potentially resulting in low demand for iCCM services. Efforts to minimize barriers to care seeking and to improve demand should be incorporated into the iCCM strategy in order to achieve reductions in child mortality and promote equity in access and child health outcomes. PMID:26608585

Does the chronic care model meet the emerging needs of people living with multimorbidity? A systematic review and thematic synthesis

PubMed Central

Abu Dabrh, Abd Moain; Gionfriddo, Michael R.; Erwin, Patricia; Montori, Victor M.

2018-01-01

Background The Chronic Care Model (CCM) emerged in the 1990s as an approach to re-organize primary care and implement critical elements that enable it to proactively attend to patients with chronic conditions. The chronic care landscape has evolved further, as most patients now present with multiple chronic conditions and increasing psychosocial complexity. These patients face accumulating and overwhelming complexity resulting from the sum of uncoordinated responses to each of their problems. Minimally Disruptive Medicine (MDM) was proposed to respond to this challenge, aiming at improving outcomes that matter to patients with the smallest burden of treatment. We sought to critically appraise the extent to which MDM constructs (e.g., reducing patient work, improving patients’ capacity) have been adopted within CCM implementations. Methods We conducted a systematic review and qualitative thematic synthesis of reports of CCM implementations published from 2011–2016. Results CCM implementations were mostly aligned with the healthcare system’s goals, condition-specific, and targeted disease-specific outcomes or healthcare utilization. No CCM implementation addressed patient work. Few reduced treatment workload without adding additional tasks. Implementations supported patient capacity by offering information, but rarely offered practical resources (e.g., financial assistance, transportation), helped patients reframe their biography with chronic illness, or assisted them in engaging with a supportive social network. Few implementations aimed at improving functional status or quality of life, and only one-third of studies were targeted for patients of low socioeconomic status. Conclusion MDM provides a lens to operationalize how to care for patients with multiple chronic conditions, but its constructs remain mostly absent from how implementations of the CCM are currently reported. Improvements to the primary care of patients with multimorbidity may benefit from the application of MDM, and the current CCM implementations that do apply MDM constructs should be considered exemplars for future implementation work. PMID:29420543

A multi-institutional quality improvement initiative to transform education for chronic illness care in resident continuity practices.

PubMed

Stevens, David P; Bowen, Judith L; Johnson, Julie K; Woods, Donna M; Provost, Lloyd P; Holman, Halsted R; Sixta, Constance S; Wagner, Ed H

2010-09-01

There is a gap between the need for patient-centered, evidence-based primary care for the large burden of chronic illness in the US, and the training of resident physicians to provide that care. To improve training for residents who provide chronic illness care in teaching practice settings. US teaching hospitals were invited to participate in one of two 18-month Breakthrough Series Collaboratives-either a national Collaborative, or a subsequent California Collaborative-to implement the Chronic Care Model (CCM) and related curriculum changes in resident practices. Most practices focused on patients with diabetes mellitus. Educational redesign strategies with related performance measures were developed for curricular innovations anchored in the CCM. In addition, three clinical measures-HbA1c <7%, LDL <100 mg/dL, and blood pressure

A Multi-Institutional Quality Improvement Initiative to Transform Education for Chronic Illness Care in Resident Continuity Practices

PubMed Central

Bowen, Judith L.; Johnson, Julie K.; Woods, Donna M.; Provost, Lloyd P.; Holman, Halsted R.; Sixta, Constance S.; Wagner, Ed H.

2010-01-01

BACKGROUND There is a gap between the need for patient-centered, evidence-based primary care for the large burden of chronic illness in the US, and the training of resident physicians to provide that care. OBJECTIVE To improve training for residents who provide chronic illness care in teaching practice settings. DESIGN US teaching hospitals were invited to participate in one of two 18-month Breakthrough Series Collaboratives—either a national Collaborative, or a subsequent California Collaborative—to implement the Chronic Care Model (CCM) and related curriculum changes in resident practices. Most practices focused on patients with diabetes mellitus. Educational redesign strategies with related performance measures were developed for curricular innovations anchored in the CCM. In addition, three clinical measures—HbA1c <7%, LDL <100 mg/dL, and blood pressure ≤130/80—and three process measures—retinal and foot examinations, and patient self-management goals—were tracked. PARTICIPANTS Fifty-seven teams from 37 self-selected teaching hospitals committed to implement the CCM in resident continuity practices; 41 teams focusing on diabetes improvement participated over the entire duration of one of the Collaboratives. INTERVENTIONS Teaching-practice teams—faculty, residents and staff—participated in Collaboratives by attending monthly calls and regular 2-day face-to-face meetings with the other teams. The national Collaborative faculty led calls and meetings. Each team used rapid cycle quality improvement (PDSA cycles) to implement the CCM and curricular changes. Teams reported education and clinical performance measures monthly. RESULTS Practices underwent extensive redesign to establish CCM elements. Education measures tracked substantial development of CCM-related learning. The clinical and process measures improved, however inconsistently, during the Collaboratives. CONCLUSIONS These initiatives suggest that systematic practice redesign for implementing the CCM along with linked educational approaches are achievable in resident continuity practices. Improvement of clinical outcomes in such practices is daunting but achievable. PMID:20737232

Improvement to the Convergence-Confinement Method: Inclusion of Support Installation Proximity and Stiffness

NASA Astrophysics Data System (ADS)

Oke, Jeffrey; Vlachopoulos, Nicholas; Diederichs, Mark

2018-05-01

The convergence-confinement method (CCM) is a method that has been introduced in tunnel construction that considers the ground response to the advancing tunnel face and the interaction with installed support. One limitation of the CCM is due to the numerically or empirically driven nature of the longitudinal displacement profile and the incomplete consideration of the longitudinal arching effect that occurs during tunnelling operations as part of the face effect. In this paper, the authors address the issue associated with when the CCM is used within squeezing ground conditions at depth. Based on numerical analysis, the authors have proposed a methodology and solution to improving the CCM in order to allow for more accurate results for squeezing ground conditions for three different excavation cases involving various excavation-support increments and distances from the face to the supported front. The tunnelling methods of consideration include: tunnel boring machine, mechanical (conventional), and drill and blast.

Is the Chronic Care Model Integrated Into Research Examining Culturally Competent Interventions for Ethnically Diverse Adults With Type 2 Diabetes Mellitus? A Review.

PubMed

Dauvrin, Marie; Lorant, Vincent; d'Hoore, William

2015-12-01

The chronic care model (CCM) concerns both the medical and the cultural and linguistic needs of patients through the inclusion of cultural competence in the delivery system design. This literature review attempted to@@ identify the domains of the CCM culturally competent (CC) interventions that the adults from ethnic minorities suffering from type 2 diabetes mellitus report. We identified the CCM and the CC components in the relevant studies published between 2005 and 2014. Thirty-two studies were included. Thirty-one articles focused on self-management and 20 on community resources. Twenty-three interventions integrated cultural norms from the patients' backgrounds. CC interventions reported the CCM at the individual level but need to address the organizational level more effectively. The scope of CC interventions should be expanded to transform health care organizations and systems. © The Author(s) 2015.

A randomized controlled trial to evaluate the safety and efficacy of cardiac contractility modulation in patients with systolic heart failure: rationale, design, and baseline patient characteristics.

PubMed

Abraham, William T; Burkhoff, Daniel; Nademanee, Koonlawee; Carson, Peter; Bourge, Robert; Ellenbogen, Kenneth A; Parides, Michael; Kadish, Alan

2008-10-01

Cardiac contractility modulation (CCM) signals are nonexcitatory electrical signals delivered during the cardiac absolute refractory period that enhance the strength of cardiac muscular contraction. Prior research in experimental and human heart failure has shown that CCM signals normalize phosphorylation of key proteins and expression of genes coding for proteins involved in regulation of calcium cycling and contraction. The results of prior clinical studies of CCM have supported its safety and efficacy. A large-scale clinical study, the FIX-HF-5 study, is currently underway to test the safety and efficacy of this treatment. In this article, we provide an overview of the system used to deliver CCM signals, the implant procedure, and the details and rationale of the FIX-HF-5 study design. Baseline characteristics for patients randomized in this trial are also presented.

Present-day Antarctic climatology of the NCAR Community Climate Model Version 1

NASA Technical Reports Server (NTRS)

Tzeng, Ren-Yow; Bromwich, David H.; Parish, Thomas R.

1993-01-01

The ability of the NCAR Community Climate Model Version 1 (CCM1) with R 15 resolution to simulate the present-day climate of Antarctica was evaluated using the five-year seasonal cycle output produced by the CCM1 and comparing the model results with observed horizontal syntheses and point data. The results showed that the CCM1 with R 15 resolution can simulate to some extent the dynamics of Antarctic climate on the synoptic scale as well as some mesoscale features. The model can also simulate the phase and the amplitude of the annual and semiannual variation of the temperature, sea level pressure, and zonally averaged zonal (E-W) wind. The main shortcomings of the CCM1 model are associated with the model's anomalously large precipitation amounts at high latitudes, due to the tendency of the scheme to suppress negative moisture values.

Applying the chronic care model to an employee benefits program: a qualitative inquiry.

