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Sample records for mycobacterium smegmatis strain

  1. Ectoine biosynthesis in Mycobacterium smegmatis.

    PubMed

    Ofer, Naomi; Wishkautzan, Marina; Meijler, Michael; Wang, Ying; Speer, Alexander; Niederweis, Michael; Gur, Eyal

    2012-10-01

    Mycobacterium smegmatis is a commonly used mycobacterial model system. Here, we show that M. smegmatis protects itself against elevated salinity by synthesizing ectoine and hydroxyectoine and characterize the phenotype of a nonproducing mutant. This is the first analysis of M. smegmatis halotolerance and of the molecular mechanism that supports it.

  2. Selective targeting of Mycobacterium smegmatis with trehalose-functionalized nanoparticles†

    PubMed Central

    Jayawardana, Kalana W.; Jayawardena, H. Surangi N.; Wijesundera, Samurdhi A.; De Zoysa, Thareendra; Sundhoro, Madanodaya

    2015-01-01

    Silica and iron oxide nanoparticles with sizes ranging from 6 to 40 nm were functionalized with trehalose. The trehalose-conjugated nanoparticles showed strong interactions with Mycobacterium smegmatis (M. smegmatis) and minimal interactions with macrophage (RAW 264.7) or A549 cells. In addition, trehalose-conjugated silica nanoparticles selectively interacted with M. smegmatis on M. smegmatis-treated A549 cells, demonstrating high potential of trehalose in developing targeted therapy for treating mycobacterial infection. PMID:26121049

  3. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis

    PubMed Central

    Flint, Jessica L.; Kowalski, Joseph C.; Karnati, Pavan K.; Derbyshire, Keith M.

    2004-01-01

    Conjugal DNA transfer occurs by an atypical mechanism in Mycobacterium smegmatis. The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer. Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M. smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using two M. smegmatis donors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region of M. tuberculosis complements the conjugation phenotype of the RD1 mutants in M. smegmatis. Our results indicate that the M. tuberculosis and M. smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus. PMID:15314236

  4. Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.

    PubMed

    Panas, Michael W; Jain, Paras; Yang, Hui; Mitra, Shimontini; Biswas, Debasis; Wattam, Alice Rebecca; Letvin, Norman L; Jacobs, William R

    2014-09-16

    Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.

  5. Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.

    PubMed

    Marshall, Darrell D; Halouska, Steven; Zinniel, Denise K; Fenton, Robert J; Kenealy, Katie; Chahal, Harpreet K; Rathnaiah, Govardhan; Barletta, Raúl G; Powers, Robert

    2017-03-03

    In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc(2)155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc(2)155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

  6. Amino Acid Transport in Mycobacterium smegmatis

    PubMed Central

    Yabu, Kunihiko

    1970-01-01

    The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion. PMID:5437732

  7. Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis

    PubMed Central

    Schultz, John C.; Elbein, Alan D.

    1974-01-01

    A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [14C]galactose from uridine 5′-diphosphate (UDP)-[14C]galactose and of [14C]glucose from UDP-[14C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [14C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described. Images PMID:4808895

  8. Intrinsic Macrolide Resistance in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)

    PubMed Central

    Nash, Kevin A.

    2003-01-01

    High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc2155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 μg/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance. PMID:14506008

  9. RNase HI Is Essential for Survival of Mycobacterium smegmatis

    PubMed Central

    Minias, Alina E.; Brzostek, Anna M.; Korycka- Machala, Malgorzata; Dziadek, Bozena; Minias, Piotr; Rajagopalan, Malini; Madiraju, Murty; Dziadek, Jaroslaw

    2015-01-01

    RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential. PMID:25965344

  10. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    PubMed

    Agrawal, Priyanka; Miryala, Sandeep; Varshney, Umesh

    2015-01-01

    Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  11. Photodynamic inactivation of the models Mycobacterium phlei and Mycobacterium smegmatis in vitro

    NASA Astrophysics Data System (ADS)

    Bruce-Micah, R.; Gamm, U.; Hüttenberger, D.; Cullum, J.; Foth, H.-J.

    2009-07-01

    Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins or their derivatives. The kinetics of porphyrin synthesis after treatment with the precursor ALA and the accumulation of the Chlorin e6 and the following illumination were studied. The goal was to estimate effectivity of the destructive power of these PS in vitro in respect of the physiological states of Mycobacteria. So the present results examine the cell destruction by PDI using ALA-induced Porphyrins and Chlorin e6 accumulated in Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. We could show that both Mycobacterium after ALA and Chlorin e6 application were killed by illumination with light of about 662 nm. A reduction of about 97% could be reached by using a lightdose of 70 mW/cm2.

  12. Importance of porins for biocide efficacy against Mycobacterium smegmatis.

    PubMed

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

    2011-05-01

    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.

  13. Mycobacterium smegmatis Erm(38) Is a Reluctant Dimethyltransferase

    PubMed Central

    Madsen, Christian Toft; Jakobsen, Lene; Douthwaite, Stephen

    2005-01-01

    The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant erm(38) in the nontuberculous species Mycobacterium smegmatis confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLSB) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLSB drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in M. smegmatis, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of M. smegmatis, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species. PMID:16127056

  14. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed Central

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-01-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed. PMID:9098072

  15. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-04-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

  16. 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.

  17. Differentiating between live and dead Mycobacterium smegmatis using autofluorescence

    PubMed Central

    Wong, Cynthia; Ha, Ngan P.; Pawlowski, Michal E.; Graviss, Edward A.; Tkaczyk, Tomasz S.

    2016-01-01

    While there have been research efforts to find faster and more efficient diagnostic techniques for tuberculosis (TB), it is equally important to monitor a patient’s response to treatment over time, especially with the increasing prevalence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB. Between sputum smear microscopy, culture, and GeneXpert, only culture can verify viability of mycobacteria. However, it may take up to six weeks to grow Mycobacterium tuberculosis (Mtb), during which time the patient may have responded to treatment or the mycobacteria are still viable because the patient has MDR or XDR TB. In both situations, treatment incurs increased patient costs and makes them more susceptible to host-drug effects such as liver damage. Coenzyme Factor 420 (F420) is a fluorescent coenzyme found naturally in mycobacteria, with an excitation peak around 420 nm and an emission peak around 470 nm. Using Mycobacterium smegmatis, we show that live and dead mycobacteria undergo different rates of photobleaching over a period of 2 min. These preliminary experiments suggest that the different photobleaching rates could be used to help monitor a patient’s response to TB treatment. In future studies, we propose to describe these experiments with Mtb as both M. smegmatis and Mtb use F420. PMID:27742463

  18. Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis

    PubMed Central

    Evangelopoulos, Dimitrios; Gupta, Antima; Lack, Nathan A.; Maitra, Arundhati; ten Bokum, Annemieke M.C.; Kendall, Sharon; Sim, Edith; Bhakta, Sanjib

    2014-01-01

    Summary MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC protein. We identified the formation of a DNA:AraC protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To characterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔaraC mutant strain where the araC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔaraC strain when compared to the wild type strain indicating an activator effect of the AraC protein on the expression of the nat operon genes. PMID:25443504

  19. Enhanced Priming of Adaptive Immunity by Mycobacterium smegmatis Mutants with High-Level Protein Secretion

    PubMed Central

    Taylor, Natalie; Bahunde, Faith; Thompson, Afton; Yu, Jae-Sung; Jacobs, William R.; Letvin, Norm L.; Haynes, Barton F.

    2012-01-01

    Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion (“high secretors”) and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8+ T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8+ T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8+ T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses. PMID:22787192

  20. Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

    PubMed Central

    Cáceres, N E; Harris, N B; Wellehan, J F; Feng, Z; Kapur, V; Barletta, R G

    1997-01-01

    D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation. PMID:9260945

  1. Identification of a multidrug efflux pump in Mycobacterium smegmatis.

    PubMed

    Bansal, Ankita; Mallik, Dhriti; Kar, Debasish; Ghosh, Anindya S

    2016-07-01

    Cell wall impermeability and active efflux of drugs are among the primary reasons for drug resistance in mycobacteria. Efflux pumps are tripartite membrane localized transport proteins that expel drug molecules outside the cells. Several of such efflux pumps are annotated in mycobacteria, but few have been characterized, like MSMEG_2991, a putative efflux pump permease of Mycobacterium smegmatis To substantiate this, we overexpressed MSMEG_2991 protein in Escherichia coli 2443. Expression of MSMEG_2991 elevated the resistance towards structurally unrelated groups of antibiotics. An active antibiotic efflux pump nature of MSMEG_2991 was revealed by assessing the acquisition of ciprofloxacin in the absence and presence of the efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazone, indicating the involvement of proton-motive force (pmf) during the efflux activity. MSMEG_2991 expression elevated biofilm formation in E. coli by 4-fold, keeping parity to some of the earlier reported efflux pumps. In silico analysis suggested the presence of 12 transmembrane helices in MSMEG_2991 resembling EmrD efflux pump of E. coli Based on in vivo and in silico analyses, MSMEG_2991 may be designated as a pmf-mediated multidrug efflux pump protein that expels diverse groups of antibiotics and might as well be involved in the biofilm enhancement.

  2. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.

    PubMed Central

    Lee, M H; Pascopella, L; Jacobs, W R; Hatfull, G F

    1991-01-01

    Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified. Images PMID:1901654

  3. A novel marRAB operon contributes to the rifampicin resistance in Mycobacterium smegmatis.

    PubMed

    Zhang, Haiwei; Gao, Long; Zhang, Jiaoling; Li, Weihui; Yang, Min; Zhang, Hua; Gao, Chunhui; He, Zheng-Guo

    2014-01-01

    The multiple-antibiotic resistance regulator (MarR) plays an important role in modulating bacterial antibiotic resistance. However, the regulatory model of the marRAB operon in mycobacteria remains to be characterized. Here we report that a MarR, encoded by Ms6508, and its marRAB operon specifically contribute to rifampicin (RIF) resistance in Mycobacterium smegmatis. We show that the MarR recognizes a conserved 21-bp palindromic motif and negatively regulates the expression of two ABC transporters in the operon, encoded by Ms6509-6510. Unlike other known drug efflux pumps, overexpression of these two ABC transporters unexpectedly increased RIF sensitivity and deletion of these two genes increased mycobacterial resistance to the antibiotic. No change can be detected for the sensitivity of recombinant mycobacterial strains to three other anti-TB drugs. Furthermore, HPLC experiments suggested that Ms6509-Ms6510 could pump RIF into the mycobacterial cells. These findings indicated that the mycobacterial MarR functions as a repressor and constitutively inhibits the expression of the marRAB operon, which specifically contributes to RIF resistance in M. smegmatis. Therefore, our data suggest a new regulatory mechanism of RIF resistance and also provide the new insight into the regulatory model of a marRAB operon in mycobacteria.

  4. 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

  5. Superoxide Generation and Its Involvement in the Growth of Mycobacterium smegmatis

    PubMed Central

    Yeware, Amar M.; Shurpali, Ketaki D.; Athalye, Meghana C.; Sarkar, Dhiman

    2017-01-01

    Superoxide generation is inevitable in aerobic organisms, most of which have developed mechanisms to detoxify superoxides. However, its significance has not been clearly understood in mycobacteria. This study demonstrates that NADH oxidase is the major source of superoxide in Mycobacterium smegmatis and elucidates the involvement of superoxide in M. smegmatis growth. The maximum inhibition of superoxide generation was observed in the presence of diphenyleneiodonium chloride (DPI), an NADH oxidase inhibitor, compared to other standard inhibitors. After incubation for 24 h, the number of colony forming units (CFUs) was reduced by 6.8 log10 compared to the untreated culture. The inhibitory effect of DPI on M. smegmatis was reversed when the same culture was supplemented with menadione and pyrogallol, which are superoxide generators. Thus, this study reports the source of superoxide generation and its involvement in the growth of M. smegmatis. PMID:28194149

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

    PubMed

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

    2016-05-07

    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.

  7. Mycobacterium smegmatis synthesizes in vitro androgens and estrogens from different steroid precursors.

    PubMed

    Dlugovitzky, Diana G; Fontela, María Sol; Martinel Lamas, Diego J; Valdez, Ricardo A; Romano, Marta C

    2015-07-01

    Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that (3)H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation. (3)H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and (3)H-testosterone was transformed to estrone and androstenedione. Incubation with (3)H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.

  8. Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

    PubMed Central

    Pelosi, Assunta; Smith, Danielle; Brammananth, Rajini; Topolska, Agnieszka; Billman-Jacobe, Helen; Nagley, Phillip

    2012-01-01

    Background Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. Methodology/Principal Findings We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. Conclusions/Significance MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria. PMID:22363734

  9. Molecular Cloning and Functional Analysis of a Novel Tetracycline Resistance Determinant, tet(V), from Mycobacterium smegmatis

    PubMed Central

    De Rossi, Edda; Blokpoel, Marian C. J.; Cantoni, Rita; Branzoni, Manuela; Riccardi, Giovanna; Young, Douglas B.; De Smet, Koen A. L.; Ciferri, Orio

    1998-01-01

    The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatis harboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to M. smegmatis and M. fortuitum. PMID:9687386

  10. Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2015-10-01

    Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.

  11. Molecular Analysis of the Gene Encoding F420-Dependent Glucose-6-Phosphate Dehydrogenase from Mycobacterium smegmatis

    PubMed Central

    Purwantini, Endang; Daniels, Lacy

    1998-01-01

    The gene fgd, which codes for F420-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from Mycobacterium smegmatis, and its sequence was determined and analyzed. A homolog of FGD which has a very high similarity to the M. smegmatis FGD-derived amino acid sequence was identified in Mycobacterium tuberculosis. FGD showed significant homology with F420-dependent N5,N10-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from M. tuberculosis and Archaeoglobus fulgidus, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases. Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences. Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences. Moderately high levels of FGD were expressed in Escherichia coli BL21(DE3) carrying fgd in pBluescript. PMID:9555906

  12. Diaryltriazenes as antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA) and Mycobacterium smegmatis.

    PubMed

    Vajs, Jure; Proud, Conor; Brozovic, Anamaria; Gazvoda, Martin; Lloyd, Adrian; Roper, David I; Osmak, Maja; Košmrlj, Janez; Dowson, Christopher G

    2017-02-15

    Diaryltriazene derivatives were synthesized and evaluated for their antimicrobial properties. Initial experiments showed some of these compounds to have activity against both methicillin-resistant strains of Staphylococus aureus (MRSA) and Mycobacterium smegmatis, with MICs of 0.02 and 0.03 μg/mL respectively. Those compounds with potent anti-staphylococcal and anti-mycobacterial activity were not found to act as growth inhibitors of mammalian cell lines or yeast. Furthermore, we demonstrated that one of the most active anti-MRSA diaryltriazene derivatives was subject to very low frequencies of resistance at <10(-9). Whole genome sequencing of resistant isolates identified mutations in the enzyme that lysylates phospholipids. This could result in the modification of phospholipid metabolism and consequently the characteristics of the staphylococcal cell membrane, ultimately modifying the sensitivity of these pathogens to triazene challenge. Our work has therefore extended the potential range of triazenes, which could yield novel antimicrobials with low levels of resistance.

  13. Quantitative mass spectrometry reveals plasticity of metabolic networks in Mycobacterium smegmatis.

    PubMed

    Chopra, Tarun; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; McKinney, John D

    2014-11-01

    Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

  14. Functional identification of MSMEG_6402 protein from Mycobacterium smegmatis in decaprenylphosphoryl-D-arabinose biosynthesis.

    PubMed

    Jiang, Tao; Cai, Lina; Zhao, Xiaojiao; He, Lianqi; Ma, Yufang; Zang, Shizhu; Zhang, Cuili; Li, Xinli; Xin, Yi

    2014-11-01

    The arabinogalactan (AG) of the mycobacterial cell wall consists of an arabinan region, a galactan region and a disaccharide linker. Decaprenylphosphoryl-D-arabinose (DPA) is the donor for arabinofuran residues, which are formed from phosphoribose diphosphate (PRPP) and decaprenyl phosphate (DP). DP is sequentially catalyzed by a three-step process that involves a transferase, a phosphatase and an epimerase. Rv3807c is a putative phospholipid phosphatase that might generate the intermediate product of decaprenyl-phosphoryl-ribose (DPR) in DPA biosynthesis. Mycobacterium smegmatis MSMEG_6402 is a homolog gene of Mycobacterium tuberculosis Rv3807c and was substituted for the functional identification of Rv3807c. Previously, we generated a conditional MSMEG_6402 gene knockout strain (M. sm-ΔM_6402) that exhibited significantly affected cell wall structure. To understand the function of MSMEG_6402 in DPA biosynthesis, this gene was amplified and expressed, and the resulting protein was identified and purified using a His-tagged approach. A MSMEG_6402 enzymatic reaction system with PRPP and DP as substrates was utilized, and the reaction products were separated using thin layer chromatography (TLC). The results revealed a specific lipid-linked sugar band that appeared in the reaction with the addition of MSMEG_6402. Furthermore, ESI-MS detection was utilized in this study, and the results revealed that the enzymatic reaction products involving MSMEG_6402 included DPPR and a sodium ion adduct of DPR. Additionally, the phosphatase activity of MSMEG_6402 was also determined through phosphate group detection using the colorimetric method. Based on our results together with the results of previous studies, including the functional identification and bioinformatics analysis of M. tuberculosis Rv3807c, we propose that MSMEG_6402, as a phosphatase, has an intimate relationship with DPA biosynthesis.

  15. Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis

    PubMed Central

    Mishra, Abhishek; Sarkar, Dhiman

    2015-01-01

    Vitamin C is a critical dietary nutrient in human which has a wide range of regulatory effects on gene expression and physiology of Mycobacterium tuberculosis that leads to a dormant drug-tolerant phenotype. In the presence of iron, vitamin C shows a high bactericidal activity even in the drug resistant phenotype of M. tuberculosis. The regulatory mechanisms underlying vitamin C induced adaptations are largely unknown due to lack of functional genomics data in this field. In this study, we attempt to characterize the direct effect of vitamin C treatment on the physiology of actively growing Mycobacterium smegmatis. The study chose M. smegmatis as it is a fast-growing bacterium and a non-pathogenic model system which shares many physiological features with the pathogenic M. tuberculosis including dormancy and its regulation. The proteomic adaptation of M. smegmatis on vitamin C treatment demonstrates the important changes in cellular and metabolic process such as reversal of tricarboxylic acid cycle, decrease in ATP synthesis, decrease in iron acquisition and storage, and induction of dormancy regulators WhiB3, PhoP, and Lsr2. PMID:26042100

  16. Rel Is Required for Morphogenesis of Resting Cells in Mycobacterium smegmatis

    PubMed Central

    Wu, Mu-Lu; Chan, Chuu Ling; Dick, Thomas

    2016-01-01

    Recently we showed that upon transfer of growing Mycobacterium smegmatis into saline, the bacilli exited the canonical cell division cycle and formed septated multi-nucleoided cells. Under shock starvation (i.e., in saline without any carbon source), differentiation terminated at this stage with internally remodeled Large Resting Cells (LARCs). Whereas under gentle starvation (i.e., in saline with trace amounts of a carbon source), the septated multi-nucleoided bacilli completed cell division and separated into mono-nucleoided Small Resting Cells (SMRCs). This demonstrated that the non-sporulating mycobacteria are in fact capable of forming morphologically differentiated resting cells when exposed to starvation. Depending on the specific starvation conditions they can form two different resting cell types, LARCs or SMRCs, which share a common cellular differentiation pathway. The mRNA encoding the (p)ppGpp synthetase Rel was found to be transiently upregulated immediately upon starvation under both conditions, suggesting a role for the stringent response factor in both LARC and SMRC development. Here, we disrupted Rel function by generating two types of mutant M. smegmatis strains: a rel nonsense mutant (relE4TAG) in which translation is prematurely terminated at codon 4, and a rel deletion mutant (Δrel) in which the entire coding sequence was deleted. Both mutants showed identical phenotypes: sparse septum formation, less DNA compaction, and failure in formation of both the septated multi-nucleoided LARCs and the small-cell morphotype SMRC under starvation conditions. All phenotypes were rescued through the introduction of a wild-type copy of rel. Therefore, we conclude that loss-of-function mutations in rel block the development of both LARCs and SMRCs by preventing the first morphogenetic step in mycobacterial resting cell development, the formation of septated multi-nucleoided cells. Interestingly, in contrast to Rel’s role in most other bacteria, starvation

  17. In vitro synergic effect of beta-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis.

    PubMed

    Silva, Joas L da; Mesquita, Amanda R C; Ximenes, Eulalia A

    2009-07-01

    Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of (2)-lapachone alone and in combination with isoniazid against Mycobacterium fortuitum and Mycobacterium smegmatis via the Time-Kill Curve method. A 2 log10 CFU/mL reduction in the M. smegmatis culture was observed 72 h after adding (2)-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For M. fortuitum, a reduction of 1.55 log10 CFU/mL occurred in 24 h, but regrowth was seen in contact with (2)-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of M. fortuitum and M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.

  18. 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

  19. Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis.

    PubMed

    Edavana, Vineetha Koroth; Pastuszak, Irena; Carroll, J D; Thampi, Prajitha; Abraham, Edathera C; Elbein, Alan D

    2004-06-15

    Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.

  20. Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis

    PubMed Central

    Singh, Albel; Varela, Cristian; Bhatt, Kiranmai; Veerapen, Natacha; Lee, Oona Y. C.; Wu, Houdini H. T.; Besra, Gurdyal S.; Minnikin, David E.; Fujiwara, Nagatoshi; Teramoto, Kanae; Bhatt, Apoorva

    2016-01-01

    Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria. PMID:27741286

  1. The crystal structure of FdxA, a 7Fe ferredoxin from Mycobacterium smegmatis

    SciTech Connect

    Ricagno, Stefano; De Rosa, Matteo; Aliverti, Alessandro; Zanetti, Giuliana; Bolognesi, Martino . E-mail: martino.bolognesi@unimi.it

    2007-08-17

    Mycobacterium smegmatis ferredoxin FdxA, which has an orthologue ferredoxin in Mycobacterium tuberculosis, FdxC, contains both one [3Fe-4S] and one [4Fe-4S] cluster. M. smegmatis FdxA has been shown to be a preferred ferredoxin substrate of FprA [F. Fischer, D. Raimondi, A. Aliverti, G. Zanetti, Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase, Eur. J. Biochem. 269 (2002) 3005-3013], an adrenodoxin reductase-like flavoprotein of M. tuberculosis, suggesting that M. tuberculosis FdxC could be the physiological partner of the enzyme in providing reducing power to the cytochromes P450. We report here the crystal structure of FdxA at 1.6 A resolution (R {sub factor} 16.5%, R {sub free} 20.2%). Besides providing an insight on protein architecture for this 106-residue ferredoxin, our crystallographic investigation highlights lability of the [4Fe-4S] center, which is shown to loose a Fe atom during crystal growth. Due to their high similarity (87% sequence identity), the structure here reported can be considered a valuable model for M. tuberculosis FdxC, thus representing a step forward in the study of the complex mycobacterial redox pathways.

  2. Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival*

    PubMed Central

    Frampton, Rebekah; Aggio, Raphael B. M.; Villas-Bôas, Silas G.; Arcus, Vickery L.; Cook, Gregory M.

    2012-01-01

    The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTAtriple, exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTAtriple mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTAtriple mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTAtriple mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTAtriple mutant may explain the survival defect in complex growth medium. PMID:22199354

  3. Cloning, expression, and transcription analysis of L-arabinose isomerase gene from Mycobacterium smegmatis SMDU.

    PubMed

    Takata, Goro; Poonperm, Wayoon; Rao, Devendar; Souda, Akane; Nishizaki, Tomoe; Morimoto, Kenji; Izumori, Ken

    2007-12-01

    The L-arabinose metabolic gene cluster, araA, araB, araD, araG, araH and araR, encoding L-arabinose isomerase (L-AI) and its accessory proteins was cloned from Mycobacterium smegmatis SMDU and sequenced. The deduced amino acid sequence of araA displayed highest identity with that of Bacillus subtilis (52%). These six genes comprised the L-arabinose operon, and its genetic arrangement was similar to that of B. subtilis. The L-AI gene (araA), encoding a 501 amino acid protein with a calculated molecular mass of 54,888 Da, was expressed in Escherichia coli. The productivity and overall enzymatic properties of the recombinant L-AI were almost same as the authentic L-AI from M. smegmatis. Although the recombinant L-AI showed high substrate specificity, as did L-AI from other organisms, this enzyme catalyzed not only isomerization of L-arabinose-L-ribulose and D-galactose-D-tagatose but also isomerization of L-altrose-L-psicose and L-erythrulose-L-threose. In combination with L-AI from M. smegmatis, L-threose and L-altrose can be produced from cheap and abundant erythritol and D-fructose respectively, indicating that this enzyme has great potential for biological application in rare sugar production. Transcription analysis using various sugars revealed that this enzyme was significantly induced not only by L-arabinose and D-galactose but also by L-ribose, galactitol, L-ribulose, and L-talitol. This different result of transcription mediated by sugars from that of E. coli suggests that the transcriptional regulation of araA from M. smegmatis against sugar is loose compared with that from E. coli, and that it depends on the hydroxyl configuration at C2, C3 and C4 positions of sugars.

  4. Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

    PubMed

    Viswanathan, Gopinath; Joshi, Shrilaxmi V; Sridhar, Aditi; Dutta, Sayantanee; Raghunand, Tirumalai R

    2015-12-10

    Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis.

  5. Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis

    PubMed Central

    García, Maria de los Angeles; 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. PMID:25548767

  6. Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

    PubMed Central

    Janardan, Neelanjana; Harijan, Rajesh K.; Wierenga, Rikkert K.; Murthy, Mathur R. N.

    2012-01-01

    An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function. PMID:22844533

  7. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  8. Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants.

    PubMed

    Mativandlela, Sannah Patience Nkami; Meyer, Jacob Jacobus Marion; Hussein, Ahmed A; Houghton, Peter J; Hamilton, Chris J; Lall, Namrita

    2008-06-01

    Seven ethnobotanically selected medicinal plants were screened for their antimycobacterial activity. The minimum inhibitory concentration (MIC) of four plants namely Artemisia afra, Dodonea angustifolia, Drosera capensis and Galenia africana ranged from 0.781 to 6.25 mg/mL against Mycobacterium smegmatis. G. africana showed the best activity exhibiting an MIC of 0.78 mg/mL and a minimum bactericidal concentration (MBC) of 1.56 mg/mL. The MICs of ethanol extracts of D. angustifolia and G. africana against M. tuberculosis were found to be 5.0 and 1.2 mg/mL respectively. The mammalian cytotoxicity IC(50) value of the most active antimycobacterial extract, from G. africana, was found to be 101.3 microg/mL against monkey kidney Vero cells. Since the ethanol G. africana displayed the best antimycobacterial activity, it was subjected to fractionation which led to the isolation of a flavone, 5,7,2'-trihydroxyflavone. The MIC of this compound was found to be 0.031 mg/mL against M. smegmatis and 0.10 mg/mL against M. tuberculosis. This study gives some scientific basis to the traditional use of these plants for TB-related symptoms.

  9. Distinct Responses of Mycobacterium smegmatis to Exposure to Low and High Levels of Hydrogen Peroxide

    PubMed Central

    Li, Xiaojing; Wu, Jun; Han, Jiao; Hu, Yongfei; Mi, Kaixia

    2015-01-01

    Hydrogen peroxide (H2O2) is a natural oxidant produced by aerobic organisms and gives rise to oxidative damage, including DNA mutations, protein inactivation and lipid damage. The genus Mycobacterium utilizes redox sensors and H2O2 scavenging enzymes for the detoxification of H2O2. To date, the precise response to oxidative stress has not been fully elucidated. Here, we compared the effects of different levels of H2O2 on transcription in M. smegmatis using RNA-sequencing. A 0.2 mM H2O2 treatment had little effect on the growth and viability of M. smegmatis whereas 7 mM H2O2 was lethal. Analysis of global transcription showed that 0.2 mM H2O2 induced relatively few changes in gene expression, whereas a large proportion of the mycobacterial genome was found to be differentially expressed after treatment with 7 mM H2O2. Genes differentially expressed following treatment with 0.2 mM H2O2 included those coding for proteins involved in glycolysis-gluconeogenesis and fatty acid metabolism pathways, and expression of most genes encoding ribosomal proteins was lower following treatment with 7 mM H2O2. Our analysis shows that M. smegmatis utilizes the sigma factor MSMEG_5214 in response to 0.2 mM H2O2, and the RpoE1 sigma factors MSMEG_0573 and MSMEG_0574 in response to 7 mM H2O2. In addition, different transcriptional regulators responded to different levels of H2O2: MSMEG_1919 was induced by 0.2 mM H2O2, while high-level induction of DevR occurred in response to 7 mM H2O2. We detected the induction of different detoxifying enzymes, including genes encoding KatG, AhpD, TrxB and Trx, at different levels of H2O2 and the detoxifying enzymes were expressed at different levels of H2O2. In conclusion, our study reveals the changes in transcription that are induced in response to different levels of H2O2 in M. smegmatis. PMID:26225431

  10. Expression, purification and functional characterization of AmiA of acetamidase operon of Mycobacterium smegmatis.

    PubMed

    Sundararaman, Balaji; Palaniyandi, Kannan; Venkatesan, Arunkumar; Narayanan, Sujatha

    2014-11-01

    Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.

  11. Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.

    PubMed Central

    Purwantini, E; Daniels, L

    1996-01-01

    A variety of Mycobacterium species contained the 5-deazaflavin coenzyme known as F420. Mycobacterium smegmatis was found to have a glucose-6-phosphate dehydrogenase that was dependent on F420 as an electron acceptor and which did not utilize NAD or NADP. The enzyme was purified by ammonium sulfate fractionation, phenyl-Sepharose column chromatography, F420-ether-linked aminohexyl-Sepharose 4B affinity chromatography, and quaternary aminoethyl-Sephadex column chromatography, and the sequence of the first 26 N-terminal amino acids has been determined. The response of enzyme activity to a range of pHs revealed a two-peak pattern, with maxima at pH 5.5 and 8.0. The apparent Km values for F420 and glucose-6-phosphate were, respectively, 0.004 and 1.6 mM. The apparent native and subunit molecular masses were 78,000 and approximately 40,000 Da, respectively. PMID:8631674

  12. Transcriptomic Characterization of an Infection of Mycobacterium smegmatis by the Cluster A4 Mycobacteriophage Kampy

    PubMed Central

    Saha, Margaret

    2015-01-01

    The mycobacteriophages, phages that infect the genus Mycobacterium, display profound genetic diversity and widespread geographical distribution, and possess significant medical and ecological importance. However, most of the majority of functions of mycobacteriophage proteins and the identity of most genetic regulatory elements remain unknown. We characterized the gene expression profile of Kampy, a cluster A4 mycobacteriophage, during infection of its host, Mycobacterium smegmatis, using RNA-Seq and mass spectrometry. We show that mycobacteriophage Kampy transcription occurs in roughly two phases, an early phase consisting of genes for metabolism, DNA synthesis, and gene regulation, and a late phase consisting of structural genes and lysis genes. Additionally, we identify the earliest genes transcribed during infection, along with several other possible regulatory units not obvious from inspection of Kampy's genomic structure. The transcriptional profile of Kampy appears similar to that of mycobacteriophage TM4 but unlike that of mycobacteriophage Giles, a result which further expands our understanding of the diversity of mycobacteriophage gene expression programs during infection. PMID:26513661

  13. Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis.