PubMed

Schauer, Gillian L; Wilson, Mark; Barrett, Barbara; Honeycutt, Sally; Hermstad, April K; Kegler, Michelle C

2013-12-01

To assess how employee benefits programs may strengthen and/or complement elements of the chronic care model (CCM), a framework used by health systems to improve chronic illness care. A qualitative inquiry consisting of semi-structured interviews with employee benefit administrators and partners from a self-insured, self-administered employee health benefits program was conducted at a large family-owned business in southwest Georgia. Results indicate that the employer adapted and used many health system-related elements of the CCM in the design of their benefit program. Data also suggest that the employee benefits program contributed to self-management skills and to informing and activating patients to interact with the health system. Findings suggest that employee benefits programs can use aspects of the CCM in their own benefit design, and can structure their benefits to contribute to patient-related elements from the CCM.

Final report on the key comparison, CCM.M.P-K15.1 in the pressure range from 1.0 × 10-4 Pa to 1.0 Pa

NASA Astrophysics Data System (ADS)

Wuethrich, Christian; Hongyan, Yu

2017-01-01

At the CCM WG-LP meeting in Berlin in April 2011 the National Institute of Metrology of the people's republic of China asked for an opportunity to be linked to the CCM.M.P-K15 comparison which just ended. METAS, which had been active as pilot for the CCM.M.P-K15, agreed to make a link with NIM. The NIM introduced a new static expansion system in his laboratory. Two spinning rotor gauges and a control electronic are used as transfer standard. The circulation of the transfer standard was made in spring 2012. The drift of the accommodation coefficient of the two SRG was measured by the pilot before and after the comparison. The stability of the transfer standards was excellent and the equivalence between METAS and NIM has been demonstrated. A link to the CCM.M.P­K15 has been established using the results from METAS. It has been possible to achieve a link with a small uncertainty due to the strong correlation of the measurements of METAS in the two comparisons. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Factors affecting the operating time for complete cyst excision and Roux-en-Y hepaticojejunostomy in paediatric cases of congenital choledochal malformation: a retrospective case study in Southeast China

PubMed Central

Guo, Wan-liang; Zhan, Yang; Fang, Fang; Deng, Yan-bing; Zhao, Jun-gang

2018-01-01

Objective The aim of this study was to evaluate factors affecting the operating time for complete cyst excision and Roux-en-Y hepaticojejunostomy in paediatric cases of congenital choledochal malformation (CCM). Design A 3-year retrospective study was undertaken between January 2013 and December 2015 in four centres in China. Setting This involved a retrospective chart review of paediatric patients with CCM in four large hospitals in Southeast China. Participants Sixty-five paediatric patients with CCM were included in this study. We derived all available information on patient demographics, clinical characteristics, preoperative complications and surgical methods from the charts of all these patients. Interventions Univariate and multivariate logistic regression analyses were used to evaluate factors significantly affecting the operating time for complete cyst excision and Roux-en-Y hepaticojejunostomy in paediatric cases of CCM. Results Twenty-three of the 65 case surgeries were performed using laparoscopic technique, and 42 surgeries were performed by conventional open surgery. The median operating time was 215 min (range 120–430 min). The morphological subtype of CCM and the presence of cholecystitis or cholangitis were the only factors found to affect the operating time (p<0.05). Logistic regression analysis confirmed cholangitis as an independent risk factor. Conclusions The morphological subtype of CMM and the presence of cholecystitis or cholangitis are factors affecting the operating time for complete cyst excision and Roux-en-Y hepaticojejunostomy in paediatric cases of CCM, whereas cholangitis is an independent risk factor. PMID:29804066

Cost-effectiveness analysis of the national implementation of integrated community case management and community-based health planning and services in Ghana for the treatment of malaria, diarrhoea and pneumonia.

PubMed

Escribano Ferrer, Blanca; Hansen, Kristian Schultz; Gyapong, Margaret; Bruce, Jane; Narh Bana, Solomon A; Narh, Clement T; Allotey, Naa-Korkor; Glover, Roland; Azantilow, Naa-Charity; Bart-Plange, Constance; Sagoe-Moses, Isabella; Webster, Jayne

2017-07-05

Ghana has developed two main community-based strategies that aim to increase access to quality treatment for malaria, diarrhoea and suspected pneumonia: the integrated community case management (iCCM) and the community-based health planning and services (CHPS). The aim of the study was to assess the cost-effectiveness of these strategies under programme conditions. A cost-effectiveness analysis was conducted. Appropriate diagnosis and treatment given was the effectiveness measure used. Appropriate diagnosis and treatment data was obtained from a household survey conducted 2 and 8 years after implementation of iCCM in the Volta and Northern Regions of Ghana, respectively. The study population was carers of children under-5 years who had fever, diarrhoea and/or cough in the last 2 weeks prior to the interview. Costs data was obtained mainly from the National Malaria Control Programme (NMCP), the Ministry of Health, CHPS compounds and from a household survey. Appropriate diagnosis and treatment of malaria, diarrhoea and suspected pneumonia was more cost-effective under the iCCM than under CHPS in the Volta Region, even after adjusting for different discount rates, facility costs and iCCM and CHPS utilization, but not when iCCM appropriate treatment was reduced by 50%. Due to low numbers of carers visiting a CBA in the Northern Region it was not possible to conduct a cost-effectiveness analysis in this region. However, the cost analysis showed that iCCM in the Northern Region had higher cost per malaria, diarrhoea and suspected pneumonia case diagnosed and treated when compared to the Volta Region and to the CHPS strategy in the Northern Region. Integrated community case management was more cost-effective than CHPS for the treatment of malaria, diarrhoea and suspected pneumonia when utilized by carers of children under-5 years in the Volta Region. A revision of the iCCM strategy in the Northern Region is needed to improve its cost-effectiveness. Long-term financing strategies should be explored including potential inclusion in the National Health Insurance Scheme (NHIS) benefit package. An acceptability study of including iCCM in the NHIS should be conducted.

Description of the NCAR Community Climate Model (CCM3). Technical note

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiehl, J.T.; Hack, J.J.; Bonan, G.B.

This repor presents the details of the governing equations, physical parameterizations, and numerical algorithms defining the version of the NCAR Community Climate Model designated CCM3. The material provides an overview of the major model components, and the way in which they interact as the numerical integration proceeds. This version of the CCM incorporates significant improvements to the physic package, new capabilities such as the incorporation of a slab ocean component, and a number of enhancements to the implementation (e.g., the ability to integrate the model on parallel distributed-memory computational platforms).

Validation of Slosh Modeling Approach Using STAR-CCM+

NASA Technical Reports Server (NTRS)

Benson, David J.; Ng, Wanyi

2018-01-01

Without an adequate understanding of propellant slosh, the spacecraft attitude control system may be inadequate to control the spacecraft or there may be an unexpected loss of science observation time due to higher slosh settling times. Computational fluid dynamics (CFD) is used to model propellant slosh. STAR-CCM+ is a commercially available CFD code. This paper seeks to validate the CFD modeling approach via a comparison between STAR-CCM+ liquid slosh modeling results and experimental, empirically, and analytically derived results. The geometries examined are a bare right cylinder tank and a right cylinder with a single ring baffle.

A demonstration of mobile phone deployment to support the treatment of acutely ill children under five in Bushenyi district, Uganda.

PubMed

Kabakyenga, Jerome; Barigye, Celestine; Brenner, Jennifer; Maling, Samuel; Buchner, Denise; Nettle-Aquirre, Alberto; Singhal, Nalini; Kyomuhangi, Teddy; Tumusiime, David; Finch, Janet; MacLeod, Stuart

2016-03-01

Benefits of mobile phone deployment for children <5 in low-resource settings remain unproven. The target population of the current demonstration study in Bushenyi District, Uganda, presented with acute fever, pneumonia, or diarrhoea and were treated by community health workers (CHWs) providing integrated community case management (iCCM). An observational study was conducted in five parishes (47 villages) served by CHWs well versed in iCCM with supplemental training in mobile phone use. Impact was assessed by quantitative measures and qualitative evaluation through household surveys, focus group discussions, and key informant interviews. CHWs in targeted sites improved child healthcare through mobile phone use coupled with iCCM. Of acutely ill children, 92.6% were correctly managed. Significant improvements in clinical outcomes compared to those obtained by CHWs with enhanced iCCM training alone were unproven in this limited demonstration. Nonetheless, qualitative evaluation showed gains in treatment planning, supply management, and logistical efficiency. Provider confidence and communications were enhanced as was ease and accuracy of record keeping. Mobile phones appear synergistic with iCCM to bolster basic supportive care for acutely ill children provided by CHWs. The full impact of expanded mobile phone deployment warrants further evaluation prior to scaling up in low-resource settings.

Cancer cell membrane-coated magnetic nanoparticles for MR/NIR fluorescence dual-modal imaging and photodynamic therapy.

PubMed

Li, Jiong; Wang, Xuandong; Zheng, Dongye; Lin, Xinyi; Wei, Zuwu; Zhang, Da; Li, Zhuanfang; Zhang, Yun; Wu, Ming; Liu, Xiaolong

2018-05-22

Theranostic nanoprobes integrated with dual-modal imaging and therapeutic functions, such as photodynamic therapy (PDT), have exhibited significant potency in cancer treatments due to their high imaging accuracy and non-invasive advantages for cancer elimination. However, biocompatibility and highly efficient accumulation of these nanoprobes in tumor are still unsatisfactory for clinical application. In this study, a photosensitizer -loaded magnetic nanobead with surface further coated with a layer of cancer cell membrane (SSAP-Ce6@CCM) was designed to improve the biocompatibility and cellular uptake and ultimately achieve enhanced MR/NIR fluorescence imaging and PDT efficacy. Compared with similar nanobeads without CCM coating, SSAP-Ce6@CCM showed significantly enhanced cellular uptake, as evidenced by Prussian blue staining, confocal laser scanning microscopy (CLSM) and flow cytometric analysis. Consequently, SSAP-Ce6@CCM displayed a more distinct MR/NIR imaging ability and more obvious photo-cytotoxicity towards cancer cells under 670 nm laser irradiation. Furthermore, the enhanced PDT effect benefited from the surface coating of cancer cell membrane was demonstrated in SMMC-7721 tumor-bearing mice through tumor growth observation and tumor tissue pathological examination. Therefore, this CCM-disguised nanobead that integrated the abilities of MR/NIR fluorescence dual-modal imaging and photodynamic therapy might be a promising theranostic platform for tumor treatment.

pH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism.

PubMed

Mangan, Niall M; Flamholz, Avi; Hood, Rachel D; Milo, Ron; Savage, David F

2016-09-06

Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3 (-) into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools.

Final report on RMO Vickers key comparison COOMET M.H-K1

NASA Astrophysics Data System (ADS)

Aslanyan, E.; Menelao, F.; Herrmann, K.; Aslanyan, A.; Pivovarov, V.; Galat, E.; Dovzhenko, Y.; Zhamanbalin, M.

2013-01-01

This report describes a COOMET key comparison on Vickers hardness scales involving five National Metrology Institutes: PTB (Germany), BelGIM (Belarus), NSC IM (Ukraine), KazInMetr (Kazakhstan) and VNIIFTRI (Russia). The pilot laboratory was VNIIFTRI, and PTB acted as the linking institute to key comparisons CCM.H-K1.b and CCM.H-K1.c conducted for the Vickers hardness scales HV1 and HV30, respectively. The comparison was also conducted for the HV5 Vickers hardness scale, since this scale is most frequently used in practice in Russia and CIS countries that work according to GOST standards. In the key comparison, two sets of hardness reference blocks for the Vickers hardness scales HV1, HV5 and HV30 consisting each of three hardness reference blocks with hardness levels of 450 HV and 750 HV were used. The measurement results and uncertainty assessments for HV1 and HV30 hardness scales, as announced by BelGIM, NSC IM, KazInMetr and VNIIFTRI, are in good agreement with the key comparison reference values of CCM.H-K1.b and CCM.H-K1.c. The comparison results for the HV5 hardness scale are viewed as additional information, since up to today no CCM key comparisons on this scale have yet been carried out. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

pH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism

PubMed Central

Flamholz, Avi; Hood, Rachel D.; Milo, Ron

2016-01-01

Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3− into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools. PMID:27551079

Magnetic Resonance Thermometry-Guided Stereotactic Laser Ablation of Cavernous Malformations in Drug-Resistant Epilepsy: Imaging and Clinical Results

PubMed Central

McCracken, D. Jay; Willie, Jon T.; Fernald, Brad; Saindane, Amit M.; Drane, Daniel L.; Barrow, Daniel L.; Gross, Robert E.

2016-01-01

BACKGROUND Surgery is indicated for cerebral cavernous malformations (CCM) that cause medically refractory epilepsy. Real-time magnetic resonance thermography (MRT)-guided stereotactic laser ablation (SLA) is a minimally invasive approach to treating focal brain lesions. SLA of CCM has not previously been described. OBJECTIVE To describe MRT-guided SLA, a novel approach to treating CCM-related epilepsy, with respect to feasibility, safety, imaging, and seizure control in 5 consecutive patients. METHODS Five patients with medically refractory epilepsy undergoing standard presurgical evaluation were found to have corresponding lesions fulfilling imaging characteristics of CCM and were prospectively enrolled. Each underwent stereotactic placement of a saline-cooled cannula containing an optical fiber to deliver 980-nm diode laser energy via twist drill craniostomy. MR anatomic imaging was used to evaluate targeting prior to ablation. MR imaging provided evaluation of targeting and near real-time feedback regarding extent of tissue thermocoagulation. Patients maintained seizure diaries, and remote imaging (6–21 months post-ablation) was obtained in all patients. RESULTS Imaging revealed no evidence of acute hemorrhage following fiber placement within presumed CCM. MRT during treatment and immediate post-procedure imaging confirmed desired extent of ablation. We identified no adverse events or neurological deficits. Four of 5 (80%) patients achieved freedom from disabling seizures after SLA alone (Engel class 1 outcome), with follow-up ranging 12–28 months. Reimaging of all subjects (6–21 months) indicated lesion diminution with surrounding liquefactive necrosis, consistent with the surgical goal of extended lesionotomy. CONCLUSION Minimally invasive MRT-guided SLA of epileptogenic CCM is a potentially safe and effective alternative to open resection. Additional experience and longer follow-up are needed. PMID:27959970

Emotion Recognition From Singing Voices Using Contemporary Commercial Music and Classical Styles.

PubMed

Hakanpää, Tua; Waaramaa, Teija; Laukkanen, Anne-Maria

2018-02-22

This study examines the recognition of emotion in contemporary commercial music (CCM) and classical styles of singing. This information may be useful in improving the training of interpretation in singing. This is an experimental comparative study. Thirteen singers (11 female, 2 male) with a minimum of 3 years' professional-level singing studies (in CCM or classical technique or both) participated. They sang at three pitches (females: a, e1, a1, males: one octave lower) expressing anger, sadness, joy, tenderness, and a neutral state. Twenty-nine listeners listened to 312 short (0.63- to 4.8-second) voice samples, 135 of which were sung using a classical singing technique and 165 of which were sung in a CCM style. The listeners were asked which emotion they heard. Activity and valence were derived from the chosen emotions. The percentage of correct recognitions out of all the answers in the listening test (N = 9048) was 30.2%. The recognition percentage for the CCM-style singing technique was higher (34.5%) than for the classical-style technique (24.5%). Valence and activation were better perceived than the emotions themselves, and activity was better recognized than valence. A higher pitch was more likely to be perceived as joy or anger, and a lower pitch as sorrow. Both valence and activation were better recognized in the female CCM samples than in the other samples. There are statistically significant differences in the recognition of emotions between classical and CCM styles of singing. Furthermore, in the singing voice, pitch affects the perception of emotions, and valence and activity are more easily recognized than emotions. Copyright © 2018 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Guided bone regeneration with asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles

PubMed Central

Zhang, Jiayu; Ma, Shiqing; Liu, Zihao; Geng, Hongjuan; Lu, Xin; Zhang, Xi; Li, Hongjie; Gao, Chenyuan; Zhang, Xu; Gao, Ping

2017-01-01

Introduction Membranes allowing the sustained release of drugs that can achieve cell adhesion are very promising for guided bone regeneration. Previous studies have suggested that aspirin has the potential to promote bone regeneration. The purpose of this study was to prepare a local drug delivery system with aspirin-loaded chitosan nanoparticles (ACS) contained in an asymmetric collagen-chitosan membrane (CCM). Methods In this study, the ACS were fabricated using different concentrations of aspirin (5 mg, 25 mg, 50 mg, and 75 mg). The drug release behavior of ACS was studied. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to examine the micromorphology of ACS and aspirin-loaded chitosan nanoparticles contained in chitosan-collagen membranes (ACS-CCM). In vitro bone mesenchymal stem cells (BMSCs) were cultured and critical-sized cranial defects on Sprague-Dawley rats were made to evaluate the effect of the ACS-CCM on bone regeneration. Results Drug release behavior results of ACS showed that the nanoparticles fabricated in this study could successfully sustain the release of the drug. TEM showed the morphology of the nanoparticles. SEM images indicated that the asymmetric membrane comprised a loose collagen layer and a dense chitosan layer. In vitro studies showed that ACS-CCM could promote the proliferation of BMSCs, and that the degree of differentiated BMSCs seeded on CCMs containing 50 mg of ACS was higher than that of other membranes. Micro-computed tomography showed that 50 mg of ACS-CCM resulted in enhanced bone regeneration compared with the control group. Conclusion This study shows that the ACS-CCM would allow the sustained release of aspirin and have further osteogenic potential. This membrane is a promising therapeutic approach to guiding bone regeneration. PMID:29276386

Small fiber neuropathy in Parkinson's disease: A clinical, pathological and corneal confocal microscopy study.