    PubMed

    Jeong, Jae-Hee; Cha, Hyung Jin; Ha, Sung-Chul; Rojviriya, Catleya; Kim, Yeon-Gil

    2014-10-03

    EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.

  14. Antibiotic Bactericidal Activity Is Countered by Maintaining pH Homeostasis in Mycobacterium smegmatis

    PubMed Central

    Bartek, I. L.; Reichlen, M. J.; Honaker, R. W.; Leistikow, R. L.; Clambey, E. T.; Scobey, M. S.; Hinds, A. B.; Born, S. E.; Covey, C. R.; Schurr, M. J.; Lenaerts, A. J.

    2016-01-01

    ABSTRACT Antibiotics target specific biosynthetic processes essential for bacterial growth. It is intriguing that several commonalities connect the bactericidal activity of seemingly disparate antibiotics, such as the numerous conditions that confer broad-spectrum antibiotic tolerance. Whether antibiotics kill in a manner unique to their specific targets or by a universal mechanism is a critical and contested subject. Herein, we demonstrate that the bactericidal activity of diverse antibiotics against Mycobacterium smegmatis and four evolutionarily divergent bacterial pathogens was blocked by conditions that worked to maintain intracellular pH homeostasis. Single-cell pH analysis demonstrated that antibiotics increased the cytosolic pH of M. smegmatis, while conditions that promoted proton entry into the cytosol prevented intracellular alkalization and antibiotic killing. These findings led to a hypothesis that posits antibiotic lethality occurs when antibiotics obstruct ATP-consuming biosynthetic processes while metabolically driven proton efflux is sustained despite the loss of proton influx via ATP synthase. Consequently, without a concomitant reduction in respiratory proton efflux, cell death occurs due to intracellular alkalization. Our findings indicate the effects of antibiotics on pH homeostasis should be considered a potential mechanism contributing to antibiotic lethality. IMPORTANCE Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our

  15. A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.

    PubMed

    Newton, Gerald L; Ta, Philong; Fahey, Robert C

    2005-11-01

    Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

  16. Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae.

    PubMed

    Santhosh, Ramachandran Sarojini; Pandian, Shunmugiah Karutha; Lini, Nirmala; Shabaana, Abdul Khader; Nagavardhini, Avuthu; Dharmalingam, Kuppamuthu

    2005-08-01

    Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of slashed circleC31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNA(Gln) amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also.

  17. Recombinant Mycobacterium smegmatis with a pMyong2 vector expressing Human Immunodeficiency Virus Type I Gag can induce enhanced virus-specific immune responses

    PubMed Central

    Kim, Byoung-Jun; Gong, Jeong-Ryeol; Kim, Ga-Na; Kim, Bo-Ram; Lee, So-Young; Kook, Yoon-Hoh; Kim, Bum-Joon

    2017-01-01

    Recently, we have developed a novel Mycobacterium-Escherichia coli shuttle vector system using pMyong2, which can provide an enhanced expression of heterologous genes in recombinant Mycobacterium smegmatis (rSmeg). To investigate the usefulness of rSmeg using pMyong2 in vaccine application, we vaccinated M. smegmatis with pMyong2 system expressing Human Immunodeficiency Virus Type I (HIV-1) Gag p24 antigen (rSmeg-pMyong2-p24) into mice and examined its cellular and humoral immune responses against HIV gag protein. We found that rSmeg-pMyong2-p24 expressed higher levels of Gag protein in bacteria, macrophage cell line (J774A.1) and mouse bone marrow derived dendritic cells (BMDCs) compared to rSmeg strains using two other vector systems, pAL5000 derived vector (rSmeg-pAL-p24) and the integrative plasmid, pMV306 (rSmeg-pMV306-p24). Inoculation of mice with rSmeg-pMyong2-p24 elicited more effective immunity compared to the other two rSmeg strains, as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, interferon gamma ELISPOT cell induction, and antibody production. Furthermore, rSmeg-pMyong2-p24 showed a higher level of cytotoxic T cell response against target cells expressing Gag p24 proteins. Our data suggest that Mycobacterium-Escherichia coli shuttle vector system with pMyong2 may provide an advantage in vaccine application of rSmeg over other vector systems. PMID:28300196

  18. 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.

  19. Ribosylative inactivation of rifampin by Mycobacterium smegmatis is a principal contributor to its low susceptibility to this antibiotic.

    PubMed Central

    Quan, S; Venter, H; Dabbs, E R

    1997-01-01

    Mycobacterium smegmatis inactivates rifampin by ribosylating this antibiotic. The gene responsible for this ability was cloned and was shown to confer low-level resistance to this antibiotic (MIC increase, about 12-fold) in related organisms. A 600-bp subclone responsible for ribosylating activity and resistance carried an open reading frame of 429 bp. Targeted disruption of the gene in M. smegmatis resulted in mutants with much increased susceptibility to rifampin (MICs of 1.5 instead of 20 microg/ml) as well as the loss of antibiotic-inactivating ability. Also, disruption of this gene led to a much lower frequency of occurrence of spontaneous high-level rifampin-resistant mutants. PMID:9371349

  20. Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

    PubMed

    Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

    2006-03-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

  1. Mycobacterium smegmatis DinB2 misincorporates deoxyribonucleotides and ribonucleotides during templated synthesis and lesion bypass

    PubMed Central

    Ordonez, Heather; Shuman, Stewart

    2014-01-01

    Mycobacterium smegmatis DinB2 is the founder of a clade of Y-family DNA polymerase that is naturally adept at utilizing rNTPs or dNTPs as substrates. Here we investigate the fidelity and lesion bypass capacity of DinB2. We report that DinB2 is an unfaithful DNA and RNA polymerase with a distinctive signature for misincorporation of dNMPs, rNMPs and oxoguanine nucleotides during templated synthesis in vitro. DinB2 has a broader mutagenic spectrum with manganese than magnesium, though low ratios of manganese to magnesium suffice to switch DinB2 to its more mutagenic mode. DinB2 discrimination against incorrect dNTPs in magnesium is primarily at the level of substrate binding affinity, rather than kpol. DinB2 can incorporate any dNMP or rNMP opposite oxo-dG in the template strand with manganese as cofactor, with a kinetic preference for synthesis of an A:oxo-dG Hoogsteen pair. With magnesium, DinB2 is adept at synthesizing A:oxo-dG or C:oxo-dG pairs. DinB2 effectively incorporates deoxyribonucleotides, but not ribonucleotides, opposite an abasic site, with kinetic preference for dATP as the substrate. We speculate that DinB2 might contribute to mycobacterial mutagenesis, oxidative stress and quiescence, and discuss the genetic challenges to linking the polymerase biochemistry to an in vivo phenotype. PMID:25352547

  2. Conversion of Mycobacterium smegmatis to a pathogenic phenotype via passage of epithelial cells during macrophage infection.

    PubMed

    Kim, Su-Young; Sohn, Hosung; Choi, Go-Eun; Cho, Sang-Nae; Oh, Taegwon; Kim, Hwa-Jung; Whang, Jake; Kim, Jong-Seok; Byun, Eui-Hong; Kim, Woo Sik; Min, Ki-Nam; Kim, Jin Man; Shin, Sung Jae

    2011-08-01

    Mycobacteria encounter many different cells during infection within their hosts. Although alveolar epithelial cells play an essential role in host defense as the first cells to be challenged upon contact with mycobacteria, they may contribute to the acquisition of mycobacterial virulence by increasing the expression of virulence or adaptation factors prior to being ingested by macrophages on the side of pathogens. From this aspect, the enhanced virulence of nonpathogenic Mycobacterium smegmatis (MSM) passed through human alveolar A549 epithelial cells (A-MSM) was compared to the direct infection of MSM (D-MSM) in THP-1 macrophages and mouse models. The intracellular growth rate and cytotoxicity of A-MSM were significantly increased in THP-1 macrophages. In addition, compared to D-MSM, A-MSM induced relatively greater interleukin (IL)-1β, IL-6, IL-8, IL-12, TNF-α, MIP-1α, and MCP-1 in THP-1 macrophages. As a next step, a more persistent A-MSM infection was observed in a murine infection model with the development of granulomatous inflammation. Finally, 58 genes induced specifically in A-MSM were partially identified by differential expression using a customized amplification library. These gene expressions were simultaneously maintained in THP-1 infection but no changes were observed in D-MSM. Bioinformatic analysis revealed that these genes are involved mainly in bacterial metabolism including energy production and conversion, carbohydrate, amino acid, and lipid transport, and metabolisms. Conclusively, alveolar epithelial cells promoted the conversion of MSM to the virulent phenotype prior to encountering macrophages by activating the genes required for intracellular survival and presenting its pathogenicity.

  3. Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model.

    PubMed

    Tsolaki, Anthony G; Nagy, Judit; Leiva, Sergio; Kishore, Uday; Rosenkrands, Ida; Robertson, Brian D

    2013-07-01

    In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.

  4. The growth and survival of Mycobacterium smegmatis is enhanced by co-metabolism of atmospheric H2.

    PubMed

    Greening, Chris; Villas-Bôas, Silas G; Robson, Jennifer R; Berney, Michael; Cook, Gregory M

    2014-01-01

    The soil bacterium Mycobacterium smegmatis is able to scavenge the trace concentrations of H2 present in the atmosphere, but the physiological function and importance of this activity is not understood. We have shown that atmospheric H2 oxidation in this organism depends on two phylogenetically and kinetically distinct high-affinity hydrogenases, Hyd1 (MSMEG_2262-2263) and Hyd2 (MSMEG_2720-2719). In this study, we explored the effect of deleting Hyd2 on cellular physiology by comparing the viability, energetics, transcriptomes, and metabolomes of wild-type vs. Δhyd2 cells. The long-term survival of the Δhyd2 mutant was significantly reduced compared to the wild-type. The mutant additionally grew less efficiently in a range of conditions, most notably during metabolism of short-chain fatty acids; there was a twofold reduction in growth rate and growth yield of the Δhyd2 strain when acetate served as the sole carbon source. Hyd1 compensated for loss of Hyd2 when cells were grown in a high H2 atmosphere. Analysis of cellular parameters showed that Hyd2 was not necessary to generate the membrane potential, maintain intracellular pH homeostasis, or sustain redox balance. However, microarray analysis indicated that Δhyd2 cells were starved for reductant and compensated by rewiring central metabolism; transcripts encoding proteins responsible for oxidative decarboxylation pathways, the urea cycle, and ABC transporter-mediated import were significantly more abundant in the Δhyd2 mutant. Metabolome profiling consistently revealed an increase in intracellular amino acids in the Δhyd2 mutant. We propose that atmospheric H2 oxidation has two major roles in mycobacterial cells: to generate reductant during mixotrophic growth and to sustain the respiratory chain during dormancy.

  5. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

    PubMed Central

    Gupta, Kajal; Singh, Sameer; van Hoek, Monique L.

    2015-01-01

    Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism

  6. Immunotherapeutic efficacy of recombinant Mycobacterium smegmatis expressing Ag85B-ESAT6 fusion protein against persistent tuberculosis infection in mice.

    PubMed

    Wang, Ping; Wang, Limei; Zhang, Wei; Bai, Yinlan; Kang, Jian; Hao, Yanfei; Luo, Tailai; Shi, Changhong; Xu, Zhikai

    2014-01-01

    The application of immunotherapy in combination with chemotherapy is considered an effective treatment strategy against persistent Mycobacterium tuberculosis (Mtb) infection. In this study, we constructed a novel recombinant Mycobacterium smegmatis (rMS) strain that expresses Ag85B and ESAT6 fusion protein (AE-rMS). Immunization of C57BL/6 mice with AE-rMS generated mainly Th1-type immune responses by strongly stimulating IFN-γ- and IL-2-producing splenocytes and increasing antigen-specific cytotoxic T lymphocyte (CTL) activity. To test the immunotherapeutic efficacy of AE-rMS, a persistent tuberculosis infection (PTBI) model was established via tail-vein injection of C57BL/6 mice with 1×10(4) colony forming units (CFU) of Mtb strain H37Rv in combination with concurrent chemotherapy drugs isoniazid (INH) and pyrazinamide (PZA). PTBI mice immunized with AE-rMS showed high levels of IFN-γ secreted by splenocytes and decreased bacteria loads in lung. Treatment with only the anti-tuberculosis (anti-TB) drugs RFP and INH (RI), decreased bacteria loads to low levels, with the Th1-type immune response further attenuated. Moreover, AE-rMS, when combined with RI treatment, further reduced the bacteria load as well as the pathological tissue damage in lung. Together, these results demonstrated the essential roles of AE-rMS-induced Th1-type responses, providing an effective treatment strategy by combining AE-rMS and RI for persistent TB.

  7. Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3.

    PubMed

    Kim, Mi-Sun; Jang, Jichan; Ab Rahman, Nurlilah Binte; Pethe, Kevin; Berry, Edward A; Huang, Li-Shar

    2015-06-05

    Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a "supercomplex" with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.

  8. Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

    PubMed Central

    Jeong, Ji-A; Hyun, Jaekyung

    2015-01-01

    ABSTRACT In the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding alanine dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism. IMPORTANCE In mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD+ pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium

  9. Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-γ priming.

    PubMed

    Huang, Ying; Zhou, Xiangmei; Bai, Yu; Yang, Lifeng; Yin, Xiaomin; Wang, Zhigang; Zhao, Deming

    2012-11-09

    Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.

  10. 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

  11. Expression, Purification And Crystallization of Native And Selenomethionine Labeled Mycobacterium Tuberculosis FDG1 (Rv0407) Using a Mycobacterium Smegmatis Expression System

    SciTech Connect

    Bashiri, G.; Squire, C.J.; Baker, E.N.; Moreland, N.J.

    2009-06-01

    FGD1 is an F{sub 420}-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGD1 using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smegmatis was used, soluble protein yields of 7 mg/L of culture were achieved. Both native and selenomethionine-substituted FGD1 were obtained by culturing M. smegmatis in autoinduction media protocols originally developed for E. coli. Using these media afforded the advantages of decreased handling, as cultures did not require monitoring of optical density and induction, and reduced cost by removing the need for expensive ADC enrichment normally used in mycobacterial cultures. Selenomethionine was efficiently incorporated at levels required for multiwavelength anomalous diffraction experiments used in crystal structure determination. As far as we are aware this is the first protocol for preparation of selenomethionine-substituted protein in mycobacteria. Native and selenomethionine-labeled FGD1 were successfully crystallized by vapor diffusion, with the crystals diffracting to 2.1 AA resolution.

  12. Mycobacterium tuberculosis PE_PGRS18 enhances the intracellular survival of M. smegmatis via altering host macrophage cytokine profiling and attenuating the cell apoptosis.

    PubMed

    Yang, Wenmin; Deng, Wanyan; Zeng, Jie; Ren, Sai; Ali, Md Kaisar; Gu, Yinzhong; Li, Yangyuling; Xie, Jianping

    2017-04-01

    Mycobacterium tuberculosis PE/PPE family proteins, named after the presence of conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains at N-terminal, are prevalent in M. tuberculosis genome. The function of most PE/PPE family proteins remains elusive. To characterize the function of PE_PGRS18, the encoding gene was heterologously expressed in M. smegmatis, a nonpathogenic mycobacterium. The recombinant PE_PGRS18 is cell wall associated. M. smegmatis PE_PGRS18 recombinant showed differential response to stresses and altered the production of host cytokines IL-6, IL-1β, IL-12p40 and IL-10, as well as enhanced survival within macrophages largely via attenuating the apoptosis of macrophages. In summary, the study firstly unveiled the role of PE_PGRS18 in physiology and pathogenesis of mycobacterium.

  13. [The role of 2-C-methylerythritol-2,4-cyclopyrophosphate in the resuscitation of the "nonculturable" forms of Mycobacterium smegmatis].

    PubMed

    Goncharenko, A V; Ershov, Iu V; Salina, E G; Wiesner, J; Vostroknutova, G N; Sandanov, A A; Kapel'iants, A S; Ostrovskiĭ, D N

    2007-01-01

    2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the biosynthesis of isoprenoid compounds in bacteria, was found to be capable of exerting a resuscitating effect on resting Mycobacterium smegmatis cells. The introduction of an additional copy of the ispE gene encoding cytidyl-methylerythritol kinase, an enzyme involved in MEC synthesis in M. smegmatis, resulted in the emergence of a capacity for spontaneous reactivation of "nonculturable" M. smegmatis cells, which is not characteristic of the wild-type cells of this species. The involvement of MEC in the transition from the "nonculturable" state to the state of active growth is indicative of a previously unknown function of MEC, assumed to consist in regulation of the bacterial genome activity.

  14. A structural and functional investigation of a novel protein from Mycobacterium smegmatis implicated in mycobacterial macrophage survivability.

    PubMed

    Shahine, Adam; Littler, Dene; Brammananath, Rajini; Chan, Phooi Y; Crellin, Paul K; Coppel, Ross L; Rossjohn, Jamie; Beddoe, Travis

    2014-09-01

    The success of pathogenic mycobacterial species is owing in part to their ability to parasitize the generally inhospitable phagosomal environment of host macrophages, utilizing a variety of strategies to avoid their antimycobacterial capabilities and thereby enabling their survival. A recently identified gene target in Mycobacterium smegmatis, highly conserved within Mycobacterium spp. and denoted MSMEG_5817, has been found to be important for bacterial survival within host macrophages. To gain insight into its function, the crystal structure of MSMEG_5817 has been solved to 2.40 Å resolution. The structure reveals a high level of structural homology to the sterol carrier protein (SCP) family, suggesting a potential role of MSMEG_5817 in the binding and transportation of biologically relevant lipids required for bacterial survival. The lipid-binding capacity of MSMEG_5817 was confirmed by ELISA, revealing binding to a number of phospholipids with varying binding specificities compared with Homo sapiens SCP. A potential lipid-binding site was probed by alanine-scanning mutagenesis, revealing structurally relevant residues and a binding mechanism potentially differing from that of the SCPs.

  15. The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan.

    PubMed

    Escuyer, V E; Lety, M A; Torrelles, J B; Khoo, K H; Tang, J B; Rithner, C D; Frehel, C; McNeil, M R; Brennan, P J; Chatterjee, D

    2001-12-28

    The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to

  16. Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

    PubMed Central

    Gupta, Kuldeepkumar Ramnaresh; Baloni, Priyanka; Indi, Shantinath S.

    2016-01-01

    ABSTRACT The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins RelMsm and DcpA, encoded by relMsm and dcpA genes, respectively. We have previously shown that the ΔrelMsm and ΔdcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571–2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated ΔrelMsm and ΔdcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in

  17. The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

    PubMed Central

    Jamet, Stevie; Slama, Nawel; Domingues, Joana; Laval, Françoise; Texier, Pauline; Eynard, Nathalie; Quémard, Annaik; Peixoto, Antonio; Lemassu, Anne; Daffé, Mamadou; Cam, Kaymeuang

    2015-01-01

    Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs. PMID:26701652

  18. Generation of recombinant bacillus Calmette-Guérin and Mycobacterium smegmatis expressing BfpA and intimin as vaccine vectors against enteropathogenic Escherichia coli.

    PubMed

    Vasconcellos, Halyka Luzorio Franzotti; Scaramuzzi, Karina; Nascimento, Ivan Pereira; Da Costa Ferreira, Jorge M; Abe, Cecilia M; Piazza, Roxane M F; Kipnis, Andre; Dias da Silva, Wilmar

    2012-09-07

    Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in children. EPEC adheres to the intestinal epithelium and causes attaching and effacing (A/E) lesions. Recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express either BfpA or intimin. The entire bfpA gene and a portion of the intimin gene were amplified by PCR from EPEC genomic DNA and inserted into the pMIP12 vector at the BamHI/KpnI sites. The pMIP_bfpA and pMIP_intimin vectors were introduced separately into Smeg and BCG. Recombinant clones were selected based on kanamycin resistance and designated rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin was detected by Immunoblotting using polyclonal anti-BfpA and anti-intimin antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice by assaying the feces and serum for the presence of anti-BfpA and anti-intimin IgA and IgG antibodies. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA, whereas TNF-γ was produced in mice immunized with recombinant intimin. The adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies from mice immunized with recombinant BfpA or intimin but not by antibodies from non-immunized mice. Immunogenic non-infectious vectors containing relevant EPEC virulence genes may be promising vaccine candidates.

  19. The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

    PubMed Central

    Chen, Xuejie; Li, Chandan; Wang, Li; Liu, Yi; Li, Chuanyou; Zhang, Junjie

    2016-01-01

    PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. It has been reported that that M. smegmatis PafA can be poly-pupylated. In this study, the mechanism of PafA self-pupylation is explored. We found that K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in M. smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop. The polymeric Pup chain formed through K7/K31 linkage is much more sensitive to Dop than the mono-Pup directly attached to PafA. Moreover, self-pupylation of PafA is involved in the regulation of its stability in vivo in a proteasome-dependent manner, suggesting that PafA self-pupylation functions as a mechanism in the auto-regulation of the Pup-proteasome system. PMID:26953889

  20. Evaluation of the humoral immune response and cross reactivity against Mycobacterium tuberculosis of mice immunized with liposomes containing glycolipids of Mycobacterium smegmatis

    PubMed Central

    2013-01-01

    Mycobacterium smegmatis (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis. PMID:23458474

  1. Crystal Structure of Reduced MsAcg, a Putative Nitroreductase from Mycobacterium smegmatis and a Close Homologue of Mycobacterium tuberculosis Acg

    PubMed Central

    Chauviac, François-Xavier; Bommer, Martin; Yan, Jun; Parkin, Gary; Daviter, Tina; Lowden, Philip; Raven, Emma L.; Thalassinos, Konstantinos; Keep, Nicholas H.

    2012-01-01

    This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria's adaptation to a dormancy state. PMID:23148223

  2. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis.

    PubMed

    Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S

    2014-10-01

    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.

  3. 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

  4. Steady-State and Pre-Steady-State Kinetic Analysis of Mycobacterium smegmatis Cysteine Ligase (MshC)

    PubMed Central

    Fan, Fan; Luxenburger, Andreas; Painter, Gavin F.; Blanchard, John S

    2008-01-01

    Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth, and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-D. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady state and pre-steady state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady state kinetic parameters were determined to be: kcat equal to 3.15 s−1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analog, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and non-competitive inhibition versus cysteine, with an inhibition constant of ~306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid quench techniques, giving rates of ~9.4 s−1 and ~5.2 s−1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC. PMID:17848100

  5. Adaptation of Mycobacterium smegmatis to an Industrial Scale Medium and Isolation of the Mycobacterial PorinMspA

    PubMed Central

    Wendel, Sebastian O; Perera, Ayomi S; Pfromm, Peter H; Czermak, Peter; Bossmann, Stefan H

    2013-01-01

    The adaptation of the organism to a simple and cost-effective growth medium is mandatory in developing a process for large scale production of the octamericporinMspA, which is isolated from Mycobacterium smegmatis. A fermentation optimization with the minimal nutrients required for growth has been performed. During the fermentation, the iron- and ammonium chloride concentrations in the medium were varied to determine their impact on the observed growth rates and cell mass yields. Common antibiotics to control contamination were eliminated in favor of copper sulfate to reduce costs. MspA has been successfully isolated from the harvested M. smegmatisusing aqueous nOPOE (n-octyloligooxyethylene) at 65°C. Because of the extraordinary stability of MspA, it is possible to denature and precipitate virtually all other proteins and contaminants by following this approach. To further purify the product, acetone is used for precipitation. Gel electrophoresis confirmed the presence and purity of MspA. A maximum of 840µg (via Bradford assay) of pure MspA per liter of the optimized simple growth medium has been obtained. This is a 40% increase with respect to the previously reported culture medium for MspA. PMID:23802026

  6. 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

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

    PubMed

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

    2013-09-01

    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.

  8. Novel Polyoxyethylene-Containing Glycolipids Are Synthesized in Corynebacterium matruchotii and Mycobacterium smegmatis Cultured in the Presence of Tween 80

    PubMed Central

    Wang, Cindy; Mahrous, Engy A.; Lee, Richard E.; Vestling, Martha M.; Takayama, Kuni

    2011-01-01

    The addition of polyoxyethylene sorbitan monooleate (Tween 80) to a culture of mycobacteria greatly influences cell permeability and sensitivity to antibiotics but very little is known regarding the underlying mechanism. Here we show that Corynebacterium matruchotii (surrogate of mycobacteria) converts Tween 80 to a structural series of polyoxyethylenic acids which are then used to form novel series-2A and series-2B glycolipids. Minor series-3 glycolipids were also synthesized. The polyoxyethylenic acids replaced corynomycolic acids in the cell wall. Correspondingly the trehalose dicorynomycolate content was reduced. MALDI mass spectrometry, MS-MS, 1H-NMR, and 13C-NMR were used to characterize the series-2 glycolipids. Series-2A glycolipid is trehalose 6-C36:2-corynomycolate-6′-polyoxyethylenate and series-2B glycolipid is trehalose 6-C36:2-corynomycolate-6′-furan ring-containing polyoxyethylenate. Mycobacterium smegmatis grown in the presence of Tween 80 also synthesizes series-2 type glycolipids. The synthesis of these novel glycolipids in corynebacteria and mycobacteria should result in gross changes in the cell wall permeability and drug sensitivity. PMID:21490808

  9. Intralesional immunotherapy of malignant melanoma with mycobacterium smegmatis cell wall skeleton combined with trehalose dimycolate (P3).

    PubMed

    Vosika, G J; Schmidtke, J R; Goldman, A; Ribi, E; Parker, R; Gray, G R

    1979-08-01

    The clinical efficacy of intralesional immunotherapy utilizing Mycobacterium smegmatis cell wall skeleton (CWS) and trehalose dimycolate attached to oil droplets was investigated in 15 patients with advanced malignant melanoma. Patients received 300 microgram to 1050 microgram of the CWS combined with one-half that amount of trehalose dimycolate every 1 to 2 weeks for a total of 8 treatments. Therapy was continued if regression of injected lesions only occurred. Therapy was discontinued if regression of noninjected disease also occurred. Six of the 15 patients had regression of at least one injected lesion. Four of these 6 patients also had regression of noninjected disease lasting 4+, 6, 16 and 18+ months. Response was highly related to immune status. Six (83%) of 7 patients who reacted to one of a battery of skin tests responded. All 8 patients who did not react to skin tests failed to respond to therapy. There was no correlation of response with sex, prior therapy, disease-free interval or presence of visceral disease. Mycobacterial CWS and trehalose dimycolate is an effective immunotherapeutic agent. Additional studies of purified immunoadjuvants are warranted.

  10. Novel Polyoxyethylene-Containing Glycolipids Are Synthesized in Corynebacterium matruchotii and Mycobacterium smegmatis Cultured in the Presence of Tween 80.

    PubMed

    Wang, Cindy; Mahrous, Engy A; Lee, Richard E; Vestling, Martha M; Takayama, Kuni

    2011-01-01

    The addition of polyoxyethylene sorbitan monooleate (Tween 80) to a culture of mycobacteria greatly influences cell permeability and sensitivity to antibiotics but very little is known regarding the underlying mechanism. Here we show that Corynebacterium matruchotii (surrogate of mycobacteria) converts Tween 80 to a structural series of polyoxyethylenic acids which are then used to form novel series-2A and series-2B glycolipids. Minor series-3 glycolipids were also synthesized. The polyoxyethylenic acids replaced corynomycolic acids in the cell wall. Correspondingly the trehalose dicorynomycolate content was reduced. MALDI mass spectrometry, MS-MS, (1)H-NMR, and (13)C-NMR were used to characterize the series-2 glycolipids. Series-2A glycolipid is trehalose 6-C(36:2)-corynomycolate-6'-polyoxyethylenate and series-2B glycolipid is trehalose 6-C(36:2)-corynomycolate-6'-furan ring-containing polyoxyethylenate. Mycobacterium smegmatis grown in the presence of Tween 80 also synthesizes series-2 type glycolipids. The synthesis of these novel glycolipids in corynebacteria and mycobacteria should result in gross changes in the cell wall permeability and drug sensitivity.

  11. A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis.

    PubMed

    Bharati, Binod K; Sharma, Indra Mani; Kasetty, Sanjay; Kumar, Manish; Mukherjee, Raju; Chatterji, Dipankar

    2012-06-01

    The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

  12. The Rv1651c-encoded PE-PGRS30 protein expressed in Mycobacterium smegmatis exhibits polar localization and modulates its growth profile.

    PubMed

    Chatrath, Shweta; Gupta, Vineet Kumar; Dixit, Aparna; Garg, Lalit C

    2011-09-01

    Sequencing analysis of the complete genome of Mycobacterium tuberculosis (Mtb) H37Rv resulted in the identification of a novel multigene, the PE family of genes. The genes of the largest PE_PGRS subfamily of the PE family are mainly restricted to pathogenic mycobacteria, and their exact role in the biology of Mtb is not clearly understood. Based on their sequence homology, PE_PGRS proteins were initially thought to serve common functions. However, studies on individual proteins reveal that the individual proteins of this subfamily could be performing several unrelated tasks. In the present study, we investigated the function of PE_PGRS30 by expressing it in Mycobacterium smegmatis. PE_PGRS30 expression in M. smegmatis resulted in phenotypic changes with altered colony morphology and growth profile. The recombinant PE_PGRS30 showed polar localization and was found to be associated with the cell wall of M. smegmatis. Thus, the present study suggests that the prolonged lag phase of growth caused by the PE_PGRS30 may, in part, contribute to the latency of Mtb.