PubMed

Kass-Iliyya, Lewis; Javed, Saad; Gosal, David; Kobylecki, Christopher; Marshall, Andrew; Petropoulos, Ioannis N; Ponirakis, Georgios; Tavakoli, Mitra; Ferdousi, Maryam; Chaudhuri, Kallol Ray; Jeziorska, Maria; Malik, Rayaz A; Silverdale, Monty A

2015-12-01

Autonomic and somatic denervation is well established in Parkinson's disease (PD). (1) To determine whether corneal confocal microscopy (CCM) can non-invasively demonstrate small nerve fiber damage in PD. (2) To identify relationships between corneal nerve parameters, intraepidermal nerve fiber density (IENFD) and clinical features of PD. Twenty-six PD patients and 26 controls underwent CCM of both eyes. 24/26 PD patients and 10/26 controls underwent skin biopsies from the dorsa of both feet. PD patients underwent assessment of parasympathetic function [deep breathing heart rate variability (DB-HRV)], autonomic symptoms [scale for outcomes in Parkinson's disease - autonomic symptoms (SCOPA-AUT)], motor symptoms [UPDRS-III "ON"] and cumulative Levodopa dose. PD patients had significantly reduced corneal nerve fiber density (CNFD) with increased corneal nerve branch density (CNBD) and corneal nerve fiber length (CNFL) compared to controls. CNBD and CNFL but not CNFD correlated inversely with UPDRS-III and SCOPA-AUT. All CCM parameters correlated strongly with DB-HRV. There was no correlation between CCM parameters and disease duration, cumulative Levodopa dose or pain. IENFD was significantly reduced in PD compared to controls and correlated with CNFD and UPDRS-III. However, unlike CCM measures, IENFD correlated with disease duration and cumulative Levodopa dose but not with autonomic dysfunction. CCM identifies corneal nerve fiber pathology, which correlates with autonomic symptoms, parasympathetic deficits and motor scores in patients with PD. IENFD is also reduced and correlates with CNFD and motor symptoms but not parasympathetic deficits, indicating it detects different aspects of peripheral nerve pathology in PD. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Improving Access to Child Health Care in Indonesia Through Community Case Management.

PubMed

Setiawan, Agus; Dignam, Denise; Waters, Cheryl; Dawson, Angela

2016-11-01

Objectives In order to reduce infant mortality in Indonesia, community case management (CCM) was introduced. CCM is a community-based service delivery model to improve children's wellness and longevity, involving the delivery of lifesaving, curative interventions to address common childhood illnesses, particularly where there are limited facility-based services. This paper reports the findings of a qualitative study that investigated the implementation of CCM in the Kutai Timur district, East Kalimantan Indonesia from the perspective of mothers who received care. Methods Seven mothers and health workers were observed during a consultation and these mothers were interviewed in their home weeks after delivery. Field notes and the interview transcriptions were analysed thematically. Findings Mothers reported that their access to care had improved, along with an increase in their knowledge of infant danger signs and when to seek care. Family compliance with care plans was also found to have improved. Mothers expressed satisfaction with the care provided under the CCM model. The mothers expressed a need for a nurse or midwife to be posted in each village, preferably someone from that village. However two mothers did not wish their children to receive health interventions as they did not believe these to be culturally appropriate. Conclusion CCM is seen by rural Indonesian mothers to be a helpful model of care in terms of increasing access to health care and the uptake of lifesaving interventions for sick children. However there is a need to modify the program to demonstrate cultural sensitivity and meet cultural needs of the target population. While CCM is a potentially effective model of care, further integrative strategies are required to embed this model into maternal and child health service delivery.

Guided bone regeneration with asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles.

PubMed

Zhang, Jiayu; Ma, Shiqing; Liu, Zihao; Geng, Hongjuan; Lu, Xin; Zhang, Xi; Li, Hongjie; Gao, Chenyuan; Zhang, Xu; Gao, Ping

2017-01-01

Membranes allowing the sustained release of drugs that can achieve cell adhesion are very promising for guided bone regeneration. Previous studies have suggested that aspirin has the potential to promote bone regeneration. The purpose of this study was to prepare a local drug delivery system with aspirin-loaded chitosan nanoparticles (ACS) contained in an asymmetric collagen-chitosan membrane (CCM). In this study, the ACS were fabricated using different concentrations of aspirin (5 mg, 25 mg, 50 mg, and 75 mg). The drug release behavior of ACS was studied. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to examine the micromorphology of ACS and aspirin-loaded chitosan nanoparticles contained in chitosan-collagen membranes (ACS-CCM). In vitro bone mesenchymal stem cells (BMSCs) were cultured and critical-sized cranial defects on Sprague-Dawley rats were made to evaluate the effect of the ACS-CCM on bone regeneration. Drug release behavior results of ACS showed that the nanoparticles fabricated in this study could successfully sustain the release of the drug. TEM showed the morphology of the nanoparticles. SEM images indicated that the asymmetric membrane comprised a loose collagen layer and a dense chitosan layer. In vitro studies showed that ACS-CCM could promote the proliferation of BMSCs, and that the degree of differentiated BMSCs seeded on CCMs containing 50 mg of ACS was higher than that of other membranes. Micro-computed tomography showed that 50 mg of ACS-CCM resulted in enhanced bone regeneration compared with the control group. This study shows that the ACS-CCM would allow the sustained release of aspirin and have further osteogenic potential. This membrane is a promising therapeutic approach to guiding bone regeneration.

Engineering of Corynebacterium glutamicum for xylitol production from lignocellulosic pentose sugars.

PubMed

Dhar, Kiran S; Wendisch, Volker F; Nampoothiri, Kesavan Madhavan

2016-07-20

Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L). Copyright © 2016 Elsevier B.V. All rights reserved.

The biodiversity of microbial cytochromes P450.

PubMed

Kelly, Steven L; Lamb, David C; Jackson, Colin J; Warrilow, Andrew G; Kelly, Diane E

2003-01-01

The cytochrome P450 (CYP) superfamily of genes and proteins are well known for their involvement in pharmacology and toxicology, but also increasingly for their importance and diversity in microbes. The extent of diversity has only recently become apparent with the emergence of data from whole genome sequencing projects and the coming years will reveal even more information on the diversity in microbial eukaryotes. This review seeks to describe the historical development of these studies and to highlight the importance of the genes and proteins. CYPs are deeply involved in the development of strategies for deterrence and attraction as well as detoxification. As such, there is intense interest in pathways of secondary metabolism that include CYPs in oxidative tailoring of antibiotics, sometimes influencing potency as bioactive compounds. Further to this is interest in CYPs in metabolism of xenobiotics for use as carbon sources for microbial growth and as biotransformation agents or in bioremediation. CYPs are also current and potential drug targets; compounds inhibiting CYP are antifungal and anti-protozoan agents, and potentially similar compounds may be useful against some bacterial diseases such as tuberculosis. Of note is the diversity of CYP requirements within an organism, ranging from Escherichia coli that has no CYPs as in many bacteria, to Mycobacterium smegmatis that has 40 representing 1% of coding genes. The basidiomycete fungus Phanerochaete chrysosporium surprised all when it was found to contain a hundred or more CYPs. The functional genomic investigation of these orphan CYPs is a major challenge for the future.

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

PubMed

Chávez-Jacobo, Víctor M; Hernández-Ramírez, Karen C; Romo-Rodríguez, Pamela; Pérez-Gallardo, Rocío Viridiana; Campos-García, Jesús; Gutiérrez-Corona, J Félix; García-Merinos, Juan Pablo; Meza-Carmen, Víctor; Silva-Sánchez, Jesús; Ramírez-Díaz, Martha I

2018-06-01

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP , for c iprofloxacin r esistance p rotein, p lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC- crpP , conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa , which involves phosphorylation of the antibiotic. Copyright © 2018 American Society for Microbiology.

Systems analysis of the CO2 concentrating mechanism in cyanobacteria

PubMed Central

Mangan, Niall M; Brenner, Michael P

2014-01-01

Cyanobacteria are photosynthetic bacteria with a unique CO2 concentrating mechanism (CCM), enhancing carbon fixation. Understanding the CCM requires a systems level perspective of how molecular components work together to enhance CO2 fixation. We present a mathematical model of the cyanobacterial CCM, giving the parameter regime (expression levels, catalytic rates, permeability of carboxysome shell) for efficient carbon fixation. Efficiency requires saturating the RuBisCO reaction, staying below saturation for carbonic anhydrase, and avoiding wasteful oxygenation reactions. We find selectivity at the carboxysome shell is not necessary; there is an optimal non-specific carboxysome shell permeability. We compare the efficacy of facilitated CO2 uptake, CO2 scavenging, and HCO3− transport with varying external pH. At the optimal carboxysome permeability, contributions from CO2 scavenging at the cell membrane are small. We examine the cumulative benefits of CCM spatial organization strategies: enzyme co-localization and compartmentalization. DOI: http://dx.doi.org/10.7554/eLife.02043.001 PMID:24842993

Examination of Daily Weather in the NCAR CCM

NASA Astrophysics Data System (ADS)

Cocke, S. D.

2006-05-01

The NCAR CCM is one of the most extensively studied climate models in the scientific community. However, most studies focus primarily on the long term mean behavior, typically monthly or longer time scales. In this study we examine the daily weather in the GCM by performing a series of daily or weekly 10 day forecasts for one year at moderate (T63) and high (T126) resolution. The model is initialized with operational "AVN" and ECMWF analyses, and model performance is compared to that of major operational centers, using conventional skill scores used by the major centers. Such a detailed look at the CCM at shorter time scales may lead to improvements in physical parameterizations, which may in turn lead to improved climate simulations. One finding from this study is that the CCM has a significant drying tendency in the lower troposphere compared to the operational analyses. Another is that the large scale predictability of the GCM is competitive with most of the operational models, particularly in the southern hemisphere.

Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin.

PubMed

Dey, Soma; Sreenivasan, K

2014-01-01

Curcumin is a potential drug for various diseases including cancer. Prime limitations associated with curcumin are low water solubility, rapid hydrolytic degradation and poor bioavailability. In order to redress these issues we developed Alginate-Curcumin (Alg-Ccm) conjugate which was characterized by FTIR and (1)H NMR spectroscopy. The conjugate self-assembled in aqueous solution forming micelles with an average hydrodynamic diameter of 459 ± 0.32 nm and negative zeta potential. The spherical micelles were visualized by TEM. The critical micelle concentration (CMC) of Alg-Ccm conjugate was determined. A significant enhancement in the aqueous solubility of curcumin was observed upon conjugation with alginate. Formation of micelles improved the stability of curcumin in water at physiological pH. The cytotoxic activity of Alg-Ccm was quantified by MTT assay using L-929 fibroblast cells and it was found to be potentially cytotoxic. Hence, Alg-Ccm could be a promising drug conjugate as well as a nanosized delivery vehicle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas

PubMed Central

Meyer, Moritz T.; Genkov, Todor; Skepper, Jeremy N.; Jouhet, Juliette; Mitchell, Madeline C.; Spreitzer, Robert J.; Griffiths, Howard

2012-01-01

The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO2-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C4 pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future. PMID:23112177

Capillary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene glycol) pyrenebutanoate.

PubMed

Horka, Marie; Ruzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

2006-12-15

The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.

Follow-up Contemporary Commercial Music (CCM) Survey: who's teaching what in nonclassical music.

PubMed

Weekly, Edrie Means; Lovetri, Jeannette L

2009-05-01

A previous study, published in the Journal of Voice in 2003, revealed that a majority of teachers of Music Theater (MT), a style of Contemporary Commercial Music (CCM), had little professional experience and little formal training in vocal pedagogy for this style. Those who did indicate that they had had training did not describe the training nor quantify it in any manner. To ascertain what type of training was available for CCM in general and MT, in particular, a follow-up study seemed warranted. A new questionnaire was developed which asked for further information from teachers of MT in several areas including performance experience, training methods, teaching philosophy, the use of terminology, knowledge of voice science and medicine, and other parameters. Responses were gathered from 145 singing teachers throughout the United States and several foreign countries. Statistical analysis obtained from the data may lead to both a better understanding of the kind of training available for teachers of CCM repertoire, and of its content and applicability.

P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces

PubMed Central

Plutoni, Cédric; Bazellieres, Elsa; Le Borgne-Rochet, Maïlys; Comunale, Franck; Brugues, Agusti; Séveno, Martial; Planchon, Damien; Thuault, Sylvie; Morin, Nathalie; Bodin, Stéphane; Trepat, Xavier

2016-01-01

Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell–cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/β-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through β-PIX, which is specifically recruited at cell–cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through β-PIX–mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM. PMID:26783302

Characteristics of stratosphere-troposphere exchange in a general circulation model

NASA Technical Reports Server (NTRS)

Mote, Philip W.; Holton, James R.; Boville, Byron A.

1994-01-01

In this study we examine mass exchange, water vapor exchange, and the behavior of idealized tracers and parcels to diagnose Stratosphere-Troposphere Exchange (STE) in the National Center for Atmospheric Research (NCAR) General Circulation Model (GCM), the Community Climate Model (CCM2). The CCM2 correctly represents the seasonality of mass exchange across 100 hPa, but values are uniformly too strong. Water vapor, however, indicates that tropical STE is not well represented in the CCM2; even though mean tropopause temperatures are colder than observed, the lower stratosphere is too moist. Most net mass flux occurs at water vapor mixing ratios of about 4-5 parts per million by volume (ppmv), about 1 ppmv too moist. Vertical resolution has little impact on the nature of tropical STE. In midlatitudes, CCM2 more successfully represents STE, which occurs in developing baroclinic waves and stationary anticyclones. Exchange from troposphere to stratosphere does occur but only influences the lowest few kilometers of the extratropical stratosphere, even for tracers with large vertical gradients.

A comparison of observed (HALOE) and modeled (CCM2) methane and stratospheric water vapor

NASA Technical Reports Server (NTRS)

Mote, Philip W.; Holton, James R.; Russell, James M., III; Boville, Byron A.

1993-01-01

Recent measurements (21 September-15 October 1992) of methane and water vapor by the Halogen Occultation Experiment (HALOE) on the Upper Atmosphere Research Satellite (UARS) are compared with model results for the same season from a troposphere-middle atmosphere version of the National Center for Atmospheric Research (NCAR) Community Climate Model (CCM2). Several important features of the two constituent fields are well reproduced by the CCM2, despite the use of simplified methane photochemistry in the CCM2 and some notable differences between the model's zonal mean circulation and climatology. Observed features simulated by the model include the following: 1) subsidence over a deep layer in the Southern Hemisphere polar vortex; 2) widespread dehydration in the polar vortex; and 3) existence of a region of low water vapor mixing ratios extending from the Antarctic into the Northern Hemisphere tropics, which suggests that Antarctic dehydration contributes to midlatitude and tropical dryness in the stratosphere.

Integrated Community Case Management of Childhood Illness in Ethiopia: Implementation Strength and Quality of Care

PubMed Central

Miller, Nathan P.; Amouzou, Agbessi; Tafesse, Mengistu; Hazel, Elizabeth; Legesse, Hailemariam; Degefie, Tedbabe; Victora, Cesar G.; Black, Robert E.; Bryce, Jennifer

2014-01-01

Ethiopia has scaled up integrated community case management of childhood illness (iCCM) in most regions. We assessed the strength of iCCM implementation and the quality of care provided by health extension workers (HEWs). Data collectors observed HEWs' consultations with sick children and carried out gold standard re-examinations. Nearly all HEWs received training and supervision, and essential commodities were available. HEWs provided correct case management for 64% of children. The proportions of children correctly managed for pneumonia, diarrhea, and malnutrition were 72%, 79%, and 59%, respectively. Only 34% of children with severe illness were correctly managed. Health posts saw an average of 16 sick children in the previous 1 month. These results show that iCCM can be implemented at scale and that community-based HEWs can correctly manage multiple illnesses. However, to increase the chances of impact on child mortality, management of severe illness and use of iCCM services must be improved. PMID:24799369

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilkey, Lindsay

This milestone presents a demonstration of the High-to-Low (Hi2Lo) process in the VVI focus area. Validation and additional calculations with the commercial computational fluid dynamics code, STAR-CCM+, were performed using a 5x5 fuel assembly with non-mixing geometry and spacer grids. This geometry was based on the benchmark experiment provided by Westinghouse. Results from the simulations were compared to existing experimental data and to the subchannel thermal-hydraulics code COBRA-TF (CTF). An uncertainty quantification (UQ) process was developed for the STAR-CCM+ model and results of the STAR UQ were communicated to CTF. Results from STAR-CCM+ simulations were used as experimental design pointsmore » in CTF to calibrate the mixing parameter β and compared to results obtained using experimental data points. This demonstrated that CTF’s β parameter can be calibrated to match existing experimental data more closely. The Hi2Lo process for the STAR-CCM+/CTF code coupling was documented in this milestone and closely linked L3:VVI.H2LP15.01 milestone report.« less

Training, supervision and quality of care in selected integrated community case management (iCCM) programmes: A scoping review of programmatic evidence.

PubMed

Bosch-Capblanch, Xavier; Marceau, Claudine

2014-12-01

To describe the training, supervision and quality of care components of integrated Community Case Management (iCCM) programmes and to draw lessons learned from existing evaluations of those programmes. Scoping review of reports from 29 selected iCCM programmes purposively provided by stakeholders containing any information relevant to understand quality of care issues. The number of people reached by iCCM programmes varied from the tens of thousands to more than a million. All programmes aimed at improving access of vulnerable populations to health care, focusing on the main childhood illnesses, managed by Community Health Workers (CHW), often selected bycommunities. Training and supervision were widely implemented, in different ways and intensities, and often complemented with tools (eg, guides, job aids), supplies, equipment and incentives. Quality of care was measured using many outcomes (eg, access or appropriate treatment). Overall, there seemed to be positive effects for those strategies that involved policy change, organisational change, standardisation of clinical practices and alignment with other programmes. Positive effects were mostly achieved in large multi-component programmes. Mild or no effects have been described on mortality reduction amongst the few programmes for which data on this outcome was available to us. Promising strategies included teaming-up of CHW, micro-franchising or social franchising. On-site training and supervision of CHW have been shown to improve clinical practices. Effects on caregivers seemed positive, with increases in knowledge, care seeking behaviour, or caregivers' basic disease management. Evidence on iCCM is often of low quality, cannot relate specific interventions or the ways they are implemented with outcomes and lacks standardisation; this limits the capacity to identify promising strategies to improve quality of care. Large, multi-faceted, iCCM programmes, with strong components of training, supervision, which included additional support of equipment and supplies, seemed to improve selected quality of care outcomes. However, current evaluation and reporting practices need to be revised in a new research agenda to address the methodological challenges of iCCM evaluations.

Formation of Fe-Mn crusts within a continental margin environment

USGS Publications Warehouse

Conrad, Tracey A.; Hein, James R.; Paytan, Adina; Clague, David A.