  13. WhmD promotes the assembly of Mycobacterium smegmatis FtsZ: A possible role of WhmD in bacterial cell division.

    PubMed

    Bhattacharya, Dipanwita; Kumar, Ashutosh; Panda, Dulal

    2017-02-01

    WhmD is considered to have a role in the septation and division of Mycobacterium smegmatis cells. Since FtsZ is the central protein of the septum, we determined the effect of WhmD on the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ) in vitro. WhmD increased both the rate and extent of the assembly of MsFtsZ in vitro. WhmD also increased the amount of polymerized MsFtsZ as evident from a sedimentation assay. Further, the assembly promoting activity of WhmD occurred in the presence of GTP. MsFtsZ polymerized to form thin filaments in the absence of WhmD while MsFtsZ formed thick filaments in the presence of WhmD suggesting that WhmD enhanced the bundling of MsFtsZ filaments. Interestingly, WhmD neither suppressed the dilution-induced disassembly of FtsZ filaments nor significantly altered the GTPase activity of FtsZ. Using size exclusion chromatography, circular dichroism and fluorescence spectroscopy, WhmD was found to bind to MsFtsZ in vitro. The results showed that WhmD can promote the assembly of FtsZ and indicated that WhmD may play a role in the division of M. smegmatis cells by assisting the polymerization of FtsZ.

  14. Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis.

    PubMed

    McNerney, R; Wilson, S M; Sidhu, A M; Harley, V S; al Suwaidi, Z; Nye, P M; Parish, T; Stoker, N G

    1998-01-01

    There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

  15. Evaluation of specific humoral immune response and cross reactivity against Mycobacterium tuberculosis antigens induced in mice immunized with liposomes composed of total lipids extracted from Mycobacterium smegmatis

    PubMed Central

    2013-01-01

    The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate. PMID:23458421

  16. 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.

  17. Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme.

    PubMed

    Balasundaram, D; Tyagi, A K

    1989-08-01

    Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.

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

    PubMed

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

    2012-12-21

    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 deoxynucleotides 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 homologues 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 from a Mycobacterium 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.

  19. Cloning, expression, purification, crystallization and preliminary X-ray studies of a pyridoxine 5′-phosphate oxidase from Mycobacterium smegmatis

    SciTech Connect

    Jackson, Colin J. Taylor, Matthew C.; Tattersall, David B.; French, Nigel G.; Carr, Paul D.; Ollis, David L.; Russell, Robyn J.; Oakeshott, John G.

    2008-05-01

    Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation. Pyridoxine 5′-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5′-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg-3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homogeneity. Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation.

  20. Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

    PubMed

    Ordonez, Heather; Unciuleac, Mihaela; Shuman, Stewart

    2012-05-01

    The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

  1. A temporal proteome dynamics study reveals the molecular basis of induced phenotypic resistance in Mycobacterium smegmatis at sub-lethal rifampicin concentrations

    PubMed Central

    Giddey, Alexander D.; de Kock, Elise; Nakedi, Kehilwe C.; Garnett, Shaun; Nel, Andrew J. M.; Soares, Nelson C.; Blackburn, Jonathan M.

    2017-01-01

    In the last 40 years only one new antitubercular drug has been approved, whilst resistance to current drugs, including rifampicin, is spreading. Here, we used the model organism Mycobacterium smegmatis to study mechanisms of phenotypic mycobacterial resistance, employing quantitative mass spectrometry-based proteomics to investigate the temporal effects of sub-lethal concentrations of rifampicin on the mycobacterial proteome at time-points corresponding to early response, onset of bacteriostasis and early recovery. Across 18 samples, a total of 3,218 proteins were identified from 31,846 distinct peptides averaging 16,250 identified peptides per sample. We found evidence that two component signal transduction systems (e.g. MprA/MprB) play a major role during initial mycobacterial adaptive responses to sub-lethal rifampicin and that, after dampening an initial SOS response, the bacteria supress the DevR (DosR) regulon and also upregulate their transcriptional and translational machineries. Furthermore, we found a co-ordinated dysregulation in haeme and mycobactin synthesis. Finally, gradual upregulation of the M. smegmatis-specific rifampin ADP-ribosyl transferase was observed which, together with upregulation of transcriptional and translational machinery, likely explains recovery of normal growth. Overall, our data indicates that in mycobacteria, sub-lethal rifampicin triggers a concerted phenotypic response that contrasts significantly with that observed at higher antimicrobial doses. PMID:28262820

  2. Genome-Wide Mapping of the Distribution of CarD, RNAP σ(A), and RNAP β on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing.

    PubMed

    Landick, Robert; Krek, Azra; Glickman, Michael S; Socci, Nicholas D; Stallings, Christina L

    2014-12-01

    CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σ(A) were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σ(A) would be predominantly enriched at promoters based on work in Escherichia coli (3), however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σ(A). The colocalization of σ(A) and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 (5) as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

  3. 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 RelMSM 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 RelMSM by isothermal titration calorimetry. Enzyme kinetics was followed where K0.5 was found to be increased with the concomitant reduction of Vmax 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.

  4. Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169

    PubMed Central

    Tokarev, Vasily; Kessler, Collin; McLimans, Christopher; Gomez-Alvarez, Vicente; Wright, Justin; King, Dawn; Lamendella, Regina

    2017-01-01

    ABSTRACT We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169T possesses unique virulence genes. PMID:28232435

  5. Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169.

    PubMed

    Pfaller, Stacy; Tokarev, Vasily; Kessler, Collin; McLimans, Christopher; Gomez-Alvarez, Vicente; Wright, Justin; King, Dawn; Lamendella, Regina

    2017-02-23

    We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169(T) was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169(T) possesses unique virulence genes.

  6. Characterization of the MSMEG_2631 Gene (mmp) Encoding a Multidrug and Toxic Compound Extrusion (MATE) Family Protein in Mycobacterium smegmatis and Exploration of Its Polyspecific Nature Using Biolog Phenotype MicroArray

    PubMed Central

    Mishra, Mukti Nath

    2013-01-01

    In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc2155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na+-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions. PMID:23292779

  7. Aggregation-based detection of M. smegmatis using D-arabinose-functionalized fluorescent silica nanoparticles.

    PubMed

    Jayawardana, Kalana W; Wijesundera, Samurdhi A; Yan, Mingdi

    2015-11-14

    Fluorescein-doped silica nanoparticles (FSNPs) functionalized with D-arabinose (Ara) showed strong interactions with Mycobacterium smegmatis (M. smegmatis) and caused the bacteria to aggregate. This aggregate formation was used as a means to detect M. smegmatis at the concentration of 10(4) CFU per mL.

  8. Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is strain dependent

    PubMed Central

    2010-01-01

    Background Two genotypically and microbiologically distinct strains of Mycobacterium avium subsp. paratuberculosis (MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions. Results We first complemented M. smegmatisΔideR with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using M. smegmatis mc2155 microarrays. In the presence of iron, sIdeR repressed expression of bfrA and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Conclusions Taken together, our study revealed

  9. 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.

  10. 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

  11. First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina.

    PubMed

    Travería, G E; Zumarraga, M; Etchechoury, I; Romano, M I; Cataldi, A; Pinedo, M F Alvarado; Pavlik, I; Pribylova, R; Romero, J R

    2013-01-01

    We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

  12. A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model of Mycobacterium smegmatis Infection

    PubMed Central

    Marcus, Sarah A.; Talaat, Adel M.; KuKanich, Butch K.; Sullivan, Ruth; Krugner-Higby, Lisa A.; Heath, Timothy D.

    2015-01-01

    Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid–loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 μg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models. PMID:26033620

  13. A New 4-Nitrotoluene Degradation Pathway in a Mycobacterium Strain

    PubMed Central

    Spiess, Tilmann; Desiere, Frank; Fischer, Peter; Spain, Jim C.; Knackmuss, Hans-Joachim; Lenke, Hiltrud

    1998-01-01

    Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (λmax, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage. PMID:9464378

  14. Characterization of a Mycobacterium intracellulare Variant Strain by Molecular Techniques

    PubMed Central

    Menendez, M. C.; Palenque, E.; Navarro, M. C.; Nuñez, M. C.; Rebollo, M. J.; Garcia, M. J.

    2001-01-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members. PMID:11724827

  15. Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.

    PubMed Central

    Gavigan, J A; Aínsa, J A; Pérez, E; Otal, I; Martín, C

    1997-01-01

    In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics. PMID:9209023

  16. Genome Sequence of Mycobacterium Phage Waterfoul

    PubMed Central

    Jackson, Paige N.; Embry, Ella K.; Johnson, Christa O.; Watson, Tiara L.; Weast, Sayre K.; DeGraw, Caroline J.; Douglas, Jessica R.; Sellers, J. Michael; D’Angelo, William A.

    2016-01-01

    Waterfoul is a newly isolated temperate siphovirus of Mycobacterium smegmatis mc2155. It was identified as a member of the K5 cluster of Mycobacterium phages and has a 61,248-bp genome with 95 predicted genes. PMID:27856585

  17. Complete Genome Sequence of the Mycobacterium immunogenum Type Strain CCUG 47286

    PubMed Central

    Jaén-Luchoro, Daniel; Seguí, Carolina; Aliaga-Lozano, Francisco; Salvà-Serra, Francisco; Busquets, Antonio; Gomila, Margarita; Ramírez, Antonio; Ruiz, Mikel; Lalucat, Jorge

    2016-01-01

    Here, we report the complete genome sequence of Mycobacterium immunogenum type strain CCUG 47286, a nontuberculous mycobacterium. The whole genome has 5,573,781 bp and covers as many as 5,484 predicted genes. This genome contributes to the task of closing the still-existing gap of genomes of rapidly growing mycobacterial type strains. PMID:27231356

  18. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    PubMed Central

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  19. The Mycobacterium phlei Genome: Expectations and Surprises

    PubMed Central

    Das, Sarbashis; Pettersson, B. M. Fredrik; Behra, Phani Rama Krishna; Ramesh, Malavika; Dasgupta, Santanu; Bhattacharya, Alok; Kirsebom, Leif A.

    2016-01-01

    Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898–1899. We present the complete genome sequence for the M. phlei CCUG21000T type strain and the draft genomes for four additional strains. The genome size for all fiveis 5.3 Mb with 69.4% Guanine-Cytosine content. This is ≈0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in the M. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phlei RIVM; 2) genes involved in polyamine metabolism and transport (potAD, potF) that are absent in other mycobacteria, and 3) strain-specific variations in the number of σ-factor genes. Moreover, M. phlei has as many as 82 mce (mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant to M. smegmatis mc2 155. PMID:26941228

  20. Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity

    PubMed Central

    Müller, Romy; Roberts, Charlotte A.; Brown, Terence A.

    2014-01-01

    The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second–nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth–nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis. PMID:24573854

  1. Draft Genome Sequence of Mycobacterium bovis Strain D-10-02315 Isolated from Wild Boar

    PubMed Central

    Branger, Maxime; Hauer, Amandine; Michelet, Lorraine; Karoui, Claudine; Cochard, Thierry; De Cruz, Krystel; Henault, Sylvie

    2016-01-01

    Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease, affecting livestock, wild animals, and sometimes humans. We report the draft genome sequence of a Mycobacterium bovis strain isolated from wild boar of spoligotype SB0120 (or BCG-like) also present in wildlife-livestock multi-host systems. PMID:27834714

  2. Virulence of two strains of Mycobacterium bovis in cattle following aerosol infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in humans, suggesting a selective advantage based upon virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e., Bovine Tuberculos...

  3. How does a Mycobacterium change its spots? Applying molecular tools to track diverse strains of Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne’s disease research. These data can now be used to define the extent of strain diversity ...

  4. Complete Genome Sequence of Carbendazim-Degrading Mycobacterium sp. Strain djl-10

    PubMed Central

    Yuan, Qiaoyun; Yang, Wei; Wang, Xinfeng

    2017-01-01

    ABSTRACT Mycobacterium sp. strain djl-10, an efficient degrader of carbendazim, was isolated from a carbendazim manufacturing wastewater treatment system. Here, we report the complete genome sequence of djl-10, which consists of a chromosome and three plasmids. PMID:28232422

  5. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

    PubMed Central

    Cywes, C; Hoppe, H C; Daffé, M; Ehlers, M R

    1997-01-01

    The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv

  6. The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1β Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp. bolletii

    PubMed Central

    Leão, Sylvia Cardoso; Matsumoto, Cristianne Kayoko; Carneiro, Adriana; Ramos, Rommel Thiago; Nogueira, Christiane Lourenço; Junior, James Daltro Lima; Lima, Karla Valéria; Lopes, Maria Luiza; Schneider, Horacio; Azevedo, Vasco Ariston; da Costa da Silva, Artur

    2013-01-01

    Background An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004–2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. Methodology/Principal Findings Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,264-bp circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc2155 could not be demonstrated. Conclusions/Significance The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease. PMID:23565273

  7. Strain specific transcriptional response in Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2012-01-01

    Background Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB. Results In spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551. In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878. Conclusions In association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell

  8. Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

    PubMed Central

    Gupta, Richa; Chatterjee, Debashree; Glickman, Michael S.; Shuman, Stewart

    2017-01-01

    RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy. PMID:27899559

  9. Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is Strain Dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Two genotypically and microbiologically distinct strains of Mycobacterium avium subsp. paratuberculosis (MAP) exist – the type I and type II strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributin...

  10. Emergence of potential superbug mycobacterium tuberculosis, lessons from new delhi mutant-1 bacterial strains.

    PubMed

    Nazir, Taha; Abraham, Suraj; Islam, Azharul

    2012-01-01

    Recent reports have shown that certain bacterial strains attain the New Delhi Metallo-beta-lactamase-1 (NDM-1) enzyme and become resistant to a broad range of antibiotics. Similarly, more dangerous "superbugs" of multi-drug resistant (MDR) and extensive drug resistant (XDR) Mycobacterium tuberculosis strains are gradually emerging through rapid genetic mutation caused by prescription non-compliance or unsupervised indiscriminate use of anti-tubercular drugs or other antibiotics. Mycobacterium tuberculosis cases have been reported in highly susceptible population groups including the aboriginal communities of US and Canada. In Canada alone, the total number of reported tuberculosis cases has decreased over the past decade. However, there is a steady increase in HIV cases in certain communities including the aboriginal communities. Reintroduction of MDR/XDR strains of tuberculosis is possible in these susceptible communities, which in turn may pose serious public health situation. MDR/XDR strains of tuberculosis are virtually untreatable using current anti-tubercular medication protocols. Thus, MDR/XDR tuberculosis presents a grave global public health threat. The unpredictable genetic mechanism involved in generating MDR/XDR resistant strains of Mycobacterium tuberculosis may pose greater challenges in developing appropriate treatment strategies. In this article, we briefly review potential genetic mechanism of emerging NDM-1 bacterial strains and draw a rationale parallel to the underlying genetic mechanism of MDR/XDR Mycobacterium tuberculosis strain development.

  11. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  12. Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1.

    PubMed Central

    Kelley, I; Freeman, J P; Evans, F E; Cerniglia, C E

    1993-01-01

    Mycobacterium sp. strain PYR-1, previously shown to extensively mineralize high-molecular-weight polycyclic aromatic hydrocarbons in pure culture and in sediments, degrades fluoranthene to 9-fluorenone-1-carboxylic acid. In this study, 10 other fluoranthene metabolites were isolated from ethyl acetate extracts of the culture medium by thin-layer and high-performance liquid chromatographic methods. On the basis of comparisons with authentic compounds by UV spectrophotometry and thin-layer chromatography as well as gas chromatography-mass spectral and proton nuclear magnetic resonance spectral analyses, the metabolites were identified as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy-1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde, benzoic acid, phenylacetic acid, and adipic acid. Authentic 9-hydroxyfluorene and 9-fluorenone were metabolized by Mycobacterium sp. strain PYR-1. A pathway for the catabolism of fluoranthene by Mycobacterium sp. strain PYR-1 is proposed. PMID:8481006

  13. 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

  14. SmoXYB1C1Z of Mycobacterium sp. strain NBB4: a soluble methane monooxygenase (sMMO)-like enzyme, active on C2 to C4 alkanes and alkenes.

    PubMed

    Martin, Kiri E; Ozsvar, Jazmin; Coleman, Nicholas V

    2014-09-01

    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 mc(2)-155. Cells of mc(2)-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc(2)-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 mc(2)-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 mc(2)-155(pSmo) provides a new model for studying sMMO-like monooxygenases.

  15. Mycobacterium avium infections of Acanthamoeba strains: host strain variability, grazing-acquired infections, and altered dynamics of inactivation with monochloramine.

    PubMed

    Berry, David; Horn, Matthias; Xi, Chuanwu; Raskin, Lutgarde

    2010-10-01

    Stable Mycobacterium avium infections of several Acanthamoeba strains were characterized by increased infection resistance of recent environmental isolates and reduced infectivity in the presence of other bacteria. Exposure of M. avium in coculture with Acanthamoeba castellanii to monochloramine yielded inactivation kinetics markedly similar to those observed for A. castellanii alone.

  16. Draft Genome Sequence of Mycobacterium acapulcensis Strain CSURP1424

    PubMed Central

    Asmar, Shady; Rascovan, Nicolás; Robert, Catherine

    2016-01-01

    Mycobacterium acapulcensis is a rapidly growing scotochromogenic acid-fast bacillus. The draft genome of M. acapulcensis CSURP1424 comprises 5,290,974 bp, exhibiting a 66.67% G+C content, 4,870 protein-coding genes, and 71 predicted RNA genes. PMID:27516522

  17. In vitro susceptibility testing of Mycobacterium tuberculosis complex strains isolated from seals to antituberculosis drugs.

    PubMed

    Bernardelli, Amelia; Morcillo, Nora; Loureiro, Julio; Quse, Viviana; Davenport, Silvana

    2004-06-01

    Mycobacteria strains belonging to the Mycobacterium tuberculosis complex were isolated from seals found in the South Atlantic. The animals were received in Mundo Marino installations and treated for Mycobacterium tuberculosis complex by conventional therapy of intensive care and enriched food supply; however, in all cases treatment failed. Necropsies of all animals revealed extensive lesions compatible with tuberculosis involving lungs, liver, spleen and lymphatic nodes. Classical biochemical methods as well as molecular techniques using the IS6110 probes were performed for mycobacterial identification. Furthermore, the LCx M. tuberculosis assay (Abbott Laboratories) identified all strains as Mycobacterium tuberculosis complex members. The in vitro susceptibility pattern was examined in mycobacterial strains isolated from seven seals and in 3 reference strains--BCG, H37Rv (M. tuberculosis) and AN5 (Mycobacterium bovis)--to 4 medications--isoniazid, rifampin, streptomycin and ethambutol. Minimal inhibitory drug concentrations were determined by the Mycobacterial Growth Indicator Tube (BD Argentina) method and a microdilution and colorimetric assay using 3-(4-5 dimethyltiazol-2)-2,5 diphenyltetrazolium bromide. All the isolates and the reference strains BCG and AN5 were inhibited by MIC values similar to those of H37Rv with good agreement obtained by both techniques. These findings suggest that a therapeutic regimen aimed to seals diagnosed with tuberculosis play an important role in the prevention of tuberculosis transmission from infected animals to humans that are in routine contact with them.

  18. Molecular Drug Susceptibility Testing and Genotyping of Mycobacterium leprae Strains from South America▿

    PubMed Central

    Singh, Pushpendra; Busso, Philippe; Paniz-Mondolfi, Alberto; Aranzazu, Nacarid; Monot, Marc; Honore, Nadine; de Faria Fernandes Belone, Andrea; Virmond, Marcos; Villarreal-Olaya, Maria Esther; Rivas, Carlos; Cole, Stewart T.

    2011-01-01

    Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%). PMID:21444694

  19. Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. Strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, ...

  20. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    PubMed Central

    de Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia. PMID:24812221

  1. Complete genome sequence of Mycobacterium fortuitum subsp. fortuitum type strain DSM46621.

    PubMed

    Ho, Yung S; Adroub, Sabir A; Aleisa, Fajr; Mahmood, Hanan; Othoum, Ghofran; Rashid, Fahad; Zaher, Manal; Ali, Shahjahan; Bitter, Wilbert; Pain, Arnab; Abdallah, Abdallah M

    2012-11-01

    Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.

  2. Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog

    PubMed Central

    Perdigão, João; Canto, Ana; Albuquerque, Teresa; Leal, Nuno; Macedo, Rita; Portugal, Isabel; Cunha, Mónica V.

    2014-01-01

    Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets. PMID:24153119

  3. Draft Genome Sequence of the Vaccination Strain Mycobacterium bovis BCG S4-Jena

    PubMed Central

    Wibberg, Daniel; Winkler, Anika; Straube, Eberhard; Karrasch, Matthias; Keller, Peter M.

    2016-01-01

    Here, we present the draft genome sequence of Mycobacterium bovis BCG S4-Jena, a tuberculosis vaccine strain. The genome of S4-Jena is represented by 48 scaffolds, consisting of 132 scaffolded contigs and amounting to a size of about 4.2 Mb. New genes potentially encoding a phage fragment were identified in the genome. PMID:27103721

  4. Complete Genome Sequence of Mycobacterium chimaera Strain AH16.

    PubMed

    Hasan, Nabeeh A; Honda, Jennifer R; Davidson, Rebecca M; Epperson, L Elaine; Bankowski, Matthew J; Chan, Edward D; Strong, Michael

    2016-11-23

    Mycobacterium chimaera is a nontuberculous mycobacterial species that causes cardiovascular, pulmonary, and postsurgical infections. Here, we report the first complete genome sequence of M. chimaera This genome is 6.33 Mbp, with a G+C content of 67.56%, and encodes 4,926 protein-coding genes, as well as 74 tRNAs, one ncRNA, and three rRNA genes.

  5. Complete Genome Sequence of Mycobacterium chimaera Strain AH16

    PubMed Central

    Honda, Jennifer R.; Davidson, Rebecca M.; Epperson, L. Elaine; Bankowski, Matthew J.; Chan, Edward D.; Strong, Michael

    2016-01-01

    Mycobacterium chimaera is a nontuberculous mycobacterial species that causes cardiovascular, pulmonary, and postsurgical infections. Here, we report the first complete genome sequence of M. chimaera. This genome is 6.33 Mbp, with a G+C content of 67.56%, and encodes 4,926 protein-coding genes, as well as 74 tRNAs, one ncRNA, and three rRNA genes. PMID:27881537

  6. Sliding Motility, Biofilm Formation, and Glycopeptidolipid Production in Mycobacterium colombiense Strains

    PubMed Central

    Maya-Hoyos, Milena; Leguizamón, John; Mariño-Ramírez, Leonardo; Soto, Carlos Y.

    2015-01-01

    Mycobacterium colombiense is a novel member of the Mycobacterium avium complex, which produces respiratory and disseminated infections in immunosuppressed patients. Currently, the morphological and genetic bases underlying the phenotypic features of M. colombiense strains remain unknown. In the present study, we demonstrated that M. colombiense strains displaying smooth morphology show increased biofilm formation on hydrophobic surfaces and sliding on motility plates. Thin-layer chromatography experiments showed that M. colombiense strains displaying smooth colonies produce large amounts of glycolipids with a chromatographic behaviour similar to that of the glycopeptidolipids (GPLs) of M. avium. Conversely, we observed a natural rough variant of M. colombiense (57B strain) lacking pigmentation and exhibiting impaired sliding, biofilm formation, and GPL production. Bioinformatics analyses revealed a gene cluster that is likely involved in GPL biosynthesis in M. colombiense CECT 3035. RT-qPCR experiments showed that motile culture conditions activate the transcription of genes possibly involved in key enzymatic activities of GPL biosynthesis. PMID:26180799

  7. Strain-dependent CNS dissemination in guinea pigs after Mycobacterium tuberculosis aerosol challenge.

    PubMed

    Be, Nicholas A; Klinkenberg, Lee G; Bishai, William R; Karakousis, Petros C; Jain, Sanjay K

    2011-09-01

    Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS disease. We therefore examined CNS dissemination by different laboratory strains (two M. tuberculosis H37Rv, one CDC1551) in a guinea pig aerosol infection model. Although all strains grew exponentially in lungs, with similar bacterial burdens at the time of extrapulmonary dissemination, M. tuberculosis CDC1551 disseminated to the CNS significantly more than the H37Rv strains. No CNS lesions were observed throughout the study, with only a modest cytokine response. These data suggest that M. tuberculosis may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB.

  8. Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population

    PubMed Central

    Pérez-Lago, Laura; Herranz, Marta; Comas, Iñaki; Ruiz-Serrano, María Jesús; López Roa, Paula; Bouza, Emilio

    2015-01-01

    A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission—despite opportunities—and challenged some of the advantages associated with Beijing strains. PMID:26719445

  9. Draft Genome Sequences of Six Mycobacterium immunogenum Strains Obtained from a Chloraminated Drinking Water Distribution System Simulator

    PubMed Central

    Revetta, Randy P.

    2016-01-01

    We report here the draft genome sequences of six Mycobacterium immunogenum strains isolated from a chloraminated drinking water distribution system simulator subjected to changes in operational parameters. M. immunogenum, a rapidly growing mycobacterium previously reported to be the cause of hypersensitivity pneumonitis from contaminated metalworking fluid aerosols, is becoming a public health concern. PMID:26744376

  10. Mycobacterium shigaense Causes Lymph Node and Cutaneous Lesions as Immune Reconstitution Syndrome in an AIDS Patient: The Third Case Report of a Novel Strain Non-tuberculous Mycobacterium

    PubMed Central

    Koizumi, Yusuke; Shimizu, Kaoru; Shigeta, Masayo; Minamiguchi, Hitoshi; Hodohara, Keiko; Andoh, Akira; Tanaka, Toshihide; Chikamatsu, Kinuyo; Mitarai, Satoshi; Mikamo, Hiroshige

    2016-01-01

    A 40-year-old man complaining of progressive body weight loss was diagnosed to have acquired immunodeficiency syndrome. Within 2 weeks after the initiation of combination antiretroviral therapy, he developed fever, massive cervical lymphadenopathy and a protruding subcutaneous abscess. A lymph node biopsy and abscess drainage revealed non-caseous granuloma and mycobacterium. The mycobacterium belonged to Runyon II group, but it showed no matches to any previously reported species. According to sequence analyses, the strain was identified as Mycobacterium shigaense. After six months of antimycobacterial treatment, the lesions were all successfully cured. This is the third case report of the novel mycobacterium, M. shigaense, presenting in associatioin with immune reconstitution syndrome. PMID:27853087

  11. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

    PubMed

    Jungblut, P R; Schaible, U E; Mollenkopf, H J; Zimny-Arndt, U; Raupach, B; Mattow, J; Halada, P; Lamer, S; Hagens, K; Kaufmann, S H

    1999-09-01

    In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

  12. A Web-Based Platform for Designing Vaccines against Existing and Emerging Strains of Mycobacterium tuberculosis

    PubMed Central

    Dhanda, Sandeep Kumar; Vir, Pooja; Singla, Deepak; Gupta, Sudheer; Kumar, Shailesh

    2016-01-01

    Development of an effective vaccine against drug-resistant Mycobacterium tuberculosis (Mtb) is crucial for saving millions of premature deaths every year due to tuberculosis. This paper describes a web portal developed for assisting researchers in designing vaccines against emerging Mtb strains using traditional and modern approaches. Firstly, we annotated 59 genomes of Mycobacterium species to understand similarity/dissimilarity between tuberculoid, non-tuberculoid and vaccine strains at genome level. Secondly, antigen-based vaccine candidates have been predicted in each Mtb strain. Thirdly, epitopes-based vaccine candidates were predicted/discovered in above antigen-based vaccine candidates that can stimulate all arms of immune system. Finally, a database of predicted vaccine candidates at epitopes as well at antigen level has been developed for above strains. In order to design vaccine against a newly sequenced genome of Mtb strain, server integrates three modules for identification of strain-, antigen-, epitope-specific vaccine candidates. We observed that 103522 unique peptides (9mers) had the potential to induce an antibody response and/or promiscuous binder to MHC alleles and/or have the capability to stimulate T lymphocytes. In summary, this web-portal will be useful for researchers working on designing vaccines against Mtb including drug-resistant strains. Availability: The database is available freely at http://crdd.osdd.net/raghava/mtbveb/. PMID:27096425

  13. Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability.

    PubMed

    Fraud, S; Hann, A C; Maillard, J-Y; Russell, A D

    2003-03-01

    The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.

  14. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

    PubMed Central

    Stinear, Timothy P.; Seemann, Torsten; Harrison, Paul F.; Jenkin, Grant A.; Davies, John K.; Johnson, Paul D.R.; Abdellah, Zahra; Arrowsmith, Claire; Chillingworth, Tracey; Churcher, Carol; Clarke, Kay; Cronin, Ann; Davis, Paul; Goodhead, Ian; Holroyd, Nancy; Jagels, Kay; Lord, Angela; Moule, Sharon; Mungall, Karen; Norbertczak, Halina; Quail, Michael A.; Rabbinowitsch, Ester; Walker, Danielle; White, Brian; Whitehead, Sally; Small, Pamela L.C.; Brosch, Roland; Ramakrishnan, Lalita; Fischbach, Michael A.; Parkhill, Julian; Cole, Stewart T.

    2008-01-01

    Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis. PMID:18403782

  15. Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

    PubMed Central

    Tientcheu, Leopold D.; Ndengane, Mthawelenga; Andoseh, Genevieve; Kampmann, Beate; Wilkinson, Robert J

    2017-01-01

    In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies. PMID:28150302

  16. [In vitro sensitivity of Mycobacterium chelonae strains to various antimicrobial agents].

    PubMed

    Hernández García, A M; Arias, A; Felipe, A; Alvarez, R; Sierra, A

    1995-12-01

    The in vitro susceptibility of 32 Mycobacterium chelonae strains to 10 antimicrobial agents was determined. The sources of the different strains were: clinical samples from patients treated at the Hospital Universitario de Canarias and Hospital del Tórax (General and Chest facilities) and from environmental sources (water supply, sewage, swimming pools and the sea). The susceptibility tests were performed by a broth microdilution method (Mueller-Hinton Broth). The results showed amikacin as the most effective antimicrobial agent against M. chelonae isolates, then ofloxacin and cefoxitin. However no statistical difference was detected among them. The least effective was imipenem, followed by ciprofloxacin and norfloxacin.

  17. Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

    PubMed Central

    van Helden, Paul D.; Wilson, Douglas; Colijn, Caroline; McLaughlin, Megan M.; Abubakar, Ibrahim; Warren, Robin M.

    2012-01-01

    Summary: Numerous studies have reported that individuals can simultaneously harbor multiple distinct strains of Mycobacterium tuberculosis. To date, there has been limited discussion of the consequences for the individual or the epidemiological importance of mixed infections. Here, we review studies that documented mixed infections, highlight challenges associated with the detection of mixed infections, and discuss possible implications of mixed infections for the diagnosis and treatment of patients and for the community impact of tuberculosis control strategies. We conclude by highlighting questions that should be resolved in order to improve our understanding of the importance of mixed-strain M. tuberculosis infections. PMID:23034327

  18. A Mycobacterium strain with extended capacities for degradation of gasoline hydrocarbons.

    PubMed

    Solano-Serena, F; Marchal, R; Casarégola, S; Vasnier, C; Lebeault, J M; Vandecasteele, J P

    2000-06-01

    A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2, 4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.

  19. Effectiveness of ultrasound for the destruction of Mycobacterium sp. strain (6PY1).

    PubMed

    Al Bsoul, Abeer; Magnin, Jean-Pierre; Commenges-Bernole, Nadine; Gondrexon, Nicolas; Willison, John; Petrier, Christian

    2010-01-01

    Ultrasound is widely used to disinfect drinking water and wastewater due to its strong physical and chemical effects on microorganisms. The aim of this study was to investigate the effect of ultrasound on the destruction of Mycobacterium strain 6PY1. Ultrasound waves (20 kHz or 612 kHz) were used to treat aqueous suspensions of Mycobacterium at different volumes, initial bacterial concentrations, and power densities. At the same power density and the same exposure time, sonication at high frequency resulted in a lower destruction of Mycobacterium sp. 6PY1 (35.5%) than sonication at low frequency (93%). The percentage of removal was not significantly affected by the volume of the irradiated suspension (150-300 ml) or the initial cell concentration (2.15 x 10(-3)-1.4 x 10(-2)mg protein L(-1)). At low frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density, with a constant level reached after a certain power density. At high frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density. The mechanism of cell killing was investigated by examining the effects of OH() radical scavengers such as sodium carbonate. At high frequency the presence of sodium carbonate suppressed the removal process. However, at low frequency the removal process was not affected, thus indicating that OH() radicals have a negligible role in this case. The latter result was supported by ten time's H(2)O(2) production at high frequency greater than that at low frequency.

  20. Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains

    PubMed Central

    2012-01-01

    Background Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. Results The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. Conclusion This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided

  1. Localization of 17beta-hydroxysteroid dehydrogenase in Mycobacterium sp. VKM Ac-1815D mutant strain.

    PubMed

    Egorova, O V; Nikolayeva, V M; Suzina, N E; Donova, M V

    2005-04-01

    The localization of mycobacterial 17beta-hydroxysteroid dehydrogenase (17beta-OH SDH) was studied using cell fractionation and cytochemical investigation. Mycobacterium sp. Et1 mutant strain derived from Mycobacterium sp. VKM Ac-1815D and characterized by increased 17beta-OH SDH activity was used as a model organism. Subcellular distribution study showed both soluble and membrane-bound forms of mycobacterial 17beta-hydroxysteroid dehydrogenase. The cytochemical method based on a copper ferrocyanide procedure followed by electron microscopic visualization was applied in order to investigate the intracellular localization of bacterial 17beta-OH SDH in more detail. The enzyme was found to be located in the peripheral cytoplasmic zone adjoining the cytoplasmic membrane (CM). 17beta-OH SDH was loosely membrane bound and easily released into the environment under the cell integrity failure.

  2. Molecular diversity of Mycobacterium tuberculosis strains in a slum area of Rio de Janeiro, Brazil.

    PubMed

    Mendes, Joycenea Matsuda; Machado, Silvia Maria Almeida; Lourenço, Maria Cristina; Ferreira, Rosa Maria Carvalho; Fonseca, Leila de Souza; Saad, Maria Helena Feres

    2008-12-01

    This retrospective molecular study involving restriction fragment length polymorphism, using insertion sequence 6110 as a marker, was conducted in order to provide an initial insight into the genetic diversity of Mycobacterium tuberculosis strains isolated in the slums of the Complexo de Manguinhos, located in the city of Rio de Janeiro, Brazil. Of the 67 strains evaluated, 23 (34.3%) were found to belong to clusters (total clusters, 10). Household and social chains of transmission were associated with clustering, in 20% and 60%, respectively. Living in the Conjunto Habitacional Programado 2 slum was associated with clustering. Although not significant, it is relevant that 26% of the clustered strains presented primary resistance. These findings, although possibly underestimating the prevalence due to the failure to analyze all strains, could help improve the local tuberculosis control program.

  3. Ecotoxicological assessment of PAHs and their dead-end metabolites after degradation by Mycobacterium sp. strain SNP11.

    PubMed

    Pagnout, Christophe; Rast, Claudine; Veber, Anne-Marie; Poupin, Pascal; Férard, Jean-François

    2006-10-01

    Mycobacterium sp. SNP11 has a high PAH biodegradation potential. In this paper, the toxicity of pyrene, fluoranthene, phenanthrene, and their dead-end metabolites, accumulated in the media after biodegradation by Mycobacterium sp. SNP11, were evaluated by a screening battery of acute, chronic, and genotoxic tests. According to the bioassays, performed on bacteria (Vibrio fischeri, Salmonella typhimurium strains TA1535/pSK1002, TA97a, TA98, TA100), algae (Pseudokirchneriella subcapitata), and crustaceans (Daphnia magna, Ceriodaphnia dubia), total disappearance or a very significant reduction of the (geno)toxic potential was observed after PAH degradation by Mycobacterium sp. SNP11.

  4. Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare.

    PubMed

    Nishimori, K; Eguchi, M; Nakaoka, Y; Onodera, Y; Ito, T; Tanaka, K

    1995-08-01

    The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR-amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex.

  5. Mapping of IS6110 Insertion Sites in Two Epidemic Strains of Mycobacterium tuberculosis

    PubMed Central

    Beggs, Marjorie L.; Eisenach, Kathleen D.; Cave, M. Donald

    2000-01-01

    A widely distributed strain designated 210 was identified in a study of the diversity of Mycobacterium tuberculosis DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS6110, and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS6110 insertions in strain 210 and compare these to IS6110 insertion sites in strain W. Our hypothesis is that an IS6110 insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the M. tuberculosis H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS6110 insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS6110 insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS6110 has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS6110 was found to reside 55 bp in the ctpD gene. This gene is expressed, indicating that IS6110 can provide a promoter sequence for the transcription of genes. PMID:10921952

  6. Mycobacterium tuberculosis strains spreading in Hanoi, Vietnam: Beijing sublineages, genotypes, drug susceptibility patterns, and host factors.

    PubMed

    Maeda, Shinji; Hang, Nguyen T L; Lien, Luu T; Thuong, Pham H; Hung, Nguyen V; Hoang, Nguyen P; Cuong, Vu C; Hijikata, Minako; Sakurada, Shinsaku; Keicho, Naoto

    2014-12-01

    Beijing genotype strains are divided into two major sublineages, ancient (atypical) and modern (typical) types, but their phenotypic variations remain largely unknown. Mycobacterium tuberculosis (MTB) isolates from Hanoi, Vietnam, were analyzed by single-nucleotide polymorphisms and spoligotyping. Patient information and drug susceptibility patterns were obtained. Genetic clustering was assessed by variable number of tandem repeat (VNTR) locus sets. Multivariate analysis was also performed to investigate factors possibly associated with these sublineages. Of the 465 strains tested, 175 (37.6%) belonged to the ancient Beijing sublineage and 97 (20.9%) were of the modern Beijing sublineage. Patients with the Beijing genotype were significantly younger and more undernourished than those with non-Beijing genotype. The proportion of clustered strains calculated from 15 locus-optimized mycobacterial interspersed repetitive units [optimized-(MIRU)15]-, optimized-MIRU24-, optimized-MIRU28-, Japan Anti-Tuberculosis Association (JATA)15-, and JATA18-VNTRs were 55.7%, 49.2%, 33.8%, 44.5%, and 32.0%, respectively. Ancient and modern Beijing genotype strains were more frequently clustered than non-Beijing genotype strains, even when using VNTR sets with high discriminatory power. Isoniazid and streptomycin resistance tended to be more frequently observed in ancient Beijing strains than in modern Beijing strains and others. Our findings may provide insight into area-dependent differences in Beijing family strain characteristics.

  7. Salicylanilide pyrazinoates inhibit in vitro multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitrate lyase.

    PubMed

    Krátký, Martin; Vinšová, Jarmila; Novotná, Eva; Stolaříková, Jiřina

    2014-03-12

    The development of antimicrobial agents represents an up-to-date topic. This study investigated in vitro antimycobacterial activity, mycobacterial isocitrate lyase inhibition and cytotoxicity of salicylanilide pyrazinoates. They may be considered being mutual prodrugs of both antimycobacterial active salicylanilides and pyrazinoic acid (POA), an active metabolite of pyrazinamide, in which these esters are likely hydrolysed without presence of pyrazinamidase/nicotinamidase. Minimum inhibitory concentrations (MICs) of the esters were within the range 0.5-8 μmol/l for Mycobacterium tuberculosis and 1-32 μmol/l for nontuberculous mycobacteria (Mycobacterium avium, Mycobacterium kansasii). All esters showed a weak inhibition (8-17%) of isocitrate lyase at the concentration of 10 μmol/l. The most active pyrazinoates showed MICs for multidrug-resistant tuberculosis strains in the range of 0.125-2 μmol/l and no cross-resistance with clinically used drugs, thus being the most in vitro efficacious salicylanilide esters with 4-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate superiority (MICs⩽0.25 μmol/l). This promising activity is likely due to an additive or synergistic effect of released POA and salicylanilides. Selectivity indexes for the most active salicylanilide pyrazinoates ranged up to 64, making some derivatives being attractive candidates for the next research; 4-bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate showed the most convenient toxicity profile.

  8. rpoB Mutations in Multidrug-Resistant Strains of Mycobacterium tuberculosis Isolated in Italy

    PubMed Central

    Pozzi, G.; Meloni, M.; Iona, E.; Orrù, G.; Thoresen, O. F.; Ricci, M. L.; Oggioni, M. R.; Fattorini, L.; Orefici, G.

    1999-01-01

    Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp). PMID:10074552

  9. Monocarbonyl analogs of curcumin inhibit growth of antibiotic sensitive and resistant strains of Mycobacterium tuberculosis

    PubMed Central

    Baldwin, Patrick R.; Reeves, Analise Z.; Powell, Kimberly R.; Napier, Ruth J.; Swimm, Alyson I.; Sun, Aiming; Giesler, Kyle; Bommarius, Bettina; Shinnick, Thomas M.; Snyder, James P.; Liotta, Dennis C.; Kalman, Daniel

    2016-01-01

    Tuberculosis (TB) is a major public health concern worldwide with over 2 billion people currently infected. The rise of strains of Mycobacterium tuberculosis (Mtb) that are resistant to some or all first and second line antibiotics, including multidrug-resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains, is of particular concern and new anti-TB drugs are urgently needed. Curcumin, a natural product used in traditional medicine in India, exhibits anti-microbial activity that includes Mtb, however it is relatively unstable and suffers from poor bioavailability. To improve activity and bioavailability, mono-carbonyl analogs of curcumin were synthesized and screened for their capacity to inhibit the growth of Mtb and the related Mycobacterium marinum (Mm). Using disk diffusion and liquid culture assays, we found several analogs that inhibit in vitro growth of Mm and Mtb, including rifampicin-resistant strains. Structure activity analysis of the analogs indicated that Michael acceptor properties are critical for inhibitory activity. However, no synergistic effects were evident between the monocarbonyl analogs and rifampicin on inhibiting growth. Together, these data provide a structural basis for the development of analogs of curcumin with pronounced anti-mycobacterial activity and provide a roadmap to develop additional structural analogs that exhibit more favorable interactions with other anti-TB drugs. PMID:25618016

  10. [Susceptibilities of Mycobacterium tuberculosis strains collected from regional tuberculosis laboratories to major antituberculous drugs].

    PubMed

    Sayğan, M Bakir; Ocak, Fatih; Cesur, Salih; Tarhan, Gülnur; Ceyhan, Ismail; Gümüişlü, Feyzullah; Beker, Gülşan; Güner, Uğur; Coşkun, Erol

    2007-07-01

    The aim of this study was to investigate the susceptibility rates of Mycobacterium tuberculosis strains sent to Refik Saydam Hygiene Center, Tuberculosis Reference and Research Laboratory, Ankara, from seven different regional tuberculosis laboratories between the 1999-2002 period against major antituberculous drugs. The sensitivities of a total 505 M. tuberculosis strains to isoniazid (INAH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) were determined by using proportion method in Lowenstein-Jensen medium. Of the strains, 385 (76.2%) were found sensitive to all of the tested drugs, while 120 strains were resistant to at least one of the antituberculous drugs. The resistant strains showed 14 different resistance patterns. The resistance rates were detected as 13.3% for INAH and RIF (67 strains of each), 9.1% for SM (46 strains), and 3.4% (17 strains) for EMB. Multidrug resistant (INAH+RIF) M. tuberculosis was 7.9% (40 strains). The highest resistance rate to INAH, RIF and EMB (21.2%, 21.2% and 10.6%, respectively) was detected in the isolates which were sent from Bursa province (located in northwestern Turkey); the highest SM (18.8%) and multidrug resistance (INAH+RIF) rates (18.8% and 10.6%, respectively) were detected in the strains sent from Elazig and Van provinces (both located in eastern Turkey). Since the inappropriate use of the first and second line antituberculous drugs leads to the development and spread of the resistant strains, "Directly Observed Therapy Shortcourse (DOTS)" is a very important practice. Therefore regional tuberculosis laboratories should be worth considering as the chains of a well-organized national laboratory network, in order to detect the antituberculous drug resistance patterns of the M. tuberculosis strains over the country.

  11. Clofazimine Prevents the Regrowth of Mycobacterium abscessus and Mycobacterium avium Type Strains Exposed to Amikacin and Clarithromycin

    PubMed Central

    Ferro, Beatriz E.; Meletiadis, Joseph; Wattenberg, Melanie; de Jong, Arjan; van Soolingen, Dick; Mouton, Johan W.

    2015-01-01

    Multidrug therapy is a standard practice when treating infections by nontuberculous mycobacteria (NTM), but few treatment options exist. We conducted this study to define the drug-drug interaction between clofazimine and both amikacin and clarithromycin and its contribution to NTM treatment. Mycobacterium abscessus and Mycobacterium avium type strains were used. Time-kill assays for clofazimine alone and combined with amikacin or clarithromycin were performed at concentrations of 0.25× to 2× MIC. Pharmacodynamic interactions were assessed by response surface model of Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA), calculating the percentage of statistically significant Bliss interactions and interaction indices (I), respectively. Monte Carlo simulations with predicted human lung concentrations were used to calculate target attainment rates for combination and monotherapy regimens. Clofazimine alone was bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M. abscessus (I = 0.41; 95% confidence interval [CI], 0.29 to 0.55) and M. avium (I = 0.027; 95% CI, 0.007 to 0.048). Based on RSBI analysis, synergistic interactions of 28.4 to 29.0% and 23.2 to 56.7% were observed at 1× to 2× MIC and 0.25× to 2× MIC for M. abscessus and M. avium, respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus (I = 0.53; 95% CI, 0.35 to 0.72) and M. avium (I = 0.16; 95% CI, 0.04 to 0.35), RSBI analysis showed 23.5% and 23.3 to 53.3% at 2× MIC and 0.25× to 0.5× MIC for M. abscessus and M. avium, respectively. Clofazimine prevented the regrowth observed with amikacin or clarithromycin alone. Target attainment rates of combination regimens were >60% higher than those of monotherapy regimens for M. abscessus and M. avium. The combination of clofazimine with amikacin or clarithromycin was synergistic in vitro. This suggests a potential role for clofazimine in treatment regimens that warrants further

  12. In vitro Anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis Strains

    PubMed Central

    2013-01-01

    Background Tuberculosis (TB) is a global burden with one –third of the world’s population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. Methods Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. Results The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25–100 μg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 μg/ml and 12.5-75 μg/mL, 25–100 μg/mL and 25–50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. Conclusions The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However

  13. Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

    PubMed Central

    Iwamoto, Tomotada; Grandjean, Louis; Arikawa, Kentaro; Nakanishi, Noriko; Caviedes, Luz; Coronel, Jorge; Sheen, Patricia; Wada, Takayuki; Taype, Carmen A.; Shaw, Marie-Anne; Moore, David A. J.; Gilman, Robert H.

    2012-01-01

    Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999. PMID:23185395

  14. Evaluation of the Antimicrobial Properties of the Essential Oil of Myrtus communis L. against Clinical Strains of Mycobacterium spp.

    PubMed Central

    Zanetti, Stefania; Cannas, Sara; Molicotti, Paola; Bua, Alessandra; Cubeddu, Marina; Porcedda, Silvia; Marongiu, Bruno; Sechi, Leonardo Antonio

    2010-01-01

    Mycobacterium tuberculosis is the etiological agent of tuberculosis. The World Health Organization has estimated that 8 million of people develop active TB every year and the situation is complicated by an increase of Mycobacterium tuberculosis strains resistant to drugs used in antitubercular therapy: MDR and XDR-TB. Myrtle leaf extracts, used as an antiseptic in Sardinian traditional medicine, have strong antibacterial activity as several investigations showed. In this study we investigated the antimicrobial properties of the essential oil of Myrtus communis against clinical strains of M. tuberculosis and M. paratuberculosis. PMID:20706606

  15. Evaluation of the Antimicrobial Properties of the Essential Oil of Myrtus communis L. against Clinical Strains of Mycobacterium spp.

    PubMed

    Zanetti, Stefania; Cannas, Sara; Molicotti, Paola; Bua, Alessandra; Cubeddu, Marina; Porcedda, Silvia; Marongiu, Bruno; Sechi, Leonardo Antonio

    2010-01-01

    Mycobacterium tuberculosis is the etiological agent of tuberculosis. The World Health Organization has estimated that 8 million of people develop active TB every year and the situation is complicated by an increase of Mycobacterium tuberculosis strains resistant to drugs used in antitubercular therapy: MDR and XDR-TB. Myrtle leaf extracts, used as an antiseptic in Sardinian traditional medicine, have strong antibacterial activity as several investigations showed. In this study we investigated the antimicrobial properties of the essential oil of Myrtus communis against clinical strains of M. tuberculosis and M. paratuberculosis.

  16. Draft Genome Sequences of Mycobacterium setense Type Strain DSM-45070 and the Nonpathogenic Strain Manresensis, Isolated from the Bank of the Cardener River in Manresa, Catalonia, Spain

    PubMed Central

    Vilaplana, Cristina; Velasco, Juan; Pluvinet, Raquel; Santín, Sheila; Prat, Cristina; Julián, Esther; Alcaide, Fernando; Comas, Iñaki; Sumoy, Lauro; Cardona, Pere-Joan

    2015-01-01

    We present here the draft genome sequences of two Mycobacterium setense strains. One of them corresponds to the M. setense type strain DSM-45070, originally isolated from a patient with a posttraumatic chronic skin abscess. The other one corresponds to the nonpathogenic M. setense strain Manresensis, isolated from the Cardener River crossing Manresa, Catalonia, Spain. A comparative genomic analysis shows a smaller genome size and fewer genes in M. setense strain Manresensis relative to those of the type strain, and it shows the genome segments unique to each strain. PMID:25657273

  17. Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.

    PubMed

    Lin, Qingqing; Zhou, Mengying; Xu, Zongkai; Khanniche, Asma; Shen, Hao; Wang, Chuan

    2015-02-20

    Bacillus Calmette-Guerin (BCG) has failed in complete control of tuberculosis (TB), thus, novel tuberculosis vaccines are urgently needed. We have constructed several TB vaccine candidates, which are characterized by the use of Listeria ivanovii (LI) strain as an antigen delivery vector. Two L. ivanovii attenuated recombinant strains L. ivanovii△actAplcB-Rv0129c and L. ivanovii△actAplcB-Rv3875 were successfully screened. Results from genome PCR and sequencing showed that the Mycobacterium tuberculosis antigen gene cassette coding for Ag85C or ESAT-6 protein respectively had been integrated into LI genome downstream of mpl gene. Western blot confirmed the secretion of Ag85C or ESAT-6 protein from the recombinant LI strains. These two recombinant strains showed similar growth curves as wide type strain in vitro. In vivo, they transiently propagated in mice spleen and liver, and induced specific CD8(+) IFN-γ secretion. Therefore, in this paper, two novel LI attenuated strains expressing specific TB antigens were successfully constructed. The promising growth characteristics in mice immune system and the capability of induction of IFN-γ secretion make them of potential interest for development of TB vaccines.

  18. Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

    PubMed

    Chaidir, Lidya; Sengstake, Sarah; de Beer, Jessica; Oktavian, Antonius; Krismawati, Hana; Muhapril, Erfin; Kusumadewi, Inri; Annisa, Jessi; Anthony, Richard; van Soolingen, Dick; Achmad, Tri Hanggono; Marzuki, Sangkot; Alisjahbana, Bachti; van Crevel, Reinout

    2016-04-01

    Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region.

  19. Pattern recognition of genomic features with microarrays: site typing of Mycobacterium tuberculosis strains

    PubMed Central

    Raychaudhuri, Soumya; Stuart, Joshua M.; Liu, Xuemin; Small, Peter M.; Altman, Russ B.

    2009-01-01

    Mycobacterium tuberculosis (M. tb.) strains differ in the number and locations of a transposon-like insertion sequence known as IS6110. Accurate detection of this sequence can be used as a fingerprint for individual strains, but can be difficult because of noisy data. In this paper, we propose a non-parametric discriminant analysis method for predicting the locations of the IS6110 sequence from microarray data. Polymerase chain reaction extension products generated from primers specific for the insertion sequence are hybridized to a microarray containing targets corresponding to each open reading frame in M. tb. To test for insertion sites, we use microarray intensity values extracted from small windows of contiguous open reading frames. Rank-transformation of spot intensities and first-order differences in local windows provide enough information to reliably determine the presence of an insertion sequence. The non-parametric approach outperforms all other methods tested in this study. PMID:10977090

  20. Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

    PubMed

    Liapi, M; Botsaris, G; Slana, I; Moravkova, M; Babak, V; Avraam, M; Di Provvido, A; Georgiadou, S; Pavlik, I

    2015-04-01

    Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains.

  1. Strain differentiation of Mycobacterium tuberculosis for epidemiology in northwest of Iran.

    PubMed

    Asgharzadeh, M; Samadi Kafil, H; Pourostadi, M; Asadi Faezi, N; Rashedi, J; Mahdavipour, B

    2016-07-31

    Tuberculosis is a major health problem throughout the world and there are still a great number of people with the disease. Planning for controlling tuberculosis is required to identify the sources of infection and screening for the disease. The aim of this study is the differentiation of Mycobacterium tuberculosis strains for better understands the spread of the disease in North West Iran. In this study, 194 positive cultures of M. tuberculosis in North West Iran were evaluated by the MIRU-VNTR method. MIRU-12 differentiated the 194 isolates into 138 different patterns, comprising 30 clusters and 108 unique patterns (HGDI=0.9930). The largest cluster contained twelve isolates. The results showed that the majority of TB cases in North West Iran are due to reactivation and the 12-MIRU typing method can be used as a first-line method for strain differentiation of M. tuberculosis.

  2. Antibacterial Activity of Rifamycins for M. Smegmatis with Comparison of Oxidation and Binding to Tear Lipocalin

    PubMed Central

    Staudinger, Tamara; Redl, Bernhard; Glasgow, Ben J.

    2014-01-01

    A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored that established for Mycobacterium tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet-visible spectrum. Oxidation of rifamycins abrogated antimycobacterial activity in Mycobacterium smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives. PMID:24530503

  3. SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes.

    PubMed

    Schürch, Anita C; Kremer, Kristin; Hendriks, Amber C A; Freyee, Benthe; McEvoy, Christopher R E; van Crevel, Reinout; Boeree, Martin J; van Helden, Paul; Warren, Robin M; Siezen, Roland J; van Soolingen, Dick

    2011-01-01

    The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.

  4. Genomic variations associated with attenuation in Mycobacterium avium subsp. paratuberculosis vaccine strains

    PubMed Central

    2013-01-01

    Background Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne’s disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. Results Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26–32 Kbp) and tandem duplicated (11–40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. Conclusions This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling. PMID:23339684

  5. Factors Associated with Recently Transmitted Mycobacterium tuberculosis strain MS0006 in Hinds County, Mississippi

    PubMed Central

    Temple, Brian; Kwara, Awewura; Sunesara, Imran; Mena, Leandro; Dobbs, Thomas; Henderson, Harold; Holcomb, Mike; Webb, Risa

    2011-01-01

    Objective The objective of this study was to investigate risk factors associated with tuberculosis (TB) transmission that was caused by Mycobacterium tuberculosis strain MS0006 from 2004 to 2009 in Hinds County, Mississippi. Methods DNA fingerprinting using spoligotyping, mycobacterial interspersed repetitive unit, and IS6110-based restriction fragment length polymorphism of culture-confirmed cases of TB was performed. Clinical and demographic factors associated with strain MS0006 were analyzed by univariate and multivariate analysis. Results Of the 144 cases of TB diagnosed during the study period, 117 were culture positive with fingerprints available. There were 48 different strains, of which 6 clustered strains were distributed among 74 patients. The MS0006 strain accounted for 46.2% of all culture-confirmed cases. Risk factors for having the MS0006 strain in a univariate analysis included homelessness, HIV co-infection, sputum smear negativity, tuberculin skin test negativity, and noninjectable drug use. Multivariate analysis identified homelessness (odds ratio 7.88, 95% confidence interval 2.90-21.35) and African American race (odds ratio 5.80, 95% confidence interval 1.37-24.55) as independent predictors of having TB caused by the MS0006 strain of M tuberculosis. Conclusions Our findings suggest that a majority of recently transmitted TB in the studied county was caused by the MS0006 strain. African American race and homelessness were significant risk factors for inclusion in the cluster. Molecular epidemiology techniques continue to provide in-depth analysis of disease transmission and play a vital role in effective contact tracing and interruption of ongoing transmission. PMID:22089361

  6. Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages

    PubMed Central

    Hinds, Jason; Malloff, Chad A.; Bains, Manjeet; Hancock, Robert E.; Lam, Wan L.

    2010-01-01

    Background H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes. Methodology/Principal Findings In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion. Conclusions/Significance Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra. PMID:20548782

  7. Virulence, immunopathology and transmissibility of selected strains of Mycobacterium tuberculosis in a murine model

    PubMed Central

    Marquina-Castillo, Brenda; García-García, Lourdes; Ponce-de-León, Alfredo; Jimenez-Corona, Maria-Eugenia; Bobadilla-del Valle, Miriam; Cano-Arellano, Bulmaro; Canizales-Quintero, Sergio; Martinez-Gamboa, Areli; Kato-Maeda, Midori; Robertson, Brian; Young, Douglas; Small, Peter; Schoolnik, Gary; Sifuentes-Osornio, Jose; Hernandez-Pando, Rogelio

    2009-01-01

    After encounter with Mycobacterium tuberculosis, a series of non-uniform immune responses are triggered that define the course of the infection. Eight M. tuberculosis strains were selected from a prospective population-based study of pulmonary tuberculosis patients (1995–2003) based on relevant clinical/epidemiological patterns and tested in a well-characterized BALB/c mouse model of progressive pulmonary tuberculosis. In addition, a new mouse model of transmissibility consisting of prolonged cohousing (up to 60 days) of infected and naïve animals was tested. Four phenotypes were defined based on strain virulence (mouse survival, lung bacillary load and tissue damage), immunology response (cytokine expression determined by real-time polymerase chain reaction) and transmissibility (lung bacillary loads and cutaneous delayed-type hypersensitivity in naïve animals).We identified four clearly defined strain phenotypes: (1) hypervirulent strain with non-protective immune response and highly transmissible; (2) virulent strain, associated with high expression of proinflammatory cytokines (tumour necrosis factor and interferon) and very low anti-inflammatory cytokine expression (interleukins 4 and 10), which induced accelerated death by immunopathology; (3) strain inducing efficient protective immunity with lower virulence, and (4) strain demonstrating strong and early macrophage activation (innate immunity) with delayed participation of acquired immunity (interferon expression). We were able to correlate virulent and transmissible phenotypes in the mouse model and markers of community transmission such as tuberculin reactivity among contacts, rapid progression to disease and cluster status. However, we were not able to find correlation with the other two phenotypes. Our new transmission model supported the hypothesis that among these strains increased virulence was linked to increased transmission. PMID:19191912

  8. Whole-Genome Sequences of Mycobacterium bovis Strain MbURU-001, Isolated from Fresh Bovine Infected Samples

    PubMed Central

    Lasserre, Moira; Berná, Luisa; Greif, Gonzalo; Díaz-Viraqué, Florencia; Naya, Hugo; Castro-Ramos, Miguel; Juambeltz, Arturo

    2015-01-01

    Bovine tuberculosis in cattle has a high incidence in Uruguay, where it is considered a disease of national importance. We present the genome sequence of Mycobacterium bovis strain MbURU-001, isolated from pectoral lymph nodes of a bovine host from a cattle farm. PMID:26543108

  9. Rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.

    PubMed

    Mulholland, Claire V; Ruthe, Ali; Cursons, Ray T; Durrant, Robert; Karalus, Noel; Coley, Kathryn; Bower, James; Permina, Elizabeth; Coleman, Megan J; Roberts, Sally A; Arcus, Vickery L; Cook, Gregory M; Aung, Htin Lin

    2017-03-23

    Despite New Zealand being a low-tuberculosis (TB) burden country, there are disproportionately high rates of TB in particular populations. Here, we report a rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.

  10. Complete Genome Sequence of Mycobacterium tuberculosis Strain MtURU-001, Isolated from a Rapidly Progressing Outbreak in Uruguay

    PubMed Central

    Greif, Gonzalo; Iraola, Gregorio; Berná, Luisa; Coitinho, Cecilia; Rivas, Carlos M.; Naya, Hugo

    2014-01-01

    Despite efficient control programs, large clonal outbreaks of tuberculosis (TB) may arise in low-risk populations. Recently, an unusual TB outbreak was reported in Uruguay, reaching an elevated disease attack rate (53 to 69%). Here, we report the genome sequence of the Mycobacterium tuberculosis strain associated with this rapidly progressing outbreak, named MtURU-001. PMID:24459279

  11. Whole-Genome Sequences of Mycobacterium bovis Strain MbURU-001, Isolated from Fresh Bovine Infected Samples.

    PubMed

    Lasserre, Moira; Berná, Luisa; Greif, Gonzalo; Díaz-Viraqué, Florencia; Iraola, Gregorio; Naya, Hugo; Castro-Ramos, Miguel; Juambeltz, Arturo; Robello, Carlos

    2015-11-05

    Bovine tuberculosis in cattle has a high incidence in Uruguay, where it is considered a disease of national importance. We present the genome sequence of Mycobacterium bovis strain MbURU-001, isolated from pectoral lymph nodes of a bovine host from a cattle farm.