2017-01-01

This study examines Fe-Mn crusts that form on seamounts along the California continental-margin (CCM), within the United States 200 nautical mile exclusive economic zone. The study area extends from approximately 30° to 38° North latitudes and from 117° to 126° West longitudes. The area of study is a tectonically active northeast Pacific plate boundary region and is also part of the North Pacific Subtropical Gyre with currents dominated by the California Current System. Upwelling of nutrient-rich water results in high primary productivity that produces a pronounced oxygen minimum zone. Hydrogenetic Fe-Mn crusts forming along the CCM show distinct chemical and mineral compositions compared to open-ocean crusts. On average, CCM crusts contain more Fe relative to Mn than open-ocean Pacific crusts. The continental shelf and slope release both Fe and Mn under low-oxygen conditions. Silica is also enriched relative to Al compared to open-ocean crusts. This is due to the North Pacific silica plume and enrichment of Si along the path of deep-water circulation, resulting in Si enrichment in bottom and intermediate waters of the eastern Pacific.The CCM Fe-Mn crusts have a higher percentage of birnessite than open-ocean crusts, reflecting lower dissolved seawater oxygen that results from the intense coastal upwelling and proximity to zones of continental slope pore-water anoxia. Carbonate fluorapatite (CFA) is not present and CCM crusts do not show evidence of phosphatization, even in the older sections. The mineralogy indicates a suboxic environment under which birnessite forms, but in which pH is not high enough to facilitate CFA deposition. Growth rates of CCM crusts generally increase with increasing water depth, likely due to deep-water Fe sources mobilized from reduced shelf and slope sediments.Many elements of economic interest including Mn, Co, Ni, Cu, W, and Te have slightly or significantly lower concentrations in CCM crusts relative to crusts from the Pacific Prime Crust Zone and other open-ocean basins. However, concentrations of total rare earth elements and yttrium average only slightly lower contents and in the future may be a strategic resource for the U.S.

Strategies of dissolved inorganic carbon use in macroalgae across a gradient of terrestrial influence: implications for the Great Barrier Reef in the context of ocean acidification

NASA Astrophysics Data System (ADS)

Diaz-Pulido, Guillermo; Cornwall, Christopher; Gartrell, Patrick; Hurd, Catriona; Tran, Dien V.

2016-12-01

Macroalgae are generally used as indicators of coral reef status; thus, understanding the drivers and mechanisms leading to increased macroalgal abundance are of critical importance. Ocean acidification (OA) due to elevated carbon dioxide (CO2) concentrations has been suggested to stimulate macroalgal growth and abundance on reefs. However, little is known about the physiological mechanisms by which reef macroalgae use CO2 from the bulk seawater for photosynthesis [i.e., (1) direct uptake of bicarbonate (HCO3 -) and/or CO2 by means of carbon concentrating mechanisms (CCM) and (2) the diffusive uptake of CO2], which species could benefit from increased CO2 or which habitats may be more susceptible to acidification-induced algal proliferations. Here, we provide the first quantitative examination of CO2-use strategies in coral reef macroalgae and provide information on how the proportion of species and the proportional abundance of species utilising each of the carbon acquisition strategies varies across a gradient of terrestrial influence (from inshore to offshore reefs) in the Great Barrier Reef (GBR). Four macroalgal groups were identified based on their carbon uptake strategies: (1) CCM-only (HCO3 - only users); (2) CCM-HCO3 -/CO2 (active uptake HCO3 - and/or CO2 use); (3) Non-CCM species (those relying on diffusive CO2 uptake); and (4) Calcifiers. δ13C values of macroalgae, confirmed by pH drift assays, show that diffusive CO2 use is more prevalent in deeper waters, possibly due to low light availability that limits activity of CCMs. Inshore shallow reefs had a higher proportion of CCM-only species, while reefs further away from terrestrial influence and exposed to better water quality had a higher number of non-CCM species than inshore and mid-shelf reefs. As non-CCM macroalgae are more responsive to increased seawater CO2 and OA, reef slopes of the outer reefs are probably the habitats most vulnerable to the impacts of OA. Our results suggest a potentially important role of carbon physiology in structuring macroalgal communities in the GBR.

Clinical marginal and internal adaptation of CAD/CAM milling, laser sintering, and cast metal ceramic crowns.

PubMed

Tamac, Ece; Toksavul, Suna; Toman, Muhittin

2014-10-01

Metal ceramic crowns are widely used in clinical practice, but comparisons of the clinical adaptation of restorations made with different processing techniques are lacking. The purpose of this study was to compare the clinical marginal and internal adaptation of metal ceramic crowns fabricated with 3 different techniques: computer-aided design and computer-aided manufacturing (CAD/CAM) milling (CCM), direct metal laser sintering (DMLS), and traditional casting (TC). Twenty CCM, 20 DMLS, and 20 TC metal ceramic crowns were fabricated for 42 patients. Before luting the crowns, silicone replicas were obtained to measure marginal gap and internal adaptation that was evaluated at 3 regions: axial wall, axio-occlusal angle, and occlusal surface. Measurements were made with a reflected light binocular stereomicroscope at 20× magnification and analyzed with 1-way analysis of variance (ANOVA) and the Bonferroni post hoc test (α=.05). The mean marginal gap values were 86.64 μm for CCM, 96.23 μm for DMLS, and 75.92 μm for TC. The means at the axial wall region were 117.5 μm for the CCM group, 139.02 μm for the DMLS group, and 121.38 μm for the TC group. One-way ANOVA revealed no statistically significant differences among the groups for measurements at the marginal gap (P=.082) and the axial wall region (P=.114). The means at the axio-occlusal region were 142.1 μm for CCM, 188.12 μm for DMLS, and 140.63 μm for TC, and those at the occlusal surface region were 265.73 μm for CCM, 290.39 μm for DMLS, and 201.09 μm for TC. The mean values of group DMLS were significantly higher at the axio-occlusal region and the occlusal surface region than those of other groups (P<.05). CCM, DMLS and TC metal ceramic crowns performed similarly in terms of clinical marginal and axial wall adaptation. The cement film thickness at the occlusal region and axio-occlusal region were higher for DMLS crowns. Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

[Development and application of component-based Chinese medicine theory].

PubMed

Zhang, Jun-Hua; Fan, Guan-Wei; Zhang, Han; Fan, Xiao-Hui; Wang, Yi; Liu, Li-Mei; Li, Chuan; Gao, Yue; Gao, Xiu-Mei; Zhang, Bo-Li

2017-11-01

Traditional Chinese medicine (TCM) prescription is the main therapies for disease prevention and treatment in Chinese medicine. Following the guidance of the theory of TCM and developing drug by composing prescriptions of TCM materials and pieces, it is a traditional application mode of TCM, and still widely used in clinic. TCM prescription has theoretical advantages and rich clinical application experience in dealing with multi-factor complex diseases, but scientific research is relatively weak. The lack of scientific cognition of the effective substances and mechanism of Chinese medicine leads to insufficient understanding of the efficacy regularity, which affects the stability of effect and hinders the improvement of quality of Chinese medicinal products. Component-based Chinese medicine (CCM) is an innovation based on inheritance, which breaks through the tradition of experience-based prescription and realize the transformation of compatibility from herbal pieces to components. CCM is an important achievement during the research process of modernization of Chinese medicine. Under the support of three national "973" projects, in order to reveal the scientific connotation of the prescription compatibility theory and develop innovative Chinese drugs, we have launched theoretical innovation and technological innovation around the "two relatively clear", and opened up the research field of CCM. CCM is an innovation based on inheritance, breaking through the tradition of experience based prescription, and realizing the transformation from compatibility of herbal pieces to component compatibility, which is an important achievement of the modernization of traditional Chinese medicine. In the past more than 10 years, with the deepening of research and the expansion of application, the theory and methods of CCM and efficacy-oriented compatibility have been continuously improved. The value of CCM is not only in developing new drug, more important is to build a communication bridge between traditional Chinese medicine and modern science and construct the system of key technologies which meet the need of innovation and development of TCM. This paper focused on the research progress, related concepts and technology development of CCM, as well as its application prospect in the theory research of Chinese medicine, development of innovative Chinese drugs, secondary development of Chinese patent medicine and upgrading of pharmaceutical technology. Copyright© by the Chinese Pharmaceutical Association.

Monitoring intensive care unit performance—impact of a novel individualised performance scorecard in critical care medicine: a mixed-methods study protocol

PubMed Central

Neilipovitz, David; Sarti, Aimee J; Rosenberg, Erin; Ishaq, Rabia; Thornton, Mary; Kim, John

2018-01-01

Introduction Patients admitted to a critical care medicine (CCM) environment, including an intensive care unit (ICU), are susceptible to harm and significant resource utilisation. Therefore, a strategy to optimise provider performance is required. Performance scorecards are used by institutions for the purposes of driving quality improvement. There is no widely accepted or standardised scorecard that has been used for overall CCM performance. We aim to improve quality of care, patient safety and patient/family experience in CCM practice through the utilisation of a standardised, repeatable and multidimensional performance scorecard, designed to provide a continuous review of ICU physician and nurse practice, as well as departmental metrics. Methods and analysis This will be a mixed-methods, controlled before and after study to assess the impact of a CCM-specific quality scorecard. Scorecard metrics were developed through expert consensus and existing literature. The study will include 19 attending CCM physicians and approximately 300 CCM nurses. Patient data for scorecard compilation are collected daily from bedside flow sheets. Preintervention baseline data will be collected for 6 months for each participant. After this, each participant will receive their scorecard measures. Following a 3-month washout period, postintervention data will be collected for 6 months. The primary outcome will be change in performance metrics following the provision of scorecard feedback to subjects. A cost analysis will also be performed, with the purpose of comparing total ICU costs prior to implementation of the scorecard with total ICU costs following implementation of the scorecard. The qualitative portion will include interviews with participants following the intervention phase. Interviews will be analysed in order to identify recurrent themes and subthemes, for the purposes of driving scorecard improvement. Ethics and dissemination This protocol has been approved by the local research ethics board. Publication of results is anticipated in 2019. If this intervention is found to improve patient- and unit-directed outcomes, with evidence of cost-effectiveness, it would support the utilisation of such a scorecard as a quality standard in CCM. PMID:29358441

How do we know? An assessment of integrated community case management data quality in four districts of Malawi.