  12. Prevention of Mycobacterium avium subsp. paratuberculosis Infection in BALB/c Mice by Feeding Lactobacillus acidophilus Strain NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immune responses of 390 BALB/c mice fed the probiotic Lactobacillus acidophilus strain NP51® and infected with Mycobacterium avium subspecies paratuberculosis (MAP) were evaluated in a 6-month trial. Mice were randomized to nine treatment groups fed either viable- or heat-killed NP51 and inocula...

  13. ISOLATION OF THE GENOME SEQUENCE STRAIN MYCOBACTERIUM AVIUM 104 FROM MULTIPLE PATIENTS OVER A 17-YEAR PERIOD

    EPA Science Inventory

    The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated form an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genoty...

  14. Strains of Mycobacterium tuberculosis differ in affinity for human osteoblasts and alveolar cells in vitro.

    PubMed

    Sarkar, Shrabanti; Dlamini, Muyalo G; Bhattacharya, Debapriya; Ashiru, Olubisi T; Sturm, A Willem; Moodley, Prashini

    2016-01-01

    Although the lung is the primary site of infection of tuberculosis, Mycobacterium tuberculosis is capable of causing infection at other sites. In 5-10 % such extra-pulmonary tuberculosis is located in bone tissue of the spine. It is unknown whether host or microbial factors are responsible for the site where extra-pulmonary tuberculosis manifests itself. One MDR isolate belonging to strain F28, one susceptible F11 and one isolate each of susceptible, MDR and XDR F15/LAM4/KZN were cultured in Middlebrook 7H9 media. Human osteoblasts (SaOS-2) and human alveolar epithelial cells (A549) were exposed to these different isolates of M. tuberculosis and invasion capacity and intra-cellular multiplication rates were established. Mouse macrophage (MHS) cells exposed to M. tuberculosis H37Rv served as control. The invasion capacity of F15/LAM4/KZN representatives increased with the level of resistance. The F28 MDR strain showed similar invasion capacity as the XDR F15/LAM4/KZN for pulmonary epthelial cells, whilst the fully susceptible F11 strain displayed a propensity for osteoblasts. The differences observed may in part explain why certain strains are able to cause infection at specific extra-pulmonary sites. We postulated that the development of extra-pulmonary tuberculosis depends on the ability of the microbe to pass effectively through the alveolar epithelial lining and its affinity for cells other than those in pulmonary tissue.

  15. Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

    PubMed

    Saelens, Joseph W; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M; Xet-Mull, Ana M; Stout, Jason E; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M

    2015-12-01

    Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

  16. PE_PGRS30 of Mycobacterium tuberculosis mediates suppression of proinflammatory immune response in macrophages through its PGRS and PE domains.

    PubMed

    Chatrath, Shweta; Gupta, Vineet Kumar; Dixit, Aparna; Garg, Lalit C

    2016-09-01

    The success of Mycobacterium tuberculosis as a pathogen relies on its ability to survive inside macrophages and evade host immune mechanisms. M. tuberculosis employs multiple strategies to confer resistance against immune system including inhibition of phago-lysosomal fusion, modulation of cytokine responses and granuloma formation. PE_PGRS proteins, uniquely present in pathogenic mycobacteria, are cell surface molecules that are suggested to interact with host cells. PE_PGRS proteins have also been implicated in its pathogenesis. In the present study, immuno-regulatory property of Rv1651c-encoded PE_PGRS30 protein was explored. Infection of PMA-differentiated human THP-1 macrophages with Mycobacterium smegmatis harbouring pVV(1651c) resulted in reduced production of IL-12, TNF-α and IL-6, as compared to infection with M. smegmatis harbouring the control plasmid pVV16. No differential effect was observed on bacterial persistence inside macrophages or on macrophage mortality upon infection with the two recombinant strains. Infection of THP-1 macrophages with recombinant M. smegmatis expressing deletion variants of PE_PGRS30 indicated that anti-inflammatory function of the protein is possessed by its PGRS and PE domains while the C-terminal domain, when expressed alone, displayed antagonistic effect in terms of TNF-α secretion. These results suggest that PE_PGRS30 interferes with macrophage immune functions important for activation of adaptive T-cell responses.

  17. Genomic and functional analyses of Mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance

    PubMed Central

    Desjardins, Christopher A.; Cohen, Keira A.; Munsamy, Vanisha; Abeel, Thomas; Maharaj, Kashmeel; Walker, Bruce J.; Shea, Terrance P.; Almeida, Deepak V.; Manson, Abigail L.; Salazar, Alex; Padayatchi, Nesri; O’Donnell, Max R.; Mlisana, Koleka P.; Wortman, Jennifer; Birren, Bruce W.; Grosset, Jacques; Earl, Ashlee M.; Pym, Alexander S.

    2016-01-01

    A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs like D-cycloserine. Here, we used whole-genome sequences from 498 strains of M. tuberculosis to identify novel resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss-of-function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss-of-function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted utilization of this toxic drug among patients with susceptible infections. PMID:27064254

  18. Application of DNA markers to estimate genetic diversity of Mycobacterium tuberculosis strains.

    PubMed

    Korzekwa, Karol; Polok, Kornelia; Zieliński, Roman

    2006-01-01

    The obligatory human pathogen, Mycobacterium tuberculosis, is the most important etiological factor of tuberculosis. Unfortunately, there is little information about genetic diversity of this pathogen. The main aim of this research was the estimation of genetic diversity of M. tuberculosis on the basis of various categories of DNA markers. The genome of 32 strains were scanned by DNA markers such RAPD, IS6110 and catalase-peroxidase katG gene. All 162 identified loci were polymorphic. The genetic diversity coefficient (HT) of M. tuberculosis was 0.32 for RAPD and 0.27 for IS 6110. There were 14 alleles in katG gene. All strains were characterised by the individual molecular pattern. Genetic similarity varied from 0.13 to 0.94 (RAPD markers) and from 0 to 1 for (IS6110). M. tuberculosis strains did not represent a clonal structure, single source of transmission and epidemiological relationships as well. The applied DNA markers proved to be highly efficient for analysis of genetic structure of M. tuberculosis.

  19. High Affinity Inha Inhibitors with Activity Against Drug-Resistant Strains of Mycobacterium Tuberculosis

    SciTech Connect

    Sullivan,T.; Truglio, J.; Boyne, M.; Novichenok, P.; Zhang, X.; Stratton, C.; Li, H.; Kaur, T.; Amin, A.; et al.

    2006-01-01

    Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki{prime} value of 1 nM for InhA and MIC{sub 99} values of 2-3 {micro}g mL{sup -1} (6-10 {micro}M) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC{sub 99}, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

  20. Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader.

    PubMed

    Coleman, Nicholas V; Yau, Sheree; Wilson, Neil L; Nolan, Laura M; Migocki, Margaret D; Ly, Mai-Anh; Crossett, Ben; Holmes, Andrew J

    2011-06-01

    Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

  1. Sublineage structure analysis of Mycobacterium tuberculosis complex strains using multiple-biomarker tensors

    PubMed Central

    2011-01-01

    Background Strains of Mycobacterium tuberculosis complex (MTBC) can be classified into major lineages based on their genotype. Further subdivision of major lineages into sublineages requires multiple biomarkers along with methods to combine and analyze multiple sources of information in one unsupervised learning model. Typically, spacer oligonucleotide type (spoligotype) and mycobacterial interspersed repetitive units (MIRU) are used for TB genotyping and surveillance. Here, we examine the sublineage structure of MTBC strains with multiple biomarkers simultaneously, by employing a tensor clustering framework (TCF) on multiple-biomarker tensors. Results Simultaneous analysis of the spoligotype and MIRU type of strains using TCF on multiple-biomarker tensors leads to coherent sublineages of major lineages with clear and distinctive spoligotype and MIRU signatures. Comparison of tensor sublineages with SpolDB4 families either supports tensor sublineages, or suggests subdivision or merging of SpolDB4 families. High prediction accuracy of major lineage classification with supervised tensor learning on multiple-biomarker tensors validates our unsupervised analysis of sublineages on multiple-biomarker tensors. Conclusions TCF on multiple-biomarker tensors achieves simultaneous analysis of multiple biomarkers and suggest a new putative sublineage structure for each major lineage. Analysis of multiple-biomarker tensors gives insight into the sublineage structure of MTBC at the genomic level. PMID:21988942

  2. Mycobacterium tuberculosis CwsA overproduction modulates cell division and cell wall synthesis.

    PubMed

    Plocinski, P; Martinez, L; Sarva, K; Plocinska, R; Madiraju, M; Rajagopalan, M

    2013-12-01

    We recently showed that two small membrane proteins of Mycobacterium tuberculosis, CwsA and CrgA, interact with each other, and that loss of CwsA in M. smegmatis is associated with defects in the cell division and cell wall synthesis processes. Here we show that CwsA overproduction also affected growth, cell division and cell shape of M. smegmatis and M. tuberculosis. CwsA overproduction in M. tuberculosis led to increased sensitivity to cefsulodin, a penicillin-binding protein (PBP) 1A/1B targeting beta (β) -lactam, but was unaffected by other β-lactams and vancomycin. A M. smegmatis cwsA overexpressing strain showed bulgy cells, increased fluorescent vancomycin staining and altered localization of Wag31-mCherry fusion protein. However, the levels of phosphorylated Wag31, important for optimal peptidoglycan synthesis and growth in mycobacteria, were not affected. Interestingly, CwsA overproduction in E. coli led to the formation of large rounded cells that eventually lysed whereas the overproduction of FtsZ along with CwsA reversed this phenotype. Together, our results emphasize that optimal levels of CwsA are required for regulated cell wall synthesis, hence maintenance of cell shape, and that CwsA likely interacts with and modulates the activities of other cell wall synthetic components including PBPs.

  3. The rpoB gene of Mycobacterium tuberculosis.

    PubMed Central

    Miller, L P; Crawford, J T; Shinnick, T M

    1994-01-01

    A portion of the Mycobacterium tuberculosis gene encoding the beta subunit of RNA polymerase (rpoB) was amplified by PCR using degenerate oligonucleotides and used as a hybridization probe to isolate plasmid clones carrying the entire rpoB gene of M. tuberculosis H37Rv, a virulent, rifampin-susceptible strain. Sequence analysis of a 5,084-bp SacI genomic DNA fragment revealed a 3,534-bp open reading frame encoding an 1,178-amino-acid protein with 57% identity with the Escherichia coli beta subunit. This SacI fragment also carried a portion of the rpoC gene located 43 bp downstream from the 3' end of the rpoB open reading frame; this organization is similar to that of the rpoBC operon of E. coli. The M. tuberculosis rpoB gene was cloned into the shuttle plasmid pMV261 and electroporated into the LR223 strain of Mycobacterium smegmatis, which is highly resistant to rifampin (MIC > 200 micrograms/ml). The resulting transformants were relatively rifampin susceptible (MIC = 50 micrograms/ml). Using PCR mutagenesis techniques, we introduced a specific rpoB point mutation (associated with clinical strains of rifampin-resistant M. tuberculosis) into the cloned M. tuberculosis rpoB gene and expressed this altered gene in the LR222 strain of M. smegmatis, which is susceptible to rifampin (MIC = 25 micrograms/ml). The resulting transformants were rifampin resistant (MIC = 200 micrograms/ml). The mutagenesis and expression strategy of the cloned M. tuberculosis rpoB gene that we have employed in this study will allow us to determine the rpoB mutations that are responsible for rifampin resistance in M. tuberculosis. PMID:8031050

  4. Metabolism of 2-methylpropene (isobutylene) by the aerobic bacterium Mycobacterium sp. strain ELW1.

    PubMed

    Kottegoda, Samanthi; Waligora, Elizabeth; Hyman, Michael

    2015-03-01

    An aerobic bacterium (Mycobacterium sp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h(-1)) with a yield of 0.38 mg (dry weight) mg 2-methylpropene(-1). Strain ELW1 also grew more slowly on both cis- and trans-2-butene but did not grow on any other C2 to C5 straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates.

  5. Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples

    PubMed Central

    Zink, A R; Nerlich, A G

    2004-01-01

    Aims: To investigate the use of different molecular analyses that can identify distinct strains of human pathogenic mycobacteria in formalin fixed and paraffin wax embedded archival tissue samples to see whether it is possible to differentiate between the members of the Mycobacterium tuberculosis complex (M tuberculosis, M bovis, M africanum, M microti, or M canettii) and/or substrains in a high number of samples. This would be of interest for identifying individual infection traits and superinfection by different mycobacterial strains. Methods: Forty nine archival tissue samples with clinically and/or histologically suspected tuberculosis infection were subjected to molecular DNA analysis. Results: The molecular analysis revealed the presence of M tuberculosis complex DNA in 20 samples, whereas acid fast bacilli could be detected by Ziehl-Neelsen staining in only eight samples. All IS6110 positive samples were further characterised by spoligotyping and seven cases provided M tuberculosis specific signatures, whereas M bovis specific signatures were obtained in four cases. The analysis of mtp40, oxyR, and pncA partial gene sequences confirmed the presence of M tuberculosis in six cases and M bovis in one case. The amplification and sequencing of four further genetic regions (katG, gyrA, TbD1, RD9) characterised six “modern” M tuberculosis strains belonging to genetic groups 2 or 3. Conclusion: This study provides clear evidence that archival paraffin wax embedded material can be used for further studies on the strain identification of M tuberculosis complex strains and can therefore unequivocally be used for the study of the epidemiology and evolution of tuberculosis pathogens. PMID:15509681

  6. Metabolism of 2-Methylpropene (Isobutylene) by the Aerobic Bacterium Mycobacterium sp. Strain ELW1

    PubMed Central

    Kottegoda, Samanthi; Waligora, Elizabeth

    2015-01-01

    An aerobic bacterium (Mycobacterium sp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h−1) with a yield of 0.38 mg (dry weight) mg 2-methylpropene−1. Strain ELW1 also grew more slowly on both cis- and trans-2-butene but did not grow on any other C2 to C5 straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates. PMID:25576605

  7. Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin.

    PubMed

    Boradia, Vishant Mahendra; Malhotra, Himanshu; Thakkar, Janak Shrikant; Tillu, Vikas Ajit; Vuppala, Bhavana; Patil, Pravinkumar; Sheokand, Navdeep; Sharma, Prerna; Chauhan, Anoop Singh; Raje, Manoj; Raje, Chaaya Iyengar

    2014-08-28

    Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.

  8. Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Rodrigues, Valnês da Silva; Pinto, Antônio Frederico Michel; Wink, Priscila Lamb; Sánchez-Quitian, Zilpa Adriana; Petersen, Guilherme Oliveira; Campos, Maria Martha; Basso, Luiz Augusto; Santos, Diógenes Santiago

    2017-01-01

    BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug

  9. Comparative functional genomics and the bovine macrophage response to strains of the mycobacterium genus.

    PubMed

    Rue-Albrecht, Kévin; Magee, David A; Killick, Kate E; Nalpas, Nicolas C; Gordon, Stephen V; MacHugh, David E

    2014-01-01

    Mycobacterial infections are major causes of morbidity and mortality in cattle and are also potential zoonotic agents with implications for human health. Despite the implementation of comprehensive animal surveillance programs, many mycobacterial diseases have remained recalcitrant to eradication in several industrialized countries. Two major mycobacterial pathogens of cattle are Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis (MAP), the causative agents of bovine tuberculosis (BTB) and Johne's disease (JD), respectively. BTB is a chronic, granulomatous disease of the respiratory tract that is spread via aerosol transmission, while JD is a chronic granulomatous disease of the intestines that is transmitted via the fecal-oral route. Although these diseases exhibit differential tissue tropism and distinct complex etiologies, both M. bovis and MAP infect, reside, and replicate in host macrophages - the key host innate immune cell that encounters mycobacterial pathogens after initial exposure and mediates the subsequent immune response. The persistence of M. bovis and MAP in macrophages relies on a diverse series of immunomodulatory mechanisms, including the inhibition of phagosome maturation and apoptosis, generation of cytokine-induced necrosis enabling dissemination of infection through the host, local pathology, and ultimately shedding of the pathogen. Here, we review the bovine macrophage response to infection with M. bovis and MAP. In particular, we describe how recent advances in functional genomics are shedding light on the host macrophage-pathogen interactions that underlie different mycobacterial diseases. To illustrate this, we present new analyses of previously published bovine macrophage transcriptomics data following in vitro infection with virulent M. bovis, the attenuated vaccine strain M. bovis BCG, and MAP, and discuss our findings with respect to the differing etiologies of BTB and JD.

  10. Bacteriological and Molecular Analysis of Rifampin-Resistant Mycobacterium tuberculosis Strains Isolated in Australia

    PubMed Central

    Yuen, Lilly K. W.; Leslie, David; Coloe, Peter J.

    1999-01-01

    To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant Mycobacterium tuberculosis strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the rpoB gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS6110, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant M. tuberculosis strains isolated within the country. PMID:10565894

  11. Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India.

    PubMed

    Lavania, Mallika; Jadhav, Rupendra; Turankar, Ravindra P; Singh, Itu; Nigam, Astha; Sengupta, U

    2015-12-01

    Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community.

  12. Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval Leprosy Hospital

    PubMed Central

    Taylor, G. Michael; Tucker, Katie; Butler, Rachel; Pike, Alistair W. G.; Lewis, Jamie; Roffey, Simon; Marter, Philip; Lee, Oona Y-C; Wu, Houdini H. T.; Minnikin, David E.; Besra, Gurdyal S.; Singh, Pushpendra; Cole, Stewart T.; Stewart, Graham R.

    2013-01-01

    Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period. PMID:23638071

  13. Transcriptional analysis of diverse strains Mycobacterium avium subspecies paratuberculosis in primary bovine monocyte derived macrophages.

    PubMed

    Zhu, Xiaochun; Tu, Zheng J; Coussens, Paul M; Kapur, Vivek; Janagama, Harish; Naser, Saleh; Sreevatsan, Srinand

    2008-10-01

    In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.

  14. Genomics Study of Mycobacterium tuberculosis Strains from Different Ethnic Populations in Taiwan

    PubMed Central

    Dou, Horng-Yunn; Chen, Yih-Yuan; Chen, Ying-Tsong; Chang, Jia-Ru; Lin, Chien-Hsing; Wu, Keh-Ming; Lin, Ming-Shian; Su, Ih-Jen; Tsai, Shih-Feng

    2016-01-01

    To better understand the transmission and evolution of Mycobacterium tuberculosis (MTB) in Taiwan, six different MTB isolates (representatives of the Beijing ancient sublineage, Beijing modern sublineage, Haarlem, East-African Indian, T1, and Latin-American Mediterranean (LAM)) were characterized and their genomes were sequenced. Discriminating among large sequence polymorphisms (LSPs) that occur once versus those that occur repeatedly in a genomic region may help to elucidate the biological roles of LSPs and to identify the useful phylogenetic relationships. In contrast to our previous LSP-based phylogeny, the sequencing data allowed us to determine actual genetic distances and to define precisely the phylogenetic relationships between the main lineages of the MTB complex. Comparative genomics analyses revealed more nonsynonymous substitutions than synonymous changes in the coding sequences. Furthermore, MTB isolate M7, a LAM-3 clinical strain isolated from a patient of Taiwanese aboriginal origin, is closely related to F11 (LAM), an epidemic tuberculosis strain isolated in the Western Cape of South Africa. The PE/PPE protein family showed a higher dn/ds ratio compared to that for all protein-coding genes. Finally, we found Haarlem-3 and LAM-3 isolates to be circulating in the aboriginal community in Taiwan, suggesting that they may have originated with post-Columbus Europeans. Taken together, our results revealed an interesting association with historical migrations of different ethnic populations, thus providing a good model to explore the global evolution and spread of MTB. PMID:27721649

  15. Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against Mycobacterium bovis Challenge

    PubMed Central

    Rizzi, Caroline; Bianco, María Verónica; Blanco, Federico Carlos; Soria, Marcelo; Gravisaco, María José; Montenegro, Valeria; Vagnoni, Lucas; Buddle, Bryce; Garbaccio, Sergio; Delgado, Fernando; Leal, Karen Silva; Cataldi, Angel Adrián; Dellagostin, Odir Antônio; Bigi, Fabiana

    2012-01-01

    Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur. PMID:23251517

  16. Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital.

    PubMed

    Taylor, G Michael; Tucker, Katie; Butler, Rachel; Pike, Alistair W G; Lewis, Jamie; Roffey, Simon; Marter, Philip; Lee, Oona Y-C; Wu, Houdini H T; Minnikin, David E; Besra, Gurdyal S; Singh, Pushpendra; Cole, Stewart T; Stewart, Graham R

    2013-01-01

    Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

  17. Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.

    PubMed

    Trombone, Ana Paula Fávaro; Pedrini, Sílvia Cristina Barbosa; Diório, Suzana Madeira; Belone, Andréa de Faria Fernandes; Fachin, Luciana Raquel Vicenzi; do Nascimento, Dejair Caitano; Rosa, Patricia Sammarco

    2014-03-23

    Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

  18. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

    PubMed

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

  19. Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

    PubMed Central

    Khan, Ashraf A.; Wang, Rong-Fu; Cao, Wei-Wen; Doerge, Daniel R.; Wennerstrom, David; Cerniglia, Carl E.

    2001-01-01

    Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits. PMID:11472934

  20. Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains.

    PubMed

    Kremer, Kristin; Arnold, Catherine; Cataldi, Angel; Gutiérrez, M Cristina; Haas, Walter H; Panaiotov, Stefan; Skuce, Robin A; Supply, Philip; van der Zanden, Adri G M; van Soolingen, Dick

    2005-11-01

    In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.

  1. Evaluation of a high-throughput repetitive-sequence-based PCR system for DNA fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium complex strains.

    PubMed

    Cangelosi, Gerard A; Freeman, Robert J; Lewis, Kaeryn N; Livingston-Rosanoff, Devon; Shah, Ketan S; Milan, Sparrow Joy; Goldberg, Stefan V

    2004-06-01

    Repetitive-sequence-based PCR (rep-PCR) is useful for generating DNA fingerprints of diverse bacterial and fungal species. Rep-PCR amplicon fingerprints represent genomic segments lying between repetitive sequences. A commercial system that electrophoretically separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been adapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis and M. avium complex (MAC) isolates was evaluated. M. tuberculosis strains (n = 56) were typed by spoligotyping with rep-PCR as a high-resolution adjunct. Results were compared with those generated by a standard approach of spoligotyping with IS6110-targeted restriction fragment length polymorphism (IS6110-RFLP) as the high-resolution adjunct. The sample included 11 epidemiologically and genotypically linked outbreak isolates and a population-based sample of 45 isolates from recent immigrants to Seattle, Wash., from the African Horn countries of Somalia, Eritrea, and Ethiopia. Twenty isolates exhibited unique spoligotypes and were not analyzed further. Of the 36 outbreak and African Horn isolates with nonunique spoligotypes, 23 fell into four clusters identified by IS6110-RFLP and rep-PCR, with 97% concordance observed between the two methods. Both approaches revealed extensive strain heterogeneity within the African Horn sample, consistent with a predominant pattern of reactivation of latent infections in this immigrant population. Rep-PCR exhibited 89% concordance with IS1245-RFLP typing of 28 M. avium subspecies avium strains. For M. tuberculosis as well as M. avium subspecies avium, the discriminative power of rep-PCR equaled or exceeded that of RFLP. Rep-PCR also generated DNA fingerprints from M. intracellulare (n = 8) and MAC(x) (n = 2) strains. It shows promise as a fast, unified method for high-throughput genotypic fingerprinting of multiple Mycobacterium species.

  2. Substrate range and enantioselectivity of epoxidation reactions mediated by the ethene-oxidising Mycobacterium strain NBB4.

    PubMed

    Cheung, Samantha; McCarl, Victoria; Holmes, Andrew J; Coleman, Nicholas V; Rutledge, Peter J

    2013-02-01

    Mycobacterium strain NBB4 is an ethene-oxidising micro-organism isolated from estuarine sediments. In pursuit of new systems for biocatalytic epoxidation, we report the capacity of strain NBB4 to convert a diverse range of alkene substrates to epoxides. A colorimetric assay based on 4-(4-nitrobenzyl)pyridine) has been developed to allow the rapid characterisation and quantification of biocatalytic epoxide synthesis. Using this assay, we have demonstrated that ethene-grown NBB4 cells epoxidise a wide range of alkenes, including terminal (propene, 1-butene, 1-hexene, 1-octene and 1-decene), cyclic (cyclopentene, cyclohexene), aromatic (styrene, indene) and functionalised substrates (allyl alcohol, dihydropyran and isoprene). Apparent specific activities have been determined and range from 2.5 to 12.0 nmol min(-1) per milligram of cell protein. The enantioselectivity of epoxidation by Mycobacterium strain NBB4 has been established using styrene as a test substrate; (R)-styrene oxide is produced in enantiomeric excesses greater than 95%. Thus, the ethene monooxygenase of Mycobacterium NBB4 has a broad substrate range and promising enantioselectivity, confirming its potential as a biocatalyst for alkene epoxidation.

  3. Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG.

    PubMed Central

    Frothingham, R

    1995-01-01

    The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Seven strains of the M. tuberculosis complex were sequenced in a region of about 300 bp which contains multiple 15-bp tandem repeats and which is part of a 1,551-bp open reading frame. Four distinct sequences were obtained, each defining a sequevar. A sequevar includes the strain or strains with a given sequence. The type strain M. tuberculosis TMC 102 (H37Rv) was designated sequevar MED-G. When compared to MED-G, sequevar LONG had an insertion of one 15-bp tandem repeat and sequevar SHORT had a deletion of one tandem repeat. Sequevar MED-C had a G-->C substitution, coding for the conservative change Ser-->Thr. BanI cuts only sequevar MED-C at the site of the substitution. PCR-restriction enzyme analysis was used to determine the sequevars of 92 M. tuberculosis complex strains. All 23 M. bovis BCG strains belonged to sequevar MED-C. The M. africanum type strain was sequevar SHORT. The remaining 68 strains of M. tuberculosis, M. bovis (not BCG), and M. microti were sequevars LONG (3 strains) or MED-G (65 strains). PCR-restriction enzyme analysis was applied to reference strains and clinical isolates with a worldwide distribution. This method provides rapid, sensitive, and specific identification of the important vaccine strain M. bovis BCG. PMID:7790448

  4. Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains.

    PubMed

    Bragin, E Yu; Shtratnikova, V Yu; Dovbnya, D V; Schelkunov, M I; Pekov, Yu A; Malakho, S G; Egorova, O V; Ivashina, T V; Sokolov, S L; Ashapkin, V V; Donova, M V

    2013-11-01

    A comparative genome analysis of Mycobacterium spp. VKM Ac-1815D, 1816D and 1817D strains used for efficient production of key steroid intermediates (androst-4-ene-3,17-dione, AD, androsta-1,4-diene-3,17-dione, ADD, 9α-hydroxy androst-4-ene-3,17-dione, 9-OH-AD) from phytosterol has been carried out by deep sequencing. The assembled contig sequences were analyzed for the presence putative genes of steroid catabolism pathways. Since 3-ketosteroid-9α-hydroxylases (KSH) and 3-ketosteroid-Δ(1)-dehydrogenase (Δ(1) KSTD) play key role in steroid core oxidation, special attention was paid to the genes encoding these enzymes. At least three genes of Δ(1) KSTD (kstD), five genes of KSH subunit A (kshA), and one gene of KSH subunit B of 3-ketosteroid-9α-hydroxylases (kshB) have been found in Mycobacterium sp. VKM Ac-1817D. Strains of Mycobacterium spp. VKM Ac-1815D and 1816D were found to possess at least one kstD, one kshB and two kshA genes. The assembled genome sequence of Mycobacterium sp. VKM Ac-1817D differs from those of 1815D and 1816D strains, whereas these last two are nearly identical, differing by 13 single nucleotide substitutions (SNPs). One of these SNPs is located in the coding region of a kstD gene and corresponds to an amino acid substitution Lys (135) in 1816D for Ser (135) in 1815D. The findings may be useful for targeted genetic engineering of the biocatalysts for biotechnological application.

  5. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    PubMed

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection.

  6. Molecular Typing of Mycobacterium Tuberculosis Strains: A Fundamental Tool for Tuberculosis Control and Elimination.

    PubMed

    Cannas, Angela; Mazzarelli, Antonio; Di Caro, Antonino; Delogu, Giovanni; Girardi, Enrico

    2016-06-24

    Tuberculosis (TB) is still an important cause of morbidity and mortality worldwide. An improvement of the strategies for disease control is necessary in both low- and high-incidence TB countries. Clinicians, epidemiologists, laboratory specialists, and public health players should work together in order to achieve a significant reduction in TB transmission and spread of drug-resistant strains. Effective TB surveillance relies on early diagnosis of new cases, appropriate therapy, and accurate detection of outbreaks in the community, in order to implement proper TB control strategies. To achieve this goal, information from classical and molecular epidemiology, together with patient clinical data need to be combined. In this review, we summarize the methodologies currently used in molecular epidemiology, namely molecular typing. We will discuss their efficiency to phylogenetically characterize Mycobacterium tuberculosis isolates, and their ability to provide information that can be useful for disease control. We will also introduce next generation sequencing as the methodology that potentially could provide in a short time both, detection of new outbreaks and identification of resistance patterns. This could envision a potential of next generation sequencing as an important tool for accurate patient management and disease control.

  7. Whole genome response in guinea pigs infected with the high virulence strain Mycobacterium tuberculosis TT372

    PubMed Central

    Aiyaz, Mohamed; Bipin, Chand; Pantulwar, Vinay; Mugasimangalam, Raja; Shanley, Crystal A.; Ordway, Diane J; Orme, Ian M.

    2014-01-01

    SUMMARY In this study we conducted a microarray-based whole genomic analysis of gene expression in the lungs after exposure of guinea pigs to a low dose aerosol of the Atypical Beijing Western Cape TT372 strain of Mycobacterium tuberculosis, after harvesting lung tissues three weeks after infection at a time that effector immunity is starting to peak. The infection resulted in a very large up-regulation of multiple genes at this time, particularly in the context of a “chemokine storm” in the lungs. Overall gene expression was considerably reduced in animals that had been vaccinated with BCG two months earlier, but in both cases strong signatures featuring gamma interferon [IFNγ] and tumor necrosis factor [TNFα] were observed indicating the potent TH1 response in these animals. Even though their effects are not seen until later in the infection, even at this early time point gene expression patterns associated with the potential emergence of regulatory T cells were observed. Genes involving lung repair, response to oxidative stress, and cell trafficking were strongly expressed, but interesting these gene patterns differed substantially between the infected and vaccinated/infected groups of animals. Given the importance of this species as a relevant and cost-effective small animal model of tuberculosis, this approach has the potential to provide new information regarding the effects of vaccination on control of the disease process. PMID:25621360

  8. Attenuated strains of Mycobacterium avium subspecies paratuberculosis as vaccine candidates against Johne's disease.

    PubMed

    Settles, Erik W; Kink, John A; Talaat, Adel

    2014-04-11

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. Johne's disease has a severe economic impact on the dairy industry in the USA and worldwide. In an effort to combat this disease, we screened several transposon mutants that were attenuated in the murine model of paratuberculosis for the potential use as live attenuated vaccines. Using the murine model, two vaccine candidates (pgs1360, pgs3965 with mutations of fabG2_2 and umaA1, respectively) were at or below the limit of detection for tissue colonization suggesting their low level persistence and hence safety. Prior to challenge, both candidates induced a M. paratuberculosis-specific IFN-γ, an indication of eliciting cell-mediated immunity. Following challenge with a virulent strain of M. paratuberculosis, the two vaccine candidates significantly reduced bacterial colonization in organs with reduced histological scores compared to control animals. In addition, one of the vaccine candidates (pgs3965) also induced IL-17a, a cytokine associated with protective immunity in mycobacterial infection. Our analysis suggested that the pgs3965 vaccine candidate is a potential live-attenuated vaccine that could be tested further in ruminant models of paratuberculosis. The analysis also validated our screening strategy to identify effective vaccine candidates against intracellular pathogens.

  9. Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

    PubMed Central

    Rodriguez, John D.; Worthy, Sue A.; Ismail, Nazir A.; Omar, Shaheed V.; Dreyer, Andries W.; Fourie, P. Bernard; Hoosen, Anwar A.; Chambers, James P.; Fischer, Gerald W.

    2012-01-01

    A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China. PMID:22972833

  10. Rapid Test for Identification of a Highly Transmissible Mycobacterium tuberculosis Beijing Strain of Sub-Saharan Origin

    PubMed Central

    Alonso, Henar; Gavín, Patricia; Hernández-Febles, Melissa; Campos-Herrero, María Isolina; Copado, Rodolfo; Cañas, Fernando; Kremer, Kristin; Caminero, José Antonio; Martín, Carlos; Samper, Sofía

    2012-01-01

    The development of a rapid test to identify Mycobacterium tuberculosis Beijing isolates and specifically strain GC1237, coming from a sub-Saharan country, is needed due to its alarming wide spread on Gran Canaria Island (Spain). A rapid test that detects IS6110 present between dnaA and dnaN in the Beijing strains and in a specific site for GC1237 (Rv2180c) has been developed. This test would be a useful tool in the surveillance of subsequent cases. PMID:22116140

  11. Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

    PubMed

    Swords, W E; Guenthner, P C; Birkness, K A; Lal, R B; Dezzutti, C S; Quinn, F D

    2006-02-01

    Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.

  12. PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

    PubMed

    Iantomasi, Raffaella; Sali, Michela; Cascioferro, Alessandro; Palucci, Ivana; Zumbo, Antonella; Soldini, Silvia; Rocca, Stefano; Greco, Emanuela; Maulucci, Giuseppe; De Spirito, Marco; Fraziano, Maurizio; Fadda, Giovanni; Manganelli, Riccardo; Delogu, Giovanni

    2012-03-01

    The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

  13. Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis*

    PubMed Central

    Zhang, Ran; Pan, Yuan T.; He, Shouming; Lam, Michael; Brayer, Gary D.; Elbein, Alan D.; Withers, Stephen G.

    2011-01-01

    Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp230 was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of kcat upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site. PMID:21840994

  14. DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

    PubMed Central

    Jensen, Ronald W.; Rivest, Jason; Li, Wei; Vissa, Varalakshmi

    2011-01-01

    The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO1, leprosy remains endemic in many countries with approximately 250,000 new cases each year.2 The entire M. leprae genome has been mapped3,4 and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites).5 Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci.5,6,7 Variable number tandem repeat (VNTR)5 analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing7,8,9, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA)10 has been used to study leprosy evolution and transmission in several countries including China11,12, Malawi8, the Philippines10,13, and Brazil14. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS).10 The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions.10 The PCR products may be subjected to agarose

  15. DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA).

    PubMed

    Jensen, Ronald W; Rivest, Jason; Li, Wei; Vissa, Varalakshmi

    2011-07-15

    The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the

  16. Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain.

    PubMed

    Marcus, Sarah A; Steinberg, Howard; Talaat, Adel M

    2015-10-13

    Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.

  17. Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

    PubMed

    Schneider, J; Grosser, R; Jayasimhulu, K; Xue, W; Warshawsky, D

    1996-01-01

    The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.

  18. Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin.

    PubMed

    Staudinger, Tamara; Redl, Bernhard; Glasgow, Ben J

    2014-04-01

    A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet-visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.

  19. Comprehensive Insights in the Mycobacterium avium subsp. paratuberculosis Genome Using New WGS Data of Sheep Strain JIII-386 from Germany

    PubMed Central

    Möbius, Petra; Hölzer, Martin; Felder, Marius; Nordsiek, Gabriele; Groth, Marco; Köhler, Heike; Reichwald, Kathrin; Platzer, Matthias; Marz, Manja

    2015-01-01

    Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP)—the etiologic agent of Johne’s disease—affects cattle, sheep, and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III], respectively, MAP-C [Type-II]), comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. This study presents the so far best assembled genome of a MAP-S-strain: Sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from the United States and Australia, and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI (National Center for Biotechnology Information) annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined by either NCBI or BacProt. A new Shine–Dalgarno sequence motif (5′-AGCTGG-3′) was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 noncoding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four of ten assumed MAP-S-specific large sequence polymorphism regions (LSPSs) are still present in MAP-C strains; new LSPSs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirms the strong similarity of MAP-C strains and shows higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to Mycobacterium intracellulare. PMID:26384038

  20. mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

    PubMed Central

    De Rossi, Edda; Branzoni, Manuela; Cantoni, Rita; Milano, Anna; Riccardi, Giovanna; Ciferri, Orio

    1998-01-01

    The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP. PMID:9811672

  1. Two genetically-related multidrug-resistant Mycobacterium tuberculosis strains induce divergent outcomes of infection in two human macrophage models.

    PubMed

    Yokobori, Noemí; López, Beatriz; Geffner, Laura; Sabio y García, Carmen; Schierloh, Pablo; Barrera, Lucía; de la Barrera, Silvia; Sakai, Shunsuke; Kawamura, Ikuo; Mitsuyama, Masao; Ritacco, Viviana; Sasiain, María del Carmen

    2013-06-01

    Mycobacterium tuberculosis has a considerable degree of genetic variability resulting in different epidemiology and disease outcomes. We evaluated the pathogen-host cell interaction of two genetically closely-related multidrug-resistant M. tuberculosis strains of the Haarlem family, namely the strain M, responsible for an extensive multidrug-resistant tuberculosis outbreak, and its kin strain 410 which caused a single case in two decades. Intracellular growth and cytokine responses were evaluated in human monocyte-derived macrophages and dU937 macrophage-like cells. In monocyte-derived macrophages, strain M grew more slowly and induced lower levels of TNF-α and IL-10 than 410, contrasting with previous studies with other strains, where a direct correlation was observed between increased intracellular growth and epidemiological success. On the other hand, in dU937 cells, no difference in growth was observed between both strains, and strain M induced significantly higher TNF-α levels than strain 410. We found that both cell models differed critically in the expression of receptors for M. tuberculosis entry, which might explain the different infection outcomes. Our results in monocyte-derived macrophages suggest that strain M relies on a modest replication rate and cytokine induction, keeping a state of quiescence and remaining rather unnoticed by the host. Collectively, our results underscore the impact of M. tuberculosis intra-species variations on the outcome of host cell infection and show that results can differ depending on the in vitro infection model.

  2. Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting

    PubMed Central

    Pérez del Molino Bernal, Inmaculada C.; Lillebaek, Troels; Pedersen, Mathias K.; Martinez-Martinez, Luis; Folkvardsen, Dorte B.; Agüero, Jesús; Rasmussen, E. Michael

    2016-01-01

    Background Tuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting. Methods DNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRU-VNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe. Results The rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants. Conclusion Transmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating. PMID:27315243

  3. Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review.

    PubMed

    Stevenson, Karen

    2015-06-19

    Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen that causes a chronic, progressive granulomatous enteritis known as Johne's disease or paratuberculosis. The disease is endemic in many parts of the world and responsible for considerable losses to the livestock and associated industries. Diagnosis and control are problematic, due mostly to the long incubation period of the disease when infected animals show no clinical signs and are difficult to detect, and the ability of the organism to survive and persist in the environment. The existence of phenotypically distinct strains of Map has been known since the 1930s but the genetic differentiation of Map strain types has been challenging and only recent technologies have proven sufficiently discriminative for strain comparisons, tracing the sources of infection and epidemiological studies. It is important to understand the differences that exist between Map strains and how they influence both development and transmission of disease. This information is required to develop improved diagnostics and effective vaccines for controlling Johne's disease. Here I review the current classification of Map strain types, the sources of the genetic variability within strains, growth characteristics and epidemiological traits associated with strain type and the influence of strain type on infection and pathogenicity.

  4. Evaluation of pathogenesis caused in cattle and guinea pig by a Mycobacterium bovis strain isolated from wild boar

    PubMed Central

    2011-01-01

    Background In many regions of the world, wild mammals act as reservoir of Mycobacterium bovis, a situation that prevents the eradication of bovine tuberculosis. In order to observe whether a strain isolated from a wild boar, previously tested as highly virulent in a mice model, is also virulent in cattle, we performed cattle experimental inoculation with this strain Results Groups of Friesian calves were either infected with the wild boar strain M. bovis 04-303 or with the bovine strain NCTC10772 as a control. We found that antigen-specific IFN-γ release in whole blood samples occurred earlier in animals infected with M. bovis 04-303. Both M. bovis strains resulted in a positive skin test, with animals infected with the wild boar isolate showing a stronger response. These results and the presence of more severe organ lesions, with granuloma and pneumonic areas in cattle demonstrate that the wild boar isolate is more virulent than the NCTC10772 strain. Additionally, we tested the infectivity of the M. bovis strains in guinea pigs and found that M. bovis 04-303 had the highest pathogenicity. Conclusions M. bovis strains isolated from wild boars may be pathogenic for cattle, producing TB lesions. PMID:21745408

  5. The effect of polycyclic aromatic hydrocarbons on the degradation of benzo[a]pyrene by Mycobacterium sp. strain RJGII-135.

    PubMed

    McLellan, Sandra L; Warshawsky, David; Shann, Jodi R

    2002-02-01

    Mycobacterium sp. strain RJGII-135 is capable of degrading a wide range of polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP). In this study, critical aspects of degradation were investigated, including compound uptake, relative rates of PAH degradation, and the effects of co-occurring PAH substrates on BaP degradation and mineralization to CO2. Mycobacterium sp. strain RJGII-135 was capable of degrading phenanthrene, anthracene, and pyrene at a 10- to 20-fold greater rate than benz[a]anthracene (BaA) and BaP. A significant amount of phenanthrene and pyrene, 30% and 10%, respectively, was completely mineralized, whereas less than 4% of anthracene, BaA, and BaP was mineralized. The PAH uptake assays demonstrated that high amounts of BaP and BaA, 81% and 75% of added compound, respectively, could be recovered from bacterial cell fractions after a 4-h incubation compared with pyrene (61%), anthracene (53%), and phenanthrene (47%). The half-saturation constant (Km) for pyrene was threefold lower for pyrene over BaP, suggesting that the degradation system in Mycobacterium sp. strain RJGII-135 has a higher affinity for pyrene, reaching maximal degradative activity at lower concentrations. No hybridization to dioxygenase gene probes nahAc, bphA1, or tolC1C2 was detected. Studies to investigate competition between different PAH substrates demonstrated that the rate of BaP metabolism was influenced by the presence of a second PAH substrate. The BaP metabolism was inhibited when coincubated with BaA, pyrene, and anthracene. Phenanthrene did not inhibit but enhanced BaP metabolism sixfold. These data suggest that induction effects of components of complex mixtures may be as important as competitive metabolism when assessing the ability of bacteria to effectively degrade high-molecular-weight PAHs in the environment.

  6. Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis.

    PubMed

    Cantrell, Sally A; Leavell, Michael D; Marjanovic, Olivera; Iavarone, Anthony T; Leary, Julie A; Riley, Lee W

    2013-10-01

    The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen's persistence.

  7. Secretome profiling of highly virulent Mycobacterium bovis 04-303 strain reveals higher abundance of virulence-associated proteins.

    PubMed

    Vargas-Romero, Fernando; Mendoza-Hernández, Guillermo; Suárez-Güemes, Francisco; Hernández-Pando, Rogelio; Castañón-Arreola, Mauricio

    2016-11-01

    Mycobacterium bovis is the causative agent of tuberculosis in farms, wildlife and causes sporadic disease in humans. Despite the high similitude in genome sequence between M. bovis strains, some strains like the wild boar 04-303 isolate show a highly virulent phenotype in animal models. Comparative studies will contribute to link protein expression with the virulence phenotype. In vitro, the 04-303 strain was more phagocytized by J774A.1 macrophages in comparison with 444 strain (a cow isolate with the same genotype) and BCG. The secretome of these strains showed a significant proportion of shared proteins (368 spots). Among the proteins only visualized in the secretome of the 04-303 strain, we identify the nine most abundant proteins by LC-MS/MS. The most relevant were EsxA and EsxB proteins, which are encoded in the RD1 region, deleted in BCG strains. These proteins are the major virulence factor of M. tuberculosis. The other proteins identified belong to functional categories of virulence, detoxification, and adaptation; lipid metabolism; and cell wall and cell processes. The relatively high proportion of proteins involved in the cell wall and cell process is consistent with the previously described variation among M. bovis genomes.

  8. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2009-06-01

    A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 +/- 36.8 min at 37 degrees C compared to 63 +/- 17.5 min for M. smegmatis mc(2) 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37 degrees C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc(2) 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

  9. Mutations in the embC-embA Intergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

    PubMed Central

    Cui, Zhenling; Li, Yuanyuan; Cheng, Song; Yang, Hua; Lu, Junmei; Hu, Zhongyi

    2014-01-01

    The rapid increase in Mycobacterium tuberculosis resistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in the embC-embA intergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767 M. tuberculosis clinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and the embC-embA IGRs of these strains were sequenced. The transcriptional activity of the embC-embA IGR mutations was examined by reporter gene assays in recombinant Mycobacterium smegmatis strains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator of embAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that the embC-embA IGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level of embA and embB mRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed into M. smegmatis strains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of the embC-embA IGR enhance the binding of EmbR to the promoter region of embAB and increase the expression of embAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST. PMID:25182646

  10. Mutations in the embC-embA intergenic region contribute to Mycobacterium tuberculosis resistance to ethambutol.

    PubMed

    Cui, Zhenling; Li, Yuanyuan; Cheng, Song; Yang, Hua; Lu, Junmei; Hu, Zhongyi; Ge, Baoxue

    2014-11-01

    The rapid increase in Mycobacterium tuberculosis resistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in the embC-embA intergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767 M. tuberculosis clinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and the embC-embA IGRs of these strains were sequenced. The transcriptional activity of the embC-embA IGR mutations was examined by reporter gene assays in recombinant Mycobacterium smegmatis strains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator of embAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that the embC-embA IGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level of embA and embB mRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed into M. smegmatis strains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of the embC-embA IGR enhance the binding of EmbR to the promoter region of embAB and increase the expression of embAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

  11. Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay.

    PubMed

    Nagai, Yuhki; Iwade, Yoshito; Nakano, Manabu; Akachi, Shigehiro; Kobayashi, Takashi; Nishinaka, Takamichi

    2016-07-01

    Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.

  12. Prevalence of Mycobacterium tuberculosis strain genotypes in Taiwan reveals a close link to ethnic and population migration.

    PubMed

    Dou, Horng-Yunn; Chen, Yih-Yuan; Kou, Shu-Chen; Su, Ih-Jen

    2015-06-01

    Taiwan is a relatively isolated island, serving as a mixing vessel for colonization by different waves of ethnic and migratory groups over the past 4 centuries. The potential transmission pattern of Mycobacterium tuberculosis in different ethnic and migratory populations remains to be elucidated. By using mycobacterial tandem repeat sequences as genetic markers, the prevalence of M. tuberculosis strains in Taiwan revealed a close link to the historical migration. Interestingly, the M. tuberculosis strain in the aborigines of Eastern and Central Taiwan had a dominance of the Haarlem (Dutch) strain while those in Southern Taiwan had a dominance of the East-African Indian (EAI) strain. The prevalence of different M. tuberculosis strains in specific ethnic populations suggests that M. tuberculosis transmission is limited and restricted to close contact. The prevalence of the Beijing modern strain in the young population causes a concern for M. tuberculosis control, because of high virulence and drug resistance. Furthermore, our data using molecular genotyping should provide valuable information on the historical study of the origin and migration of aborigines in Taiwan.

  13. Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion.

    PubMed

    Jiang, Yi; Wei, Jianhao; Liu, Haican; Li, Guilian; Guo, Qian; Qiu, Yan; Zhao, Lili; Li, Machao; Zhao, Xiuqin; Dou, Xiangfeng; Wan, Kanglin

    2016-01-01

    Previous studies have demonstrated that the Pro‑Glu/Pro‑Pro‑Glu (PE/PPE) genes in strains of Mycobacterium tuberculosis exhibit high sequence variation and may be involved in antigenic variation and immune evasion. Region of Difference 1 (RD1), encoding genes from Rv3871 to Rv3879, was observed to be lost during the original derivation of Bacillus Calmette‑Guérin between 1908 and 1921. It has been previously demonstrated that two PE/PPE proteins, PE35 (Rv3872) and PPE68 (Rv3873), are encoded by RD1 and exhibit immunodominance. To explore the genetic diversity of PE35 and PPE68, and to evaluate the impact of sequence variation on the immune recognition of these proteins, 161 clinical M. tuberculosis strains were selected from China and comparative sequence analysis of PE35 and PPE68 was performed. The results indicated that polymorphisms in PE35 and PPE68 may lead to alterations in the function of these proteins, which may potentially affect strain virulence. In addition, the human T‑cell epitopes of PE35 and PPE68 were highly variable, suggesting that the two antigens may be involved in diversifying selection to evade host immunity. The prevalence of strains with PE35 mutations in the non‑Beijing family was significantly greater compared with the Beijing family, indicating that Beijing strains may be more conservative than non‑Beijing strains in this gene.

  14. Cloning and sequence analysis of a class A beta-lactamase from Mycobacterium tuberculosis H37Ra.

    PubMed Central

    Hackbarth, C J; Unsal, I; Chambers, H F

    1997-01-01

    A cosmid library from Mycobacterium tuberculosis H37Ra was introduced into Mycobacterium smegmatis, and eight recombinant clones with increased resistance to cefoxitin were identified. Isoelectric focusing detected an M. tuberculosis-derived beta-lactamase in one of these recombinant clones. A sequence analysis identified it as a class A beta-lactamase whose expression correlated with the increased resistance phenotype. PMID:9145897

  15. Importance of confirming data on the in vivo efficacy of novel antibacterial drug regimens against various strains of Mycobacterium tuberculosis.

    PubMed

    De Groote, Mary A; Gruppo, Veronica; Woolhiser, Lisa K; Orme, Ian M; Gilliland, Janet C; Lenaerts, Anne J

    2012-02-01

    In preclinical testing of antituberculosis drugs, laboratory-adapted strains of Mycobacterium tuberculosis are usually used both for in vitro and in vivo studies. However, it is unknown whether the heterogeneity of M. tuberculosis stocks used by various laboratories can result in different outcomes in tests of antituberculosis drug regimens in animal infection models. In head-to-head studies, we investigated whether bactericidal efficacy results in BALB/c mice infected by inhalation with the laboratory-adapted strains H37Rv and Erdman differ from each other and from those obtained with clinical tuberculosis strains. Treatment of mice consisted of dual and triple drug combinations of isoniazid (H), rifampin (R), and pyrazinamide (Z). The results showed that not all strains gave the same in vivo efficacy results for the drug combinations tested. Moreover, the ranking of HRZ and RZ efficacy results was not the same for the two H37Rv strains evaluated. The magnitude of this strain difference also varied between experiments, emphasizing the risk of drawing firm conclusions for human trials based on single animal studies. The results also confirmed that the antagonism seen within the standard HRZ regimen by some investigators appears to be an M. tuberculosis strain-specific phenomenon. In conclusion, the specific identity of M. tuberculosis strain used was found to be an important variable that can change the apparent outcome of in vivo efficacy studies in mice. We highly recommend confirmation of efficacy results in late preclinical testing against a different M. tuberculosis strain than the one used in the initial mouse efficacy study, thereby increasing confidence to advance potent drug regimens to clinical trials.

  16. Experimental infection of lambs with C and S-type strains of Mycobacterium avium subspecies paratuberculosis: immunological and pathological findings.

    PubMed

    Fernández, Miguel; Benavides, Julio; Sevilla, Iker A; Fuertes, Miguel; Castaño, Pablo; Delgado, Laetitia; García Marín, J Francisco; Garrido, Joseba M; Ferreras, M Carmen; Pérez, Valentín

    2014-01-16

    The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C- (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C-type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C-type strains in lambs show a regressive character and tend to decrease as the infection progresses.

  17. Experimental infection of lambs with C and S-type strains of Mycobacterium avium subspecies paratuberculosis: immunological and pathological findings

    PubMed Central

    2014-01-01

    The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C– (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C–type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C–type strains in lambs show a regressive character and tend to decrease as the infection progresses. PMID:24428881

  18. Reduced virulence of an extensively drug-resistant outbreak strain of Mycobacterium tuberculosis in a murine model.

    PubMed

    Smith, Kristen L Jurcic; Saini, Divey; Bardarov, Svetoslav; Larsen, Michelle; Frothingham, Richard; Gandhi, Neel R; Jacobs, William R; Sturm, A Willem; Lee, Sunhee

    2014-01-01

    Bacterial drug resistance is often associated with a fitness cost. Large outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB have been described that predominately affect persons with HIV infection. We obtained four closely-related Mycobacterium tuberculosis strains (genotype F15/LAM4/KZN) from an outbreak in KwaZulu-Natal (KZN), South Africa, including drug-sensitive, MDR, and XDR clinical isolates. We compared the virulence of these strains in a murine model of aerosol M. tuberculosis infection for four phenotypes: (1) competitive in vivo growth in lung and spleen, (2) non-competitive in vivo growth in lung and spleen, (3) murine survival time, and (4) lung pathology. When mixtures of sensitive, MDR, and XDR KZN strains were aerosolized (competitive model), lung CFUs were similar at 60 days after infection, and spleen CFUs were ordered as follows: sensitive > MDR > XDR. When individual strains were aerosolized (non-competitive model), modest differences in lung and spleen CFUs were observed with the same ordering. C57BL/6, C3H/FeJ, and SCID mice all survived longer after infection with MDR as compared to sensitive strains. SCID mice infected with an XDR strain survived longer than those infected with MDR or sensitive strains. Lung pathology was reduced after XDR TB infection compared to sensitive or MDR TB infection. In summary, increasing degrees of drug resistance were associated with decreasing murine virulence in this collection of KZN strains as measured by all four virulence phenotypes. The predominance of HIV-infected patients in MDR and XDR TB outbreaks may be explained by decreased virulence of these strains in humans.

  19. 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

  20. Mycobacterium tuberculosis outbreak strain of Danish origin spreading at worrying rates among greenland-born persons in Denmark and Greenland.

    PubMed

    Lillebaek, T; Andersen, A B; Rasmussen, E M; Kamper-Jørgensen, Z; Pedersen, M K; Bjorn-Mortensen, K; Ladefoged, K; Thomsen, V O

    2013-12-01

    Transmission of Mycobacterium tuberculosis continues at high rates among Greenland-born persons in Greenland and Denmark, with 203 and 450 notified cases per 10(5) population, respectively, in the year 2010. Here, we document that the predominant M. tuberculosis outbreak strain C2/1112-15 of Danish origin has been transmitted to Greenland-born persons in Denmark and subsequently to Greenland, where it is spreading at worrying rates and adding to the already heavy tuberculosis burden in this population group. It is now clear that the C2/1112-15 strain is able to gain new territories using a new population group as the "vehicle." Thus, it might have the ability to spread even further, considering the potential clinical consequences of strain diversity such as that seen in the widely spread Beijing genotype. The introduction of the predominant M. tuberculosis outbreak strain C2/1112-15 into the Arctic circumpolar region is a worrying tendency which deserves attention. We need to monitor whether this strain already has, or will, spread to other countries.

  1. Comparative genome analyses of Mycobacterium avium reveal genomic features of its subspecies and strains that cause progression of pulmonary disease

    PubMed Central

    Uchiya, Kei-ichi; Tomida, Shuta; Nakagawa, Taku; Asahi, Shoki; Nikai, Toshiaki; Ogawa, Kenji

    2017-01-01

    Pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing worldwide. Mycobacterium avium is the most clinically significant NTM species in humans and animals, and comprises four subspecies: M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), M. avium subsp. paratuberculosis (MAP), and M. avium subsp. hominissuis (MAH). To improve our understanding of the genetic landscape and diversity of M. avium and its role in disease, we performed a comparative genome analysis of 79 M. avium strains. Our analysis demonstrated that MAH is an open pan-genome species. Phylogenetic analysis based on single nucleotide variants showed that MAH had the highest degree of sequence variability among the subspecies, and MAH strains isolated in Japan and those isolated abroad possessed distinct phylogenetic features. Furthermore, MAP strains, MAS and MAA strains isolated from birds, and many MAH strains that cause the progression of pulmonary disease were grouped in each specific cluster. Comparative genome analysis revealed the presence of genetic elements specific to each lineage, which are thought to be acquired via horizontal gene transfer during the evolutionary process, and identified potential genetic determinants accounting for the pathogenic and host range characteristics of M. avium. PMID:28045086

  2. Whole-Genome Sequencing of an Isoniazid-Resistant Clinical Isolate of Mycobacterium tuberculosis Strain MtURU-002 from Uruguay

    PubMed Central

    Berná, Luisa; Iraola, Gregorio; Greif, Gonzalo; Coitinho, Cecilia; Rivas, Carlos M.; Naya, Hugo

    2014-01-01

    The incidence of tuberculosis in Uruguay has been effectively reduced to <30 per 100,000 population, although an increase in nonrisk populations in the last few years is evident. Here, we present the genome sequence of Mycobacterium tuberculosis strain MtURU-002 isolated from a patient showing bilateral pulmonary tuberculosis that was resistant to isoniazid. PMID:25035326

  3. Whole-Genome Sequences of Four Strains Closely Related to Members of the Mycobacterium chelonae Group, Isolated from Biofilms in a Drinking Water Distribution System Simulator

    PubMed Central

    Revetta, Randy P.

    2016-01-01

    We report here the draft genome sequences of four Mycobacterium chelonae strains from biofilms subjected to a “chlorine burn” in a chloraminated drinking water distribution system simulator. These opportunistic pathogens have been detected in hospital and municipal water distribution systems, in which biofilms have been recognized as an important factor for their persistence. PMID:26798093

  4. Whole-Genome Sequences of Four Strains Closely Related to Members of the Mycobacterium chelonae Group, Isolated from Biofilms in a Drinking Water Distribution System Simulator

    EPA Science Inventory

    We report the draft genome sequences of four Mycobacterium chelonae group strains from biofilms obtained after a ‘chlorine burn’ in a chloraminated drinking water distribution system simulator. These opportunistic pathogens have been detected in drinking and hospital water distr...

  5. Whole-Genome Sequencing of an Isoniazid-Resistant Clinical Isolate of Mycobacterium tuberculosis Strain MtURU-002 from Uruguay.

    PubMed

    Berná, Luisa; Iraola, Gregorio; Greif, Gonzalo; Coitinho, Cecilia; Rivas, Carlos M; Naya, Hugo; Robello, Carlos

    2014-07-17

    The incidence of tuberculosis in Uruguay has been effectively reduced to <30 per 100,000 population, although an increase in nonrisk populations in the last few years is evident. Here, we present the genome sequence of Mycobacterium tuberculosis strain MtURU-002 isolated from a patient showing bilateral pulmonary tuberculosis that was resistant to isoniazid.

  6. [Epidemiology of resistance to antituberculosis drugs in Mycobacterium tuberculosis complex strains isolated from adenopathies in Djibouti. Prospective study carried out in 1999].

    PubMed

    Koeck, J L; Bernatas, J J; Gerome, P; Fabre, M; Houmed, A; Herve, V; Teyssou, R

    2002-01-01

    Tuberculosis is a major cause of death in the Republic of Djibouti. Tuberculous lymphadenitis represents about 25% of the clinical forms of tuberculosis in this country. Between January 1999 and April 1999, 196 lymph node specimens were consecutively collected from 153 patients living in Djibouti. Testing of susceptibility to the major anti-tuberculosis drugs was performed by the proportion method. Growth of Mycobacterium tuberculosis complex strains was obtained from specimens of 85 patients including 9 with prior treatment. Strains were identified as Mycobacterium tuberculosis in 78 cases, Mycobacterium canetti in 3, Mycobacterium africanum in 3, and Mycobacterium bovis in 1. Prevalence of HIV infection was 15%. Assessment of primary resistance demonstrated that the overall resistance rate, i.e., resistance to 1 or more drugs, was 18 (21.2%). Results showed resistance to isoniazid (H) in 6 cases (7.1%), rifampicin (R) in 3 (3.5%), ethambutol (E) in 1 (1.2%), streptomycin (S) in 13 (15.3%) and pyrazinamide (Z) in 1 (1.2%). Multidrug resistance (MDR) was found in 2 cases (2.4%). Assessment of acquired resistance demonstrated resistance to H in 4 cases (44%), R in 2 (22%), S in 2 (22%), E in 0, Z in 0 and MDR in 1 (11%). These findings were not significantly different from data obtained from sputum samples analysed between 1997 and 2000 or from those described in a study conducted in 1985.