PubMed

Yourkavitch, Jennifer; Zalisk, Kirsten; Prosnitz, Debra; Luhanga, Misheck; Nsona, Humphreys

2016-11-01

The World Health Organization contracted annual data quality assessments of Rapid Access Expansion (RAcE) projects to review integrated community case management (iCCM) data quality and the monitoring and evaluation (M&E) system for iCCM, and to suggest ways to improve data quality. The first RAcE data quality assessment was conducted in Malawi in January 2014 and we present findings pertaining to data from the health management information system at the community, facility and other sub-national levels because RAcE grantees rely on that for most of their monitoring data. We randomly selected 10 health facilities (10% of eligible facilities) from the four RAcE project districts, and collected quantitative data with an adapted and comprehensive tool that included an assessment of Malawi's M&E system for iCCM data and a data verification exercise that traced selected indicators through the reporting system. We rated the iCCM M&E system across five function areas based on interviews and observations, and calculated verification ratios for each data reporting level. We also conducted key informant interviews with Health Surveillance Assistants and facility, district and central Ministry of Health staff. Scores show a high-functioning M&E system for iCCM with some deficiencies in data management processes. The system lacks quality controls, including data entry verification, a protocol for addressing errors, and written procedures for data collection, entry, analysis and management. Data availability was generally high except for supervision data. The data verification process identified gaps in completeness and consistency, particularly in Health Surveillance Assistants' record keeping. Staff at all levels would like more training in data management. This data quality assessment illuminates where an otherwise strong M&E system for iCCM fails to ensure some aspects of data quality. Prioritizing data management with documented protocols, additional training and approaches to create efficient supervision practices may improve iCCM data quality. © The Author 2016. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Training, supervision and quality of care in selected integrated community case management (iCCM) programmes: A scoping review of programmatic evidence

PubMed Central

Bosch–Capblanch, Xavier; Marceau, Claudine

2014-01-01

Aim To describe the training, supervision and quality of care components of integrated Community Case Management (iCCM) programmes and to draw lessons learned from existing evaluations of those programmes. Methods Scoping review of reports from 29 selected iCCM programmes purposively provided by stakeholders containing any information relevant to understand quality of care issues. Results The number of people reached by iCCM programmes varied from the tens of thousands to more than a million. All programmes aimed at improving access of vulnerable populations to health care, focusing on the main childhood illnesses, managed by Community Health Workers (CHW), often selected bycommunities. Training and supervision were widely implemented, in different ways and intensities, and often complemented with tools (eg, guides, job aids), supplies, equipment and incentives. Quality of care was measured using many outcomes (eg, access or appropriate treatment). Overall, there seemed to be positive effects for those strategies that involved policy change, organisational change, standardisation of clinical practices and alignment with other programmes. Positive effects were mostly achieved in large multi–component programmes. Mild or no effects have been described on mortality reduction amongst the few programmes for which data on this outcome was available to us. Promising strategies included teaming–up of CHW, micro–franchising or social franchising. On–site training and supervision of CHW have been shown to improve clinical practices. Effects on caregivers seemed positive, with increases in knowledge, care seeking behaviour, or caregivers’ basic disease management. Evidence on iCCM is often of low quality, cannot relate specific interventions or the ways they are implemented with outcomes and lacks standardisation; this limits the capacity to identify promising strategies to improve quality of care. Conclusion Large, multi–faceted, iCCM programmes, with strong components of training, supervision, which included additional support of equipment and supplies, seemed to improve selected quality of care outcomes. However, current evaluation and reporting practices need to be revised in a new research agenda to address the methodological challenges of iCCM evaluations. PMID:25520793

Integrated community case management in Malawi: an analysis of innovation and institutional characteristics for policy adoption.

PubMed

Rodríguez, Daniela C; Banda, Hastings; Namakhoma, Ireen

2015-12-01

In 2007, Malawi became an early adopter of integrated community case management for childhood illnesses (iCCM), a policy aimed at community-level treatment for malaria, diarrhoea and pneumonia for children below 5 years. Through a retrospective case study, this article explores critical issues in implementation that arose during policy formulation through the lens of the innovation (i.e. iCCM) and of the institutions involved in the policy process. Data analysis is founded on a documentary review and 21 in-depth stakeholder interviews across institutions in Malawi. Findings indicate that the characteristics of iCCM made it a suitable policy to address persistent challenges in child mortality, namely that ill children were not interacting with health workers on a timely basis and consequently were dying in their communities. Further, iCCM was compatible with the Malawian health system due to the ability to build on an existing community health worker cadre of health surveillance assistants (HSAs) and previous experiences with treatment provision at the community level. In terms of institutions, the Ministry of Health (MoH) demonstrated leadership in the overall policy process despite early challenges of co-ordination within the MoH. WHO, United Nations Children's Fund (UNICEF) and implementing organizations played a supportive role in their position as knowledge brokers. Greater challenges were faced in the organizational capacity of the MoH. Regulatory issues around HSA training as well as concerns around supervision and overburdening of HSAs were discussed, though not fully addressed during policy development. Similarly, the financial sustainability of iCCM, including the mechanisms for channelling funding flows, also remains an unresolved issue. This analysis highlights the role of implementation questions during policy development. Despite several outstanding concerns, the compatibility between iCCM as a policy alternative and the local context laid the foundation for Malawi's road to early adoption of iCCM. © The Author 2015. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Access to integrated community case management of childhood illnesses services in rural Ethiopia: a qualitative study of the perspectives and experiences of caregivers.

PubMed

Shaw, Bryan; Amouzou, Agbessi; Miller, Nathan P; Tafesse, Mengistu; Bryce, Jennifer; Surkan, Pamela J

2016-06-01

In 2010, Ethiopia began scaling up the integrated community case management (iCCM) of childhood illness strategy throughout the country allowing health extension workers (HEWs) to treat children in rural health posts. After 2 years of iCCM scale up, utilization of HEWs remains low. Little is known about factors related to the use of health services in this setting. This research aimed to elicit perceptions and experiences of caregivers to better understand reasons for low utilization of iCCM services. A rapid ethnographic assessment was conducted in eight rural health post catchment areas in two zones: Jimma and West Hararghe. In total, 16 focus group discussions and 78 in-depth interviews were completed with mothers, fathers, HEWs and community health volunteers. In spite of the HEW being a core component of iCCM, we found that the lack of availability of HEWs at the health post was one of the most common barriers to the utilization of iCCM services mentioned by caregivers. Financial and geographic challenges continue to influence caregiver decisions despite extension of free child health services in communities. Acceptability of HEWs was often low due to a perceived lack of sensitivity of HEWs and concerns about medicines given at the health post. Social networks acted both to facilitate and hinder use of HEWs. Many mothers stated a preference for using the health post, but some were unable to do so due to objections or alternative care-seeking preferences of gatekeepers, often mothers-in-law and husbands. Caregivers in Ethiopia face many challenges in using HEWs at the health post, potentially resulting in low demand for iCCM services. Efforts to minimize barriers to care seeking and to improve demand should be incorporated into the iCCM strategy in order to achieve reductions in child mortality and promote equity in access and child health outcomes. © The Author 2015. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine.

GLYOXALASE I A111E, PARAOXONASE 1 Q192R AND L55M POLYMORPHISMS IN ITALIAN PATIENTS WITH SPORADIC CEREBRAL CAVERNOUS MALFORMATIONS: A PILOT STUDY.

PubMed

Rinaldi, C; Bramanti, P; Famà, A; Scimone, C; Donato, L; Antognelli, C; Alafaci, C; Tomasello, F; D'Angelo, R; Sidoti, A

2015-01-01

It is already known that the conditions of increased oxidative stress are associated to a greater susceptibility to vascular malformations including cerebral cavernous malformations (CCMs). These are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities that can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1(Krit1), CCM2 (MGC4607) and CCM3 (PDCD10). Polymorphisms in the genes encoding for enzymes involved in the antioxidant systems such as glyoxalase I (GLO I) and paraoxonase I (PON I) could influence individual susceptibility to the vascular malformations. A single nucleotide polymorphism was identified in the exon 4 of GLO 1 gene that causes an amino acid substitution of Ala for Glu (Ala111Glu). Two common polymorphisms have been described in the coding region of PON1, which lead to glutamine → arginine substitution at 192 (Q192R) and a leucine → methionine substitution at 55 (L55M). The polymorphisms were characterized in 59 patients without mutations in the CCM genes versus 213 healthy controls by PCR/RFLP methods using DNA from lymphocytes. We found that the frequency of patients carrying the GLO1 A/E genotype among the case group (56%) was four-fold higher than among the controls (14.1%). In the cohort of CCM patients, an increase in the frequency of PON192 Q/R genotype was observed (39% in the CCM group versus 3.7% in the healthy controls). Similarly, an increase was observed in the proportion of individuals with the genotype R/R in the disease group (5%) in respect to the normal healthy cohort (0.5%). Finally, the frequency of the PON55 heterozygotes L/M genotype was 29% in patients with CCMs and 4% in the healthy controls. The same trend was observed in PON55 homozygous M/M genotype frequency (CCMs 20% vs controls 10%). The present study aimed to investigate the possible association of GLO1 A111E, PON1 Q192R and L55M polymorphisms with the risk of CCMs. We found that individuals with the GLO1 A /E genotype, PON192/QR-RR genotypes and PON55/LM-MM genotypes had a significantly higher risk of CCMs compared with the other genotypes. However, because CCM is a heterogeneous disease, other additional factors might be involved in the initiation and progression of CCM disease.