  7. Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

    PubMed Central

    Schneider, J; Grosser, R; Jayasimhulu, K; Xue, W; Warshawsky, D

    1996-01-01

    The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH. PMID:8572690

  8. Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey

    PubMed Central

    Zhou, Yang; van den Hof, Susan; Wang, Shengfen; Pang, Yu; Zhao, Bing; Xia, Hui; Anthony, Richard; Ou, Xichao; Li, Qiang; Zheng, Yang; Song, Yuanyuan; Zhao, Yanlin; van Soolingen, Dick

    2017-01-01

    We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading. PMID:28333978

  9. Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain.

    PubMed

    Lee, Jong Seok; Krause, Roland; Schreiber, Jörg; Mollenkopf, Hans-Joachim; Kowall, Jane; Stein, Robert; Jeon, Bo-Young; Kwak, Jeong-Yeon; Song, Min-Kyong; Patron, Juan Pablo; Jorg, Sabine; Roh, Kyoungmin; Cho, Sang-Nae; Kaufmann, Stefan H E

    2008-02-14

    Attenuated strains of mycobacteria can be exploited to determine genes essential for their pathogenesis and persistence. To this goal, we sequenced the genome of H37Ra, an attenuated variant of Mycobacterium tuberculosis H37Rv strain. Comparison with H37Rv revealed three unique coding region polymorphisms. One polymorphism was located in the DNA-binding domain of the transcriptional regulator PhoP, causing the protein's diminished DNA-binding capacity. Temporal gene expression profiles showed that several genes with reduced expression in H37Ra were also repressed in an H37Rv phoP knockout strain. At later time points, genes of the dormancy regulon, typically expressed in a state of nonreplicating persistence, were upregulated in H37Ra. Complementation of H37Ra with H37Rv phoP partially restored its persistence in a murine macrophage infection model. Our approach demonstrates the feasibility of identifying minute but distinct differences between isogenic strains and illustrates the consequences of single point mutations on the survival stratagem of M. tuberculosis.

  10. Mapping IS6110 in high-copy number Mycobacterium tuberculosis strains shows specific insertion points in the Beijing genotype

    PubMed Central

    2013-01-01

    Background Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. Results In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. Conclusions This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains. PMID:23800083

  11. The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages

    PubMed Central

    Saikolappan, Sankaralingam; Estrella, Jaymie; Sasindran, Smitha J.; Khan, Arshad; Armitige, Lisa Y.; Jagannath, Chinnaswamy; Dhandayuthapani, Subramanian

    2012-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine. PMID:22574140

  12. The fbpA/sapM double knock out strain of Mycobacterium tuberculosis is highly attenuated and immunogenic in macrophages.

    PubMed

    Saikolappan, Sankaralingam; Estrella, Jaymie; Sasindran, Smitha J; Khan, Arshad; Armitige, Lisa Y; Jagannath, Chinnaswamy; Dhandayuthapani, Subramanian

    2012-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.

  13. Molecular Basis of Intrinsic Macrolide Resistance in the Mycobacterium tuberculosis Complex

    PubMed Central

    Buriánková, Karolína; Doucet-Populaire, Florence; Dorson, Olivier; Gondran, Anne; Ghnassia, Jean-Claude; Weiser, Jaroslav; Pernodet, Jean-Luc

    2004-01-01

    The intrinsic resistance of the Mycobacterium tuberculosis complex (MTC) to most antibiotics, including macrolides, is generally attributed to the low permeability of the mycobacterial cell wall. However, nontuberculous mycobacteria (NTM) are much more sensitive to macrolides than members of the MTC. A search for macrolide resistance determinants within the genome of M. tuberculosis revealed the presence of a sequence encoding a putative rRNA methyltransferase. The deduced protein is similar to Erm methyltransferases, which confer macrolide-lincosamide-streptogramin (MLS) resistance by methylation of 23S rRNA, and was named ErmMT. The corresponding gene, ermMT (erm37), is present in all members of the MTC but is absent in NTM species. Part of ermMT is deleted in some vaccine strains of Mycobacterium bovis BCG, such as the Pasteur strain, which lack the RD2 region. The Pasteur strain was susceptible to MLS antibiotics, whereas MTC species harboring the RD2 region were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium smegmatis and BCG Pasteur conferred MLS resistance. The resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT, srmA (monomethyltransferase from Streptomyces ambofaciens), and ermE (dimethyltransferase from Saccharopolyspora erythraea) were compared, and the ones conferred by ErmMT were similar to those conferred by SrmA, corresponding to the MLS type I phenotype. These results suggest that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC and could be the first example of a gene conferring resistance by target modification in mycobacteria. PMID:14693532

  14. Molecular basis of intrinsic macrolide resistance in the Mycobacterium tuberculosis complex.

    PubMed

    Buriánková, Karolína; Doucet-Populaire, Florence; Dorson, Olivier; Gondran, Anne; Ghnassia, Jean-Claude; Weiser, Jaroslav; Pernodet, Jean-Luc

    2004-01-01

    The intrinsic resistance of the Mycobacterium tuberculosis complex (MTC) to most antibiotics, including macrolides, is generally attributed to the low permeability of the mycobacterial cell wall. However, nontuberculous mycobacteria (NTM) are much more sensitive to macrolides than members of the MTC. A search for macrolide resistance determinants within the genome of M. tuberculosis revealed the presence of a sequence encoding a putative rRNA methyltransferase. The deduced protein is similar to Erm methyltransferases, which confer macrolide-lincosamide-streptogramin (MLS) resistance by methylation of 23S rRNA, and was named ErmMT. The corresponding gene, ermMT (erm37), is present in all members of the MTC but is absent in NTM species. Part of ermMT is deleted in some vaccine strains of Mycobacterium bovis BCG, such as the Pasteur strain, which lack the RD2 region. The Pasteur strain was susceptible to MLS antibiotics, whereas MTC species harboring the RD2 region were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium smegmatis and BCG Pasteur conferred MLS resistance. The resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT, srmA (monomethyltransferase from Streptomyces ambofaciens), and ermE (dimethyltransferase from Saccharopolyspora erythraea) were compared, and the ones conferred by ErmMT were similar to those conferred by SrmA, corresponding to the MLS type I phenotype. These results suggest that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC and could be the first example of a gene conferring resistance by target modification in mycobacteria.

  15. Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice.

    PubMed

    Liu, Laicheng; Fu, Ruiling; Yuan, Xuefeng; Shi, Chunwei; Wang, Shuling; Lu, Xianyu; Ma, Zhao; Zhang, Xiaoming; Qin, Weiyan; Fan, Xionglin

    2015-07-01

    Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.

  16. 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.

  17. 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

  18. 2-(Quinolin-4-yloxy)acetamides Are Active against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains

    PubMed Central

    2016-01-01

    2-(Quinolin-4-yloxy)acetamides have been described as potent in vitro inhibitors of Mycobacterium tuberculosis growth. Herein, additional chemical modifications of lead compounds were carried out, yielding highly potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.05 μM. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to Vero and HaCat cells (IC50s ≥ 20 μM). In addition, the 2-(quinolin-4-yloxy)acetamides showed intracellular activity against the bacilli in infected macrophages with action similar to rifampin, low risk of drug–drug interactions, and no sign of cardiac toxicity in zebrafish (Danio rerio) at 1 and 5 μM. Therefore, these data indicate that this class of compounds may furnish candidates for future development to, hopefully, provide drug alternatives for tuberculosis treatment. PMID:26985307

  19. Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

    PubMed

    Bigi, María Mercedes; Lopez, Beatriz; Blanco, Federico Carlos; Sasiain, María Del Carmen; De la Barrera, Silvia; Marti, Marcelo A; Sosa, Ezequiel Jorge; Fernández Do Porto, Darío Augusto; Ritacco, Viviana; Bigi, Fabiana; Soria, Marcelo Abel

    2017-03-01

    Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8(+) cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.

  20. Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

    PubMed Central

    Maiga, Mamoudou; Abeel, Thomas; Shea, Terrance; Desjardins, Christopher A.; Diarra, Bassirou; Baya, Bocar; Sanogo, Moumine; Diallo, Souleymane; Earl, Ashlee M.; Bishai, William R.

    2016-01-01

    Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity. PMID:26751217

  1. Synchronous Comparison of Mycobacterium tuberculosis Epidemiology Strains by "MIRU-VNTR" and "MIRU-VNTR and Spoligotyping" Technique.

    PubMed

    Jafarian, Mehdi; Aghali-Merza, Muayed; Farnia, Parissa; Ahmadi, Mojtaba; Masjedi, Mohammad Reza; Velayati, Ali Akbar

    2010-07-01

    Molecular epidemiology analyses are frequently used in determining epidemiology of tuberculosis. Recently, Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) and Spoligotyping has become an important method, as it allows high-through put, discriminatory and reproducible analysis of clinical isolate. The purpose of this study is to compare techniques of "MIRU-VNTR" versus "MIRU-VNTR and Spoligotyping" together for study of genetic pattern of Mycobacterium tuberculosis (M. tuberculosis) strains. Sixty M. tuberculosis (MTB) isolates were selected (30 susceptible, 30 multi-drug resistant) for this study. Thereafter, the "MIRU-VNTR and Spoligotyping" were performed to identify their genetic patterns. The frequency of unknown genetic pattern of MTB was compared using technique of "MIRU-VNTR" alone versus "MIRU-VNTR and Spoligotyping" together. The MIRU-VNTR allelic diversity at each of the loci was calculated by Hunter - Gaston Discriminatory Index (HGDI). Based on differentiation index of all strains 10, 16, 26, 31 and 40 loci were identified as the most distinctive (HGI ≥0.6) and 2, 4, 20 and 24 as the weakest distinctive locus (HGI ≤0.3). By using MIRU-VNTR technique 38% (n = 23) of isolates could not be typed, whereas by applying "MIRU-VNTR and Spoligotyping" together only 15% (n = 9) of isolates remained unknown (p = 0.004). For sensitive strains, the difference was significant (67% vs. 90%, p = 0.028), but only marginally significant for drug resistant strains (57% vs. 80%, p = 0.052). The discrimination power of 12-locus MIRU-VNTR and Spoligotyping was equal to that of MIRU-VNTR analysis. If appropriate loci are added to the standard MIRU analysis, MIRU-VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis. With this approach a more clear understanding about genetic pattern of MTB can be achieved.

  2. Beijing strains of Mycobacterium tuberculosis in smear-positive tuberculosis patients in North-West and West of Iran

    PubMed Central

    Sahebi, Leyla; Ansarin, Khalil; Hoffner, Sven; Mohajeri, Parviz; Mohammadi, Abolghasem

    2016-01-01

    Background: Tuberculosis (TB) is the leading cause of morbidity and mortality among chronic infectious diseases. The goal of this cross-sectional study (2012–2014) was to examine the prevalence of Mycobacterium TB (MTB) Beijing strains in regions near the Iranian border and to identify any epidemiological links. Materials and Methods: To this end, MTB isolates were harvested, from 64 HIV-negative, pulmonary smear-positive TB patients from the Iranian border provinces of East Azerbaijan (North-West), Kurdistan (West), and Kermanshah (West) (2012–2014). Isolates were subjected to restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 as a probe (IS6110 RFLP), and drug susceptibility testing by the proportion method. We gathered demographic and clinical data using a questionnaire and reviewing patient records. Results were analyzed with Gel Compare II 6.6 and SPSS-18. Results: The mean age of the patients was 54.4 years and 46.9% were male. The prevalence of Beijing strains among the isolates was 9.4% (17.6% in the Western provinces and 0% in East Azerbaijan). There was a statistically significant relationship between the Beijing strains and drug resistance and also between these strains, and the recurrence of TB in patients that had previously received treatment (P = 0.02 and P = 0.04, respectively). Conclusions: Finally, the prevalence of Beijing strains in Western Iran was greater than expected. Our results therefore indicate that regional and cross-border tracing may be necessary to control spread of this organism. PMID:28028521

  3. Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

    PubMed Central

    2016-01-01

    Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications. PMID:27709842

  4. The viable Mycobacterium tuberculosis H37Ra strain induces a stronger mouse macrophage response compared to the heat-inactivated H37Rv strain.

    PubMed

    He, Zong-Lin; Du, Fa-Wang; Du, Xian-Zhi

    2013-05-01

    Macrophages are the target cells for Mycobacterium tuberculosis (M. tuberculosis) as well as key effector cells for clearance of this pathogen. The aim of the present study was to measure and compare the responses of mouse peritoneal macrophages following exposure to the live M. tuberculosis H37Ra and heat-inactivated H37Rv strains. In vitro phagocytosis assays indicated that the macrophages had a higher capacity to engulf the live H37Ra strain compared to the inactivated H37Rv strain. Enzyme-linked immunosorbent assay (ELISA) demonstrated that H37Ra‑stimulated macrophages produced significantly increased concentrations of interleukin‑12p40 (IL‑12p40), tumor necrosis factor-α (TNF‑α) and interferon‑γ (IFN‑γ) compared to the untreated control cells. However, H37Rv exposure induced little to no increase in the levels of the cytokines examined. The results from ELISA were confirmed by reverse transcription-polymerase chain reaction (RT‑PCR) at the mRNA level. There was a dose-dependent increase in nitric oxide (NO) and hydrogen peroxide (H2O2) production from the H37Ra‑stimulated macrophages compared to the H37Rv‑stimulated ones. Confocal microscopy and flow cytometric analysis indicated that the IFN‑γ‑stimulated macrophages from viable H37Ra‑immunized mice had an enhanced surface expression of CD40 ligand (CD40L) compared to those from inactivated H37Rv‑immunized mice. Our data collectively indicate that exposure to the viable H37Ra strain induces a stronger macrophage response compared to exposure to the heat-inactivated H37Rv strain, which may be associated with the increased surface expression of CD40L in activated macrophages.

  5. Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance.

    PubMed

    Zimic, Mirko; Fuentes, Patricia; Gilman, Robert H; Gutiérrez, Andrés H; Kirwan, Daniela; Sheen, Patricia

    2012-01-01

    Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M. smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux

  6. Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

    PubMed

    Verma, Renu; Pinto, Sneha Maria; Patil, Arun Hanumana; Advani, Jayshree; Subba, Pratigya; Kumar, Manish; Sharma, Jyoti; Dey, Gourav; Ravikumar, Raju; Buggi, Shashidhar; Satishchandra, Parthasarathy; Sharma, Kusum; Suar, Mrutyunjay; Tripathy, Srikanth Prasad; Chauhan, Devendra Singh; Gowda, Harsha; Pandey, Akhilesh; Gandotra, Sheetal; Prasad, Thottethodi Subrahmanya Keshava

    2017-03-20

    Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

  7. Detection of RD(Rio) strain of Mycobacterium tuberculosis in tapirs (Tapirus terrestris) from a zoo in Brazil.

    PubMed

    Murakami, Patricia Sayuri; Monego, Fernanda; Ho, John L; Gibson, Andrea; Javorouski, Manoel Lucas; Bonat, Marcelo; Lacerda, Oneida; Brockelt, Sonia Regina; Biesdorf, Sonia Maria; Nakatani, Sueli Massumi; Riediger, Irina Nastassja; Fuverki, Renata Benício Neves; Biava, Janaína Socolovski; Vieira, Rafael Felipe Costa; do Santos, Andrea Pires; de Barros Filho, Ivan Roque; Biondo, Alexander Welker

    2012-12-01

    Tuberculosis is a chronic infection caused by strains of the Mycobacterium tuberculosis complex and occurs in both animal and human populations. The death of a tapir showing purulent material and a hard mass in the lungs at necropsy raised suspicion of a potential disease caused by mycobacteria species in a Brazilian zoo. Later, two other tapirs with similar signs died and were further investigated. Polymerase chain reaction (PCR) from bronco-alveolar lavages was performed, and both animals tested positive for the RD(Rio) strain of M. tuberculosis, which is a recently discovered Latin American-Mediterranean sublineage and the main cause of human tuberculosis in Rio de Janeiro, Brazil. To investigate the possibility of human infection and the source of transmission, all 50 zoo employees underwent tuberculin skin testing; four were reactive, but radiographic exams and direct sample staining did not suggest tuberculosis. Thus, direct human to animal transmission was not proven. However, the presence of RD(Rio) M. tuberculosis in tapirs highlights the lack of attention to diseases that human beings may transmit to wildlife.

  8. Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

    PubMed

    Manson, Abigail L; Cohen, Keira A; Abeel, Thomas; Desjardins, Christopher A; Armstrong, Derek T; Barry, Clifton E; Brand, Jeannette; Chapman, Sinéad B; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A A; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E; Cassell, Gail H; Dorman, Susan E; Ellner, Jerrold; Farnia, Parissa; Galagan, James E; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R; Cohen, Ted; Hoffner, Sven; Birren, Bruce W; Earl, Ashlee M

    2017-03-01

    Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

  9. First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains.

    PubMed

    de Beer, Jessica L; Kremer, Kristin; Ködmön, Csaba; Supply, Philip; van Soolingen, Dick

    2012-03-01

    Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.

  10. Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

    PubMed Central

    Davidson, Rebecca M.; Hasan, Nabeeh A.; Reynolds, Paul R.; Totten, Sarah; Garcia, Benjamin; Levin, Adrah; Ramamoorthy, Preveen; Heifets, Leonid; Daley, Charles L.

    2014-01-01

    Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977T. Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen. PMID:25056330

  11. Unexpectedly High Proportion of Ancestral Manu Genotype Mycobacterium tuberculosis Strains Cultured from Tuberculosis Patients in Egypt ▿

    PubMed Central

    Helal, Zeinab H.; El-Din Ashour, Mohamed Seif; Eissa, Somaia A.; Abd-Elatef, Ghanem; Zozio, Thierry; Babapoor, Sankhiros; Rastogi, Nalin; Khan, Mazhar I.

    2009-01-01

    Tuberculosis is one of the important public health problems in Egypt. However, limited information on the Mycobacterium tuberculosis genotypes circulating in Egypt is available. A total of 151 M. tuberculosis strains were characterized by spoligotyping. The results revealed that 74.8% of M. tuberculosis isolates grouped into 13 different clusters, while 25.2% had unique spoligotype patterns. Comparison with an international spoligotyping database (the SITVIT2 database) showed that types SIT53 (T1 variant) and SIT54 (Manu2 variant) were the most common types between cluster groups. In addition, new shared types SIT2977, SIT2978, and SIT2979 were observed. The results identified for the first time an unusually high proportion of ancestral Manu strains of M. tuberculosis from patients in Egypt. The percentage of the Manu clade in this study (27.15%) was significantly higher than its overall representation of 0.4% in the SITVIT2 database. We show that in Egypt tuberculosis is caused by a predominant M. tuberculosis genotype belonging to the ancestral Manu lineage which could be a missing link in the split between ancestral and modern tubercle bacilli during the evolution of M. tuberculosis. PMID:19553569

  12. Effect of Lagerstroemia tomentosa and Diospyros virginiana methanolic extracts on different drug-resistant strains of Mycobacterium tuberculosis

    PubMed Central

    Esfahani, B. Nasr; Hozoorbakhsh, F.; Rashed, Kh.; Havaei, S.A.; Heidari, K.; Moghim, Sh.

    2014-01-01

    Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis. The increasing incidence of multi drug resistance tuberculosis (MDR-TB) and extensively drug resistance tuberculosis (XDR-TB) worldwide highlighted the urgent need to search for alternative antimycobacterial agents. More and more people in developing countries utilize traditional medicine for their major primary health care needs. It has been determined that pharmaceutical plant, Lagerstroemia tomentosa and Diospyros virginiana, possesses some antibacterial effect. In this study, the antimycobacterial effects of L. tomentosa and D. virginiana methanolic extracts on sensitive and resistant isolates of MTB were examined. Leaf methanolic extract was prepared using methanol 70%. Sensitivity and resistance of isolates was determined by proportion method. The effects of two different methonolic extract concentrations (20 and 40 μg/ml) of the plants were examined against 6 sensitive and resistant strains of MTB with different patterns of drug resistance. MTB H37Rv (ATCC 27294) was set as control in all culturing and sensitivity testing processes. The results showed that L. tomentosa and D. virginiana methanolic extracts had weak inhibitory effect on different strains of MTB. The highest percentage of inhibition for L. tomentosa and D. virginiana was observed 38% and 33.3%, respectively. PMID:25657789

  13. Novel phage amplified multichannel series piezoelectric quartz crystal sensor for rapid and sensitive detection of Mycobacterium tuberculosis.

    PubMed

    Mi, Xianwen; He, Fengjiao; Xiang, Meiyu; Lian, Yan; Yi, Songlin

    2012-01-17

    The key factors that control the spread and mortality rate of tuberculosis (TB) are rapid detection and diagnosis. However, the current detection of Mycobacterium tuberculosis (M. tuberculosis) cannot meet the recommended requirements for clinical diagnosis in turnaround time. In this paper, the feature of phage D29 that infects M. tuberculosis and Mycobacterium smegmatis (M. smegmatis) was combined with the sensitivity of multichannel series piezoelectric quartz crystal sensor (MSPQC) to detect M. tuberculosis. The phage D29 played a role of inhibiting the growth of M. tuberculosis and M. smegmatis. M. tuberculosis is used to protect phage D29 from being killed by ferrous ammonium sulfate (FAS) and carries phage D29 into the detection medium containing M. smegmatis. The action of M. smegmatis indicated the existence state of phage D29 in the detection medium. The growth curve of M. smegmatis obtained by MSPQC indicated the state of the growth of M. tuberculosis. Therefore, M. tuberculosis in the sample could be rapidly detected by evaluating the extent of inhibiting the growth of M. smegmatis compared with the normal growth of M. smegmatis. The detection of M. tuberculosis was transformed into the detection of M. smegmatis, which is more rapid and sensitive than that of M. tuberculosis. For 10(2) cfu/mL of M. tuberculosis in clinical sample, the turnaround time was less than 30 h. Although statistical analysis showed that no significant difference existed between the results of the proposed method here and the BACTEC960 MGIT method in clinical M. tuberculosis detection, the phage amplified MSPQC (PA MSPQC) method presented here was faster and more economical.

  14. 2-[4-(4-Methoxyphenylcarbonyloxy)benzylidene]-6-dimethylaminomethyl cyclohexanone hydrochloride: a Mannich base which inhibits the growth of some drug-resistant strains of Mycobacterium tuberculosis.

    PubMed

    Das, S; Das, U; Bandy, B; Gorecki, D K J; Dimmock, J R

    2010-11-01

    2-[4-(4-Methoxyphenylcarbonyloxy)benzylidene]-6-dime-thylaminomethyl cyclohexanone hydrochloride 1 has a MIC value of 0.78 microg/mL towards Mycobacterium tuberculosis H37Rv and displays similar or identical MIC figures towards various drug-resistant strains of this microorganism. The enone 1 along with a partial structure 2-dimethylaminomethylcyclohexanone hydrochloride 3 affected respiration in isolated rat liver mitochondria differently which may contribute to the variation in toxicity to both normal cells and M. tuberculosis.

  15. Lymphadenitis caused by infection with an isoniazid- and rifampin-resistant strain of Mycobacterium bovis BCG in an infant with IFN-γ/IL-12 pathway defect*

    PubMed Central

    Diniz, Lilian Martins Oliveira; Guimarães, Tiago; de Oliveira, Maria das Graças Rodrigues; Pinto, Jorge Andrade; de Miranda, Silvana Spindola

    2014-01-01

    We report a rare case in a female infant (age, 3.5 months) with primary immunodeficiency (IFN-γ/IL-12 pathway defect) who presented with suppurative lymphadenitis after Mycobacterium bovis BCG vaccination. The strain of M. bovis BCG identified was found to be resistant to isoniazid and rifampin. The patient was treated with a special pharmacological regimen involving isoniazid (in a limited, strategic manner), ethambutol, streptomycin, and IFN-γ, after which there was complete resolution of the lesions. PMID:24831405

  16. Draft Genome Sequence of the Mycobacterium tuberculosis Strain 43-16836, Belonging to the Indo-Oceanic Lineage, Isolated From Tuberculous Meningitis in Thailand.

    PubMed

    Viratyosin, W; Kulawonganunchai, S; Smittipat, N; Juthayothin, T; Penpassakarn, P; Pasomsub, E; Chantratita, W; Chaiprasert, A; Palittapongarnpim, P

    2013-10-03

    We present the draft genome sequence of Mycobacterium tuberculosis strain 43-16836, belonging to the Indo-Oceanic lineage, isolated from a tuberculous meningitis patient in Thailand. The genome is 4,381,942 bp long with 4,316 protein-coding genes and contains new single nucleotide polymorphisms (SNPs), including SNPs of genes that may encode cell wall components and possibly influence virulence.

  17. Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White-tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. The cause for many faltering eradication programs is the presence of wildlife reservoirs of M. bovis. One approach in dealing with this wildlife reservoir is to vaccinate ...

  18. Phenotypic and genotypic characterization of drug-resistant Mycobacterium tuberculosis strains.

    PubMed

    Clemente, Wanessa Trindade; Soares Lima, Stella Sala; Palaci, Moises; Silva, Márcia S N; Sumnienski Rodrigues, Vivian F; Dalla Costa, Elis R; Possuelo, Lia; Cafrune, Patrícia Izquierdo; Ribeiro, Fabíola Karla; Gomes, Harisson M; Serufo, José Carlos

    2008-10-01

    Of 142 pulmonary tuberculosis patients, 76 were considered high risk for the development of resistance, and 24 were confirmed as resistant strain carriers. Resistant isoniazid strains presented a high frequency of katG and ahpC mutations (90%) correlated with an MIC >4 microg/mL (94%). inhA mutations were not seen. rpoB mutations were identified in 78.6% of rifampicin-resistant strains, usually in codon 531 (72.7%), and 75% had an MIC >16 microg/mL. katG and rpoB mutations recognized 88.2% of multidrug-resistant strains and proved more efficient than the katG and rpoB mutations alone. Seventy percent of resistant pyrazinamide strains had pncA mutations between genes 136 and 188, 62.5% of them with an MIC >900 microg/mL. Pyrazinamidase inactivity was not an efficient resistance marker because 60% of pncA-mutated strains maintained enzymatic activity despite displaying good correlation with high resistance levels. Resistant ethambutol strains had embB mutations in codon 306, with MIC >16 microg/mL.

  19. Non-exponential growth of Mycobacterium leprae Thai-53 strain cultured in vitro.

    PubMed

    Amako, Kazunobu; Iida, Ken-Ichiro; Saito, Mitsumasa; Ogura, Yoshitoshi; Hayashi, Tetsuya; Yoshida, Shin-Ichi

    2016-12-01

    In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell-associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells.

  20. Non‐exponential growth of Mycobacterium leprae Thai‐53 strain cultured in vitro

    PubMed Central

    Iida, Ken‐ichiro; Saito, Mitsumasa; Ogura, Yoshitoshi; Hayashi, Tetsuya; Yoshida, Shin‐ichi

    2017-01-01

    ABSTRACT In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell‐associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells. PMID:27925336

  1. Relationship between IS901 in the Mycobacterium avium Complex Strains Isolated from Birds, Animals, Humans, and the Environment and Virulence for Poultry

    PubMed Central

    Pavlik, Ivo; Svastova, Petra; Bartl, Jiri; Dvorska, Lenka; Rychlik, Ivan

    2000-01-01

    A total of 738 strains of Mycobacterium avium complex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901 was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901 was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901 positive. PMID:10702495

  2. Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis.

    PubMed

    Deng, Lingyi Lynn; Humphries, Donald E; Arbeit, Robert D; Carlton, Laura E; Smole, Sandra C; Carroll, J David

    2005-02-14

    Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.

  3. Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains.

    PubMed

    Zeng, Jun; Lin, Xiangui; Zhang, Jing; Zhu, Hong; Chen, Hong; Wong, Ming Hung

    2013-04-01

    We previously hypothesized that polycyclic aromatic hydrocarbon (PAH)-degrading bacteria that produce laccase may enhance the degree of benzo[a]pyrene mineralization. However, whether the metabolites of benzo[a]pyrene oxidized by laccase can be further transformed by PAH degraders remains unknown. In this study, pyrene-degrading mycobacteria with diverse degradation properties were isolated and employed for investigating the subsequent transformation on the metabolites of benzo[a]pyrene oxidized by fungal laccase of Trametes versicolor. The results confirm the successive transformation of benzo[a]pyrene metabolites, 6-benzo[a]pyrenyl acetate, and quinones by Mycobacterium strains, and report the discovery of the involvement of a O-methylation mediated pathway in the process. In detail, the vast majority of metabolite 6-benzo[a]pyrenyl acetate was transformed into benzo[a]pyrene quinones or methoxybenzo[a]pyrene, via two distinct steps that were controlled by the catechol-O-methyltransferase mediated O-methylation, while quinones were reduced to dihydroxybenzo[a]pyrene and further transformed into dimethoxy derivatives.

  4. Isolation and characterization of Mycobacterium bovis strains from indigenous Zambian cattle using Spacer oligonucleotide typing technique

    PubMed Central

    2009-01-01

    Background Bovine tuberculosis (BTB), caused by Mycobacterium bovis, has remained a major source of concern to public health officials in Zambia. Previous investigations have used traditional epidemiological methods that are unable to identify the causative agent and from which dynamics of disease dispersion is difficult to discern. The objective of this study was to isolate, characterize and determine the genetic diversity and relatedness of M. bovis from major cattle rearing districts in Zambia by spoligotyping. A total of 695 carcasses were examined and 98 tissues had gross post-mortem lesions compatible with BTB. Results Forty-two out of the ninety-eight suspected tissues examined had culture properties characteristic of mycobacteria from which 31 isolates yielded interpretable spoligotypes. This technique showed good discriminatory power (HGDI = 0.98), revealing 10 different spoligotype patterns. Twenty-seven isolates belonged to one cluster with more than 95% similarity and inside the cluster, one predominant spoligotype was found in 20 (64.5%) of the isolates tested. The highest number of spoligotypes was observed among samples from Namwala district. Spoligotypes from 26 (83.9%) of the isolates belonged to five spoligotypes that have been reported before while the remaining 5 (16.1%) isolates had unique spoligotypes that are being reported for the first time; these have been assigned numbers SB1763 to SB1767. Five of the 6 districts had the predominant spoligotype (SB0120). Conclusion The study has described the dispersion patterns of M. bovis in Zambian cattle for the first time and has identified 5 spoligotype patterns specific to Zambia. The observation of an overlap in the spoligotype pattern SB0120 in 5 of the 6 districts suggests the probability of sharing a common source of infection. PMID:19619309

  5. Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 2012.

    PubMed

    François, Alan; Mathis, Hugues; Godefroy, Davy; Piveteau, Pascal; Fayolle, Françoise; Monot, Frédéric

    2002-06-01

    A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.

  6. Inhibition of the multiplication of Mycobacterium leprae by vaccination with a recombinant M. bovis BCG strain that secretes major membrane protein II in mice.

    PubMed

    Maeda, Y; Tamura, T; Matsuoka, M; Makino, M

    2009-10-01

    The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-gamma) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8(+) T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-gamma on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.

  7. Draft Genome Sequences of Six Mycobacterium immunogenum, Strains Obtained from a Chloraminated Drinking Water Distribution System Simulator

    EPA Science Inventory

    We report the draft genome sequences of six Mycobacterium immunogenum isolated from a chloraminated drinking water distribution system simulator subjected to changes in operational parameters. M. immunogenum, a rapidly growing mycobacteria previously reported as the cause of hyp...

  8. Whole-Genome Sequences of Mycobacterium tuberculosis TB282 and TB284, a Widespread and a Unique Strain, Respectively, Identified in a Previous Study of Tuberculosis Transmission in Central Los Angeles, California, USA

    PubMed Central

    Yang, Zhenhua

    2017-01-01

    ABSTRACT We report here the genome sequences of two Mycobacterium tuberculosis clinical isolates previously identified in central Los Angeles, CA, in the 1990s using a PacBio platform. Isolate TB282 represents a large-cluster strain that caused 27% of the tuberculosis cases, while TB284 represents a strain that caused disease in only one patient. PMID:28082486

  9. Human Genetic Ancestral Composition Correlates with the Origin of Mycobacterium leprae Strains in a Leprosy Endemic Population.

    PubMed

    Cardona-Castro, Nora; Cortés, Edwin; Beltrán, Camilo; Romero, Marcela; Badel-Mogollón, Jaime E; Bedoya, Gabriel

    2015-01-01

    Recent reports have suggested that leprosy originated in Africa, extended to Asia and Europe, and arrived in the Americas during European colonization and the African slave trade. Due to colonization, the contemporary Colombian population is an admixture of Native-American, European and African ancestries. Because microorganisms are known to accompany humans during migrations, patterns of human migration can be traced by examining genomic changes in associated microbes. The current study analyzed 118 leprosy cases and 116 unrelated controls from two Colombian regions endemic for leprosy (Atlantic and Andean) in order to determine possible associations of leprosy with patient ancestral background (determined using 36 ancestry informative markers), Mycobacterium leprae genotype and/or patient geographical origin. We found significant differences between ancestral genetic composition. European components were predominant in Andean populations. In contrast, African components were higher in the Atlantic region. M. leprae genotypes were then analyzed for cluster associations and compared with the ancestral composition of leprosy patients. Two M. leprae principal clusters were found: haplotypes C54 and T45. Haplotype C54 associated with African origin and was more frequent in patients from the Atlantic region with a high African component. In contrast, haplotype T45 associated with European origin and was more frequent in Andean patients with a higher European component. These results suggest that the human and M. leprae genomes have co-existed since the African and European origins of the disease, with leprosy ultimately arriving in Colombia during colonization. Distinct M. leprae strains followed European and African settlement in the country and can be detected in contemporary Colombian populations.

  10. Human Genetic Ancestral Composition Correlates with the Origin of Mycobacterium leprae Strains in a Leprosy Endemic Population

    PubMed Central

    Cardona-Castro, Nora; Cortés, Edwin; Beltrán, Camilo; Romero, Marcela; Badel-Mogollón, Jaime E.; Bedoya, Gabriel

    2015-01-01

    Recent reports have suggested that leprosy originated in Africa, extended to Asia and Europe, and arrived in the Americas during European colonization and the African slave trade. Due to colonization, the contemporary Colombian population is an admixture of Native-American, European and African ancestries. Because microorganisms are known to accompany humans during migrations, patterns of human migration can be traced by examining genomic changes in associated microbes. The current study analyzed 118 leprosy cases and 116 unrelated controls from two Colombian regions endemic for leprosy (Atlantic and Andean) in order to determine possible associations of leprosy with patient ancestral background (determined using 36 ancestry informative markers), Mycobacterium leprae genotype and/or patient geographical origin. We found significant differences between ancestral genetic composition. European components were predominant in Andean populations. In contrast, African components were higher in the Atlantic region. M. leprae genotypes were then analyzed for cluster associations and compared with the ancestral composition of leprosy patients. Two M. leprae principal clusters were found: haplotypes C54 and T45. Haplotype C54 associated with African origin and was more frequent in patients from the Atlantic region with a high African component. In contrast, haplotype T45 associated with European origin and was more frequent in Andean patients with a higher European component. These results suggest that the human and M. leprae genomes have co-existed since the African and European origins of the disease, with leprosy ultimately arriving in Colombia during colonization. Distinct M. leprae strains followed European and African settlement in the country and can be detected in contemporary Colombian populations. PMID:26360617

  11. MspA-Mycobacterium tuberculosis-transformant with reduced virulence: the "unbirthday paradigm".

    PubMed

    Lamrabet, Otmane; Ghigo, Eric; Mège, Jean-Louis; Lepidi, Hubert; Nappez, Claude; Raoult, Didier; Drancourt, Michel

    2014-11-01

    Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical "unbirthday" gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity.

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

    SciTech Connect

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

    2011-08-24

    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.

  13. Persistent Infection by a Mycobacterium tuberculosis Strain That Was Theorized To Have Advantageous Properties, as It Was Responsible for a Massive Outbreak

    PubMed Central

    Pérez-Lago, Laura; Navarro, Yurena; Montilla, Pedro; Comas, Iñaki; Herranz, Marta; Rodríguez-Gallego, Carlos; Ruiz Serrano, María Jesús; Bouza, Emilio

    2015-01-01

    The strains involved in tuberculosis outbreaks are considered highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain, who was persistently infected over an 8-year period by the same Beijing Mycobacterium tuberculosis strain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case provides us with a unique opportunity to challenge our assumptions about M. tuberculosis Beijing strains. No clinical/radiological findings consistent with a virulent strain were documented, and the in vitro growth rate of the strain in macrophages was only moderate. No secondary cases stemming from this prolonged active case were detected in the host population. The strain did not acquire resistance mutations, despite constant treatment interruptions, and it remained extremely stable, as demonstrated by the lack of single-nucleotide-polymorphism (SNP)-based differences between the sequential isolates. Our data suggest that the general assumption about M. tuberculosis Beijing strains having advantageous properties (in terms of virulence, transmissibility, and the tendency to acquire mutations and resistance) is not always accurate. PMID:26269618

  14. Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

    PubMed Central

    Alonso-Hearn, Marta; Abendaño, Naiara; Ruvira, Maria A.; Aznar, Rosa; Landin, Mariana; Juste, Ramon A.

    2017-01-01

    Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two Map isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of Map for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each Map isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two Map strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of Map. In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both Map

  15. Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

    PubMed

    Sequeira, Patrícia Carvalho de; Fonseca, Leila de Souza; Silva, Marlei Gomes da; Saad, Maria Helena Féres

    2005-11-01

    Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

  16. Virulence attenuation of a Mycobacterium avium subspecies paratuberculosis S-type strain prepared from intestinal mucosa after bacterial culture. Evaluation in an experimental ovine model.

    PubMed

    Fernández, Miguel; Delgado, Laetitia; Sevilla, Iker A; Fuertes, Miguel; Castaño, Pablo; Royo, Marcos; Ferreras, M Carmen; Benavides, Julio; Pérez, Valentín

    2015-04-01

    The differences in pathogenicity between an inoculum derived directly from an intestinal tissue homogenate from a paratuberculosis affected sheep and the S-type Mycobacterium avium subsp. partuberculosis (Map) strain isolated in laboratory media from the mentioned homogenate were assessed in two experiments in lambs. Specific peripheral immune responses were significantly lower in animals inoculated with the cultured organisms that showed only granulomatous lesions in the intestinal lymphoid tissue. However, in the homogenate group, more abundant granulomata also occurred in the lamina propria. Map was isolated only in lambs infected with the culture strain. Map DNA was demonstrated by nested-PCR in all the lambs but in a lower proportion (57.1% vs 100%) in those from the culture group. Under these particular experimental conditions, the results suggest that an attenuation of Map virulence has occurred in the cultured strain compared to the initial tissue homogenate, even after a low number of passages.

  17. Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

    PubMed Central

    González-Pérez, Mónica; Mariño-Ramírez, Leonardo; Parra-López, Carlos Alberto; Murcia, Martha Isabel; Marquina, Brenda; Mata-Espinoza, Dulce; Rodriguez-Míguez, Yadira; Baay-Guzman, Guillermina J.; Huerta-Yepez, Sara

    2013-01-01

    The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue. PMID:23959717

  18. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli

    PubMed Central

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  19. Functional characterization delineates that a Mycobacterium tuberculosis specific protein kinase (Rv3080c) is responsible for the growth, phagocytosis and intracellular survival of avirulent mycobacteria.

    PubMed

    Kumari, Ruma; Singh, Susmita K; Singh, Diwakar K; Singh, Pramod K; Chaurasiya, Shivendra K; Srivastava, Kishore K

    2012-10-01

    Serine/threonine protein kinases (STPKs) are predominantly involved in growth, development, division, differentiation, and in regulating immune responses in mycobacteria. A wide variety of functions of mycobacterial STPKs persuade mycobacterial growth and further its survival in the hosts. The polymorphic studies have shown that a full length gene of Rv3080c (pknK) is present in the slow growing mycobacteria. The wild type Mycobacterium smegmatis containing only vector (M. smegmatis) and M. smegmatis containing Rv3080c (pknK) cloned in pMV261 vector (M. smegmatis::K) were cultured in different growth media. The studies have shown that M. smegmatis did not differ in the growth and in survival while a substantial reduction in the growth (four-ten-folds) and a significant delay in the colony formation were observed in M. smegmatis::K. In order to look for the stage specific and modulated expression of PknK, the study was comprehended to quantitate pknK transcripts at different phases of cultures. The mycobacterium, containing high copy number of pknK specific RNA was unable to multiply. The study thus highlights that Rv3080c is largely accountable for changing the fate of avirulent mycobacteria and hence the protein can be utilized as an important molecule to target pathogenesis.

  20. Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

    PubMed

    Wright, Douglas G; Castore, Reneau; Shi, Runhua; Mallick, Amrita; Ennis, Don G; Harrison, Lynn

    2016-09-09

    Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis.

  1. Assessment of the Immune Responses Induced in Cattle after Inoculation of a Mycobacterium bovis Strain Deleted in Two mce2 Genes

    PubMed Central

    Blanco, Federico Carlos; Soria, Marcelo; Gravisaco, María José; Bianco, María Verónica; Meikle, Virginia; Garbaccio, Sergio; Vagnoni, Lucas; Cataldi, Angel Adrián; Bigi, Fabiana

    2012-01-01

    The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis. PMID:22719207

  2. Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes.

    PubMed

    Café Oliveira, Luita Nice; Muniz-Sobrinho, Jairo da Silva; Viana-Magno, Luiz Alexandre; Oliveira Melo, Sônia Cristina; Macho, Antonio; Rios-Santos, Fabrício

    2016-01-01

    Multidrug-resistant tuberculosis (MDRTB) is a serious world health problem that limits public actions to control tuberculosis, because the most used anti-tuberculosis first-line drugs fail to stop mycobacterium spread. Consequently, a quick detection through molecular diagnosis is essential to reduce morbidity and medical costs. Despite the availability of several molecular-based commercial-kits to diagnose multidrug-resistant tuberculosis, their diagnostic value might diverge worldwide since Mycobacterium tuberculosis genetic variability differs according to geographic location. Here, we studied the predictive value of four common mycobacterial mutations in strains isolated from endemic areas of Brazil. Mutations were found at the frequency of 41.9% for katG, 25.6% for inhA, and 69.8% for rpoB genes in multidrug-resistant strains. Multimarker analysis revealed that combination of only two mutations ("katG/S315T+rpoB/S531L") was a better surrogate of multidrug-resistant tuberculosis than single-marker analysis (86% sensitivity vs. 62.8%). Prediction of multidrug-resistant tuberculosis was not improved by adding a third or fourth mutation in the model. Therefore, rather than using diagnostic kits detecting several mutations, we propose a simple dual-marker panel to detect multidrug-resistant tuberculosis, with 86% sensitivity and 100% specificity. In conclusion, this approach (previous genetic study+analysis of only prevalent markers) would considerably decrease the processing costs while retaining diagnostic accuracy.

  3. Actions of Mycobacterium sp. strain AP1 on the saturated- and aromatic-hydrocarbon fractions of fuel oil in a marine medium.

    PubMed

    Vila, Joaquim; Grifoll, Magdalena

    2009-10-01

    The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C(12) to C(40)) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.

  4. Predominance of multi-drug-resistant LAM and Beijing family strains among Mycobacterium tuberculosis isolates recovered from prison inmates in Tula Region, Russia.

    PubMed

    Ignatova, Anna; Dubiley, Svetlana; Stepanshina, Valentina; Shemyakin, Igor

    2006-10-01

    The genotypic characteristics and drug susceptibility profiles of clinical isolates of Mycobacterium tuberculosis recovered from prison hospital patients in the Tula region (central Russia) during 2001 and 2002 are reported. The emergence of multi-drug-resistant tuberculosis (TB) poses a major health risk to the population, with economic implications for TB control. Prisons serve as a continuous source of TB transmission. The results showed that members of the LAM and Beijing families are major contributors to the epidemiological picture of TB in the population studied. The two families of strains accounted for most of the drug-resistant TB in the population. The genotypic characteristics of the M. tuberculosis predominant LAM strain that was responsible for 31 % of TB cases in this setting are presented.

  5. Investigation of the population structure of Mycobacterium abscessus complex strains using 17-locus variable number tandem repeat typing and the further distinction of Mycobacterium massiliense hsp65 genotypes.

    PubMed

    Yoshida, Shiomi; Arikawa, Kentaro; Tsuyuguchi, Kazunari; Kurashima, Atsuyuki; Harada, Toshiyuki; Nagai, Hideaki; Suzuki, Katsuhiro; Iwamoto, Tomotada; Hayashi, Seiji

    2015-03-01

    Mycobacterium abscessus complex is a significant pathogen in patients with non-cystic fibrosis (non-CF). Nevertheless, there is little description of the genetic diversity of this species. The aims of this study were to investigate the distribution of M. abscessus complex isolated from respiratory specimens by variable number tandem repeat (VNTR) typing. The results of 104 clinical isolates from 104 non-CF patients were compared using PFGE, hsp65 genotypes and clarithromycin susceptibility. The allelic diversity (Hunter-Gaston Discriminatory Index) of the 17 loci examined by VNTR typing was high (0.977). We determined that C28 sequevar erm(41) genotypes and clarithromycin-acquired resistance isolates were scattered in the minimum spanning tree. Intriguingly, VNTR typing and PFGE were highly congruent and revealed that there were clear examples of grouping of isolates from different individuals amongst both M. abscessus and M. massiliense, and showed five clusters of distinct identical isolates. Within these clusters, M. massiliense hsp65 type I formed three different clusters. Although the distribution of M. massiliense hsp65 type II-1 was low (9.3 %), M. massiliense hsp65 type II-1 isolates separated from clusters contained hsp65 type I isolates. Thus, M. massiliense hsp65 genotypes could be discriminated by analysing VNTRs with sufficient genetic distance for intra-species-level discrimination.

  6. 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

  7. Novel cyclic lipodepsipeptide from Pseudomonas syringae pv. lachrymans strain 508 and syringopeptin antimicrobial activities.

    PubMed

    Grgurina, Ingeborg; Bensaci, Mekki; Pocsfalvi, Gabriella; Mannina, Luisa; Cruciani, Oscar; Fiore, Alberto; Fogliano, Vincenzo; Sorensen, Kevin N; Takemoto, Jon Y

    2005-12-01

    The syringopeptins are a group of antimicrobial cyclic lipodepsipeptides produced by several plant-associated pseudomonads. A novel syringopeptin, SP508, was shown to be produced as two homologs (A and B) by Pseudomonas syringae pv. lachrymans strain 508 from apple and to structurally resemble syringopeptin SP22. SP508 differed from SP22 and other syringopeptins by having three instead of four alpha,beta-unsaturated amino acids and a longer beta-hydroxy acyl chain. Both SP508 and SP22 displayed growth-inhibitory activities against Mycobacterium smegmatis, other gram-positive bacteria, and yeasts but not against gram-negative bacteria. Structure-activity analyses of the SP508 and SP22 homologs indicated chemical structural features that lead to enhanced antimycobacterial activity by these pseudomonad cyclic lipodepsipeptides.

  8. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  9. Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages

    PubMed Central

    Vázquez, Cristina Lourdes; Bianco, María Verónica; Blanco, Federico Carlos; Forrellad, Marina Andrea; Gutierrez, Maximiliano Gabriel

    2016-01-01

    ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host. PMID:28031264

  10. PPE38 Protein of Mycobacterium tuberculosis Inhibits Macrophage MHC Class I Expression and Dampens CD8+ T Cell Responses

    PubMed Central

    Meng, Lu; Tong, Jingfeng; Wang, Hui; Tao, Chengwu; Wang, Qinglan; Niu, Chen; Zhang, Xiaoming; Gao, Qian

    2017-01-01

    Suppression of CD8+ T cell activation is a critical mechanism used by Mycobacterium tuberculosis (MTB) to escape protective host immune responses. PPE38 belongs to the unique PPE family of MTB and in our previous study, PPE38 protein was speculated to participate in manipulating macrophage MHC class I pathway. To test this hypothesis, the function of mycobacterial PPE38 protein was assessed here using macrophage and mouse infection models. Decreased amount of MHC class I was observed on the surface of macrophages infected with PPE38-expressing mycobacteria. The transcript of genes encoding MHC class I was also inhibited by PPE38. After infection of C57BL/6 mice with Mycobacterium smegmatis expressing PPE38 (Msmeg-PPE38), decreased number of CD8+ T cells was found in spleen, liver, and lungs through immunohistochemical analysis, comparing to the control strain harboring empty vector (Msmeg-V). Consistently, flow cytometry assay showed that fewer effector/memory CD8+ T cells (CD44highCD62Llow) were activated in spleen from Msmeg-PPE38 infected mice. Moreover, Msmeg-PPE38 confers a growth advantage over Msmeg-V in C57BL/6 mice, indicating an effect of PPE38 to favor mycobacterial persistence in vivo. Overall, this study shows a unique biological function of PPE38 protein to facilitate mycobacteria to escape host immunity, and provides hints for TB vaccine development. PMID:28348981

  11. Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice

    PubMed Central

    Yabe, Idalia; Morris, Sheldon; Cowley, Siobhan

    2016-01-01

    Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4+ and CD8+ T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6′-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ−/− mice depleted of CD4+, CD8+, and NK1.1+ T cells against an aerosol challenge with M. tuberculosis. These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4+, CD8+, NK1.1+, and TCRγδ T cells and could exhibit memory. Focusing on CD4− CD8− double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells. PMID:27226281

  12. Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains.

    PubMed Central

    Gutacker, Michaela M; Smoot, James C; Migliaccio, Cristi A Lux; Ricklefs, Stacy M; Hua, Su; Cousins, Debby V; Graviss, Edward A; Shashkina, Elena; Kreiswirth, Barry N; Musser, James M

    2002-01-01

    Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range. PMID:12524330

  13. Single nucleotide polymorphism in Ag85 genes of Mycobacterium tuberculosis complex: analysis of 178 clinical isolates from China and 13 BCG strains.

    PubMed

    Jiang, Yi; Liu, Haican; Li, Machao; Li, Guilian; Pang, Hui; Dou, Xiangfeng; Zhao, Xiuqin; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. Human T-cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) were evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. However, in our previous studies, proteins MPT64, PstS1, Rv0309 and Rv2945c all harbored higher numbers of amino acid substitutions in their T cell epitopes, which suggests their roles in ongoing immune evasion. Here, we used the same set of 180 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Ag85 complex, and compared the sequences. The results showed that Ag85 were hyperconserved in T/B cell epitopes and the genes were more likely to be under purifying selection. The divergence of host immune selection on different proteins may result from different function of the proteins. In addition, A312G of Ag85A and T418C of Ag85B may represent special mutations in BCG strains, which may be used to differentiate M.bovis and BCG strains from MTB strains. Also, C714A in Ag85B seems to be a valuable phylogenetic marker for Beijing strains.

  14. Mefloquine and its oxazolidine derivative compound are active against drug-resistant Mycobacterium tuberculosis strains and in a murine model of tuberculosis infection.

    PubMed

    Rodrigues-Junior, Valnês S; Villela, Anne D; Gonçalves, Raoni S B; Abbadi, Bruno Lopes; Trindade, Rogério Valim; López-Gavín, Alexandre; Tudó, Griselda; González-Martín, Julian; Basso, Luiz Augusto; de Souza, Marcus V N; Campos, Maria Martha; Santos, Diógenes Santiago

    2016-08-01

    Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5-8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.

  15. Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

    PubMed Central

    Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Rubin, Daniel H. F.; Kajee, Afsana; Msibi, Zama N. P.; Larsen, Michelle H.; Jacobs, William R.; Lawrence, Jeffrey G.; Hendrix, Roger W.

    2014-01-01

    ABSTRACT Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc2155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host. PMID:25467442

  16. Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth

    PubMed Central

    Lee, Kang-In; Whang, Jake; Choi, Han-Gyu; Son, Yeo-Jin; Jeon, Haet Sal; Back, Yong Woo; Park, Hye-Soo; Paik, Seungwha; Park, Jeong-Kyu; Choi, Chul Hee; Kim, Hwa-Jung

    2016-01-01

    Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication. PMID:27901051

  17. Tuberculous Lymphadenitis in Ethiopia Predominantly Caused by Strains Belonging to the Delhi/CAS Lineage and Newly Identified Ethiopian Clades of the Mycobacterium tuberculosis Complex

    PubMed Central

    Biadglegne, Fantahun; Merker, Matthias; Sack, Ulrich; Rodloff, Arne C.; Niemann, Stefan

    2015-01-01

    Background Recently, newly defined clades of Mycobacterium tuberculosis complex (MTBC) strains, namely Ethiopia 1–3 and Ethiopia H37Rv-like strains, and other clades associated with pulmonary TB (PTB) were identified in Ethiopia. In this study, we investigated whether these new strain types exhibit an increased ability to cause TB lymphadenitis (TBLN) and raised the question, if particular MTBC strains derived from TBLN patients in northern Ethiopia are genetically adapted to their local hosts and/or to the TBLN. Methods Genotyping of 196 MTBC strains isolated from TBLN patients was performed by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing. A statistical analysis was carried out to see possible associations between patient characteristics and phylogenetic MTBC strain classification. Results Among 196 isolates, the majority of strains belonged to the Delhi/CAS (38.8%) lineage, followed by Ethiopia 1 (9.7%), Ethiopia 3 (8.7%), Ethiopia H37RV-like (8.2%), Ethiopia 2 and Haarlem (7.7% each), URAL (3.6%), Uganda l and LAM (2% each), S-type (1.5%), X-type (1%), and 0.5% isolates of TUR, EAI, and Beijing genotype, respectively. Overall, 15 strains (7.7%) could not be allocated to a previously described phylogenetic lineage. The distribution of MTBC lineages is similar to that found in studies of PTB samples. The cluster rate (35%) in this study is significantly lower (P = 0.035) compared to 45% in the study of PTB in northwestern Ethiopia. Conclusion In the studied area, lymph node samples are dominated by Dehli/CAS genotype strains and strains of largely not yet defined clades based on MIRU-VNTR 24-loci nomenclature. We found no indication that strains of particular genotypes are specifically associated with TBLN. However, a detailed analysis of specific genetic variants of the locally contained Ethiopian clades by whole genome sequencing may reveal new insights into the host-pathogen co

  18. Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice.

    PubMed

    Almeida, Fabrício M; Ventura, Thatiana L B; Amaral, Eduardo P; Ribeiro, Simone C M; Calixto, Sanderson D; Manhães, Marcelle R; Rezende, Andreza L; Souzal, Giliane S; de Carvalho, Igor S; Silva, Elisangela C; Silva, Juliana Azevedo da; Carvalho, Eulógio C Q; Kritski, Afranio L; Lasunskaia, Elena B

    2017-01-01

    Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions.

  19. Genomic variability of Mycobacterium tuberculosis strains of the Euro-American lineage based on large sequence deletions and 15-locus MIRU-VNTR polymorphism.

    PubMed

    Rindi, Laura; Medici, Chiara; Bimbi, Nicola; Buzzigoli, Andrea; Lari, Nicoletta; Garzelli, Carlo

    2014-01-01

    A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RD(Rio) deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the "Cameroon" family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification.

  20. Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice

    PubMed Central

    Almeida, Fabrício M.; Ventura, Thatiana L. B.; Amaral, Eduardo P.; Ribeiro, Simone C. M.; Calixto, Sanderson D.; Manhães, Marcelle R.; Rezende, Andreza L.; Souzal, Giliane S.; de Carvalho, Igor S.; Silva, Elisangela C.; da Silva, Juliana Azevedo; Carvalho, Eulógio C. Q.; Kritski, Afranio L.

    2017-01-01

    Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions. PMID:28306733

  1. Real-time PCR and high-resolution melt analysis for rapid detection of Mycobacterium leprae drug resistance mutations and strain types.

    PubMed

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A; Brennan, Patrick J; Vissa, Varalakshmi

    2012-03-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.

  2. Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

    PubMed

    Chandra, Pallavi; Rajmani, R S; Verma, Garima; Bhavesh, Neel Sarovar; Kumar, Dhiraj

    2016-01-01

    In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a

  3. Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages.

    PubMed

    Pires, David; Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard; Constantino, Luís; Anes, Elsa

    2015-12-01

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257-263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

  4. Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages

    PubMed Central

    Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard

    2015-01-01

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development. PMID:26438493

  5. Occurrence, diagnosis, and strain typing of Mycobacterium avium subspecies paratuberculosis infection in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in southwestern Alberta.

    PubMed

    Forde, Taya; Kutz, Susan; De Buck, Jeroen; Warren, Amy; Ruckstuhl, Kathreen; Pybus, Margo; Orsel, Karin

    2012-01-01

    The role that wildlife may play in the transmission of Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne's disease (JD), and the potential consequences of infection in these populations are being given increasing consideration. A yearling male Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from southwestern Alberta, Canada, was found infected with Map in August 2009. Clinical signs of emaciation and diarrhea and histologic findings of diffuse granulomatous enteritis of the distal ileum, lymphadenitis of the mesenteric lymph nodes, and lymphangitis of the ileum were similar to previously described cases of JD in bighorn sheep. Infection with Map was confirmed by bacterial isolation through fecal culture, acid-fast staining, and polymerase chain reaction (PCR) of IS900. The Map1506 gene was sequenced, and the isolate was identified as a Cattle (Type II) strain. In a follow-up herd-level survey, three of 44 fecal samples (7%) from individual bighorn sheep from the same herd as the index case were PCR-positive and identified as Type II Map strains. Twenty-five samples from a distant bighorn population were negative. Additional strain typing of the isolates from the index case and the positive fecal samples was done by sequencing three discriminatory short sequence repeat (SSR) regions. All four SSR profiles differed from one another, suggesting multiple introductions or a long-existing circulation of Map within this bighorn population. Detailed molecular analyses are essential for understanding and managing diseases at the wildlife-livestock interface.

  6. Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

    PubMed Central

    Tyagi, Priyanka; Dharmaraja, Allimuthu T.; Bhaskar, Ashima; Chakrapani, Harinath; Singh, Amit

    2015-01-01

    Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. PMID:25819161

  7. Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

    PubMed

    Garzelli, Carlo; Lari, Nicoletta; Rindi, Laura

    2016-03-01

    The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey.

  8. Analysis of Mutations in Streptomycin-Resistant Strains Reveals a Simple and Reliable Genetic Marker for Identification of the Mycobacterium tuberculosis Beijing Genotype

    PubMed Central

    Villellas, Cristina; Aristimuño, Liselotte; Vitoria, María-Asunción; Prat, Cristina; Blanco, Silvia; García de Viedma, Darío; Domínguez, José; Samper, Sofía

    2013-01-01

    The Mycobacterium tuberculosis pandemic is a major health problem, further complicated by an increasing incidence of drug-resistant isolates and the existence of highly transmissible strains, such as those in the Beijing family. Streptomycin (STR)-resistant M. tuberculosis clinical isolates have been analyzed to look for mutations in the rpsL, rrs, and gidB genes. In addition, the Rv1258c gene, which encodes Tap, an efflux pump that transports STR, has been sequenced. Mutations affecting codons 43 and 88 of the rpsL gene were found in 44.4% of the strains, and 16.7% of the strains carried mutations in the rrs gene, both of which probably contribute to STR resistance. Many strains presented with mutations in the gidB gene, but the implication of those mutations in STR resistance remains unclear. Interestingly, a cytosine nucleotide insertion between positions 580 and 581 (denominated Tap580) in the Rv1258c gene has been found in all Beijing isolates included in this study, suggesting that it might be a novel polymorphism specific to the Beijing family of M. tuberculosis. A simple and fast restriction fragment length polymorphism (RFLP)-PCR method for detecting the Tap580 insertion has been developed and used to screen a collection of 220 DNA samples obtained from cultures of M. tuberculosis isolates and 30 respiratory specimens. In all cases, the Beijing and non-Beijing representative samples were identified correctly. Tap580 is a novel polymorphism specific to the highly transmissible Beijing family, which allows for fast detection of these strains even at the very early stages of infection. PMID:23616454

  9. Application of four molecular typing methods for analysis of Mycobacterium fortuitum group strains causing post-mammaplasty infections.

    PubMed

    Sampaio, J L M; Chimara, E; Ferrazoli, L; da Silva Telles, M A; Del Guercio, V M F; Jericó, Z V N; Miyashiro, K; Fortaleza, C M C B; Padoveze, M C; Leão, S C

    2006-02-01

    A cluster of cases of post-augmentation mammaplasty surgical site infections occurred between 2002 and 2004 in Campinas, in the southern region of Brazil. Rapidly growing mycobacteria were isolated from samples from 12 patients. Eleven isolates were identified as Mycobacterium fortuitum and one as Mycobacterium porcinum by PCR-restriction digestion of the hsp65 gene. These 12 isolates, plus six additional M. fortuitum isolates from non-related patients, were typed by pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: 16S-23S rRNA internal transcribed spacer (ITS) genotyping; randomly amplified polymorphic DNA (RAPD) PCR; and enterobacterial repetitive intergenic consensus (ERIC) PCR. Four novel M. fortuitum allelic variants were identified by restriction