Monitoring iCCM referral systems: Bugoye Integrated Community Case Management Initiative (BIMI) in Uganda.

PubMed

English, Lacey; Miller, James S; Mbusa, Rapheal; Matte, Michael; Kenney, Jessica; Bwambale, Shem; Ntaro, Moses; Patel, Palka; Mulogo, Edgar; Stone, Geren S

2016-04-29

In Uganda, over half of under-five child mortality is attributed to three infectious diseases: malaria, pneumonia and diarrhoea. Integrated community case management (iCCM) trains village health workers (VHWs) to provide in-home diagnosis and treatment of these common childhood illnesses. For severely ill children, iCCM relies on a functioning referral system to ensure timely treatment at a health facility. However, referral completion rates vary widely among iCCM programmes and are difficult to monitor. The Bugoye Integrated Community Case Management Initiative (BIMI) is an iCCM programme operating in Bugoye sub-county, Uganda. This case study describes BIMI's experience with monitoring referral completion at Bugoye Health Centre III (BHC), and outlines improvements to be made within iCCM referral systems. This study triangulated multiple data sources to evaluate the strengths and gaps in the BIMI referral system. Three quantitative data sources were reviewed: (1) VHW report of referred patients, (2) referral forms found at BHC, and (3) BHC patient records. These data sources were collated and triangulated from January-December 2014. The goal was to determine if patients were completing their referrals and if referrals were adequately documented using routine data sources. From January-December 2014, there were 268 patients referred to BHC, as documented by VHWs. However, only 52 of these patients had referral forms stored at BHC. Of the 52 referral forms found, 22 of these patients were also found in BHC register books recorded by clinic staff. Thus, the study found a mismatch between VHW reports of patient referrals and the referral visits documented at BHC. This discrepancy may indicate several gaps: (1) referred patients may not be completing their referral, (2) referral forms may be getting lost at BHC, and, (3) referred patients may be going to other health facilities or drug shops, rather than BHC, for their referral. This study demonstrates the challenges of effectively monitoring iCCM referral completion, given identified limitations such as discordant data sources, incomplete record keeping and lack of unique identifiers. There is a need to innovate and improve the ways by which referral compliance is monitored using routine data, in order to improve the percentage of referrals completed. Through research and field experience, this study proposes programmatic and technological solutions to rectify these gaps within iCCM programmes facing similar challenges. With improved monitoring, VHWs will be empowered to increase referral completion, allowing critically ill children to access needed health services.

The potential for co-evolution of CO2-concentrating mechanisms and Rubisco in diatoms.

PubMed

Young, Jodi N; Hopkinson, Brian M

2017-06-01

Diatoms are a diverse group of unicellular algae that contribute significantly to global photosynthetic carbon fixation and export in the modern ocean, and are an important source of microfossils for paleoclimate reconstructions. Because of their importance in the environment, diatoms have been a focus of study on the physiology and ecophysiology of carbon fixation, in particular their CO2-concentrating mechanisms (CCMs) and Rubisco characteristics. While carbon fixation in diatoms is not as well understood as in certain model aquatic photoautotrophs, a greater number of species have been examined in diatoms. Recent work has highlighted a large diversity in the function, physiology, and kinetics of both the CCM and Rubisco between different diatom species. This diversity was unexpected since it has generally been assumed that CCMs and Rubiscos were similar within major algal lineages as the result of selective events deep in evolutionary history, and suggests a more recent co-evolution between the CCM and Rubisco within diatoms. This review explores our current understanding of the diatom CCM and highlights the diversity of both the CCM and Rubisco kinetics. We will suggest possible environmental, physiological, and evolutionary drivers for the co-evolution of the CCM and Rubisco in diatoms. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Bilateral key comparison CCM.P-K3.1 for absolute pressure measurements from 3 × 10-6 Pa to 9 × 10-4 Pa

NASA Astrophysics Data System (ADS)

Fedchak, J. A.; Bock, Th; Jousten, K.

2014-01-01

This report describes the bilateral key comparison CCM.P-K3.1 between the National Institute of Standards and Technology (NIST) and Physikalisch-Technische Bundesanstalt (PTB) for absolute pressure in the range from 3 × 10-6 Pa to 9 × 10-4 Pa. This comparison was a follow-up to the comparison CCM.P-K3. Two ionization gauges and two spinning rotor gauges (SRGs) were used as the transfer standards for the comparison. The SRGs were used to compare the standards at a pressure of 9 × 10-4 Pa and to normalize the ionization gauge readings. The two ionization gauges were used to compare the standards in the pressure range of from 3 × 10-6 Pa to 3 × 10-4 Pa. Both laboratories used dynamic expansion chambers as standards in the comparison. The two labs showed excellent agreement with each other and with the CCM.P-K3 key comparison reference value (KCRV) over the entire range. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Critical Communicative Methodology: Informing Real Social Transformation through Research

ERIC Educational Resources Information Center

Gomez, Aitor; Puigvert, Lidia; Flecha, Ramon

2011-01-01

The critical communicative methodology (CCM) is a methodological response to the dialogic turn of societies and sciences that has already had an important impact in transforming situations of inequality and exclusion. Research conducted with the CCM implies continuous and egalitarian dialogue among researchers and the people involved in the…

CCM key comparison CCM.D-K4 'Hydrometer'

NASA Astrophysics Data System (ADS)

Lorefice, S.; Becerra, L. O.; Lenard, E.; Lee, Y. J.; Lee, W. G.; Madec, T.; Meury, P. A.; Caceres, J.; Santos, C.; Vamossy, C.; Man, J.; Fen, K.; Toda, K.; Wright, J.; Bettin, H.; Toth, H.

2016-01-01

This report presents the comparison philosophy, methodology, analysis and the results of the designed CCM.D-K4 key comparison that covered the calibration of high resolution hydrometers for liquid and alcoholometers in the density range 600 kg/m3 to 2000 kg/m3 at the temperature of 20°C. The main purpose of this comparison was not only to evaluate the degree of equivalence in the calibration of high accuracy hydrometers between NMI participants, but also to link, were it is possible, the results of previous comparisons to Key Comparison Reference Values (KCRVs) of CCM.D-K4. Eleven NMI laboratories took part in the CCM.D-K4 divided in two groups (petals). With the CCM.D-K4 purpose, two similar sets consisting of three hydrometers for liquid density determinations and an alcoholometer were circulated to the NMI participants as a travelling standard in the time interval from January 2011 to April 2012. Twelve Key Comparison Reference Values (KCRVs) for each petal have been obtained at the density values related to the tested density marks of the transfer standards by the results of participants. The KCRVs and corresponding uncertainties were calculated by the weighted mean in the case of consistent results, otherwise the median was used. The degree of equivalence (DoE) with respect to the corresponding KCRV was determined for each participant and, in this particular comparison, the Weighted Least Squares (WLS) method was used to link the individual DoE of each participant by a continuous function. Significant drift of the transfer standards was not detected. This report also gives instructions on calculating pair-wise degrees of equivalence, with the addition of any information on correlations that may be necessary to estimate more accurately as well as the procedure for linking international comparisons to the CCM.D-K4. Finally an example of linkage to the CCM.D-K4 is given by dealing with the results of the bilateral comparison between INRiM and NMIA, which was added to this comparison so that all participants were engaged after the breakage of the 9340171 artefact. A particularly good agreement was found among the results provided by most of the participants, even if some systematic differences and either underestimated or overestimated uncertainties of the submitted results can be identified with respect to the KCRVs. In general the deviations of the laboratory results to the KCRVs are within of 1/3rd to 1/4th of a scale division and the uncertainty at 95% is usually within half a division. During the analysis of the submitted results, a systematic difference between the first and last immersed mark was also noted, possibly due to a temperature gradient along the stem and/or wetting of the stem around the tested mark, and therefore a corrected claimed uncertainty from each laboratory is expected. However this comparison may help the laboratories to solve some residual or marginal problems as well as to better understand the uncertainty components. The comparison fully supports the calibration measurement capabilities table in the BIPM key comparison database (KCDB). The results can be used to link regional comparisons to this CCM key comparison Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Convergent Cross Mapping: Basic concept, influence of estimation parameters and practical application.

PubMed

Schiecke, Karin; Pester, Britta; Feucht, Martha; Leistritz, Lutz; Witte, Herbert

2015-01-01

In neuroscience, data are typically generated from neural network activity. Complex interactions between measured time series are involved, and nothing or only little is known about the underlying dynamic system. Convergent Cross Mapping (CCM) provides the possibility to investigate nonlinear causal interactions between time series by using nonlinear state space reconstruction. Aim of this study is to investigate the general applicability, and to show potentials and limitation of CCM. Influence of estimation parameters could be demonstrated by means of simulated data, whereas interval-based application of CCM on real data could be adapted for the investigation of interactions between heart rate and specific EEG components of children with temporal lobe epilepsy.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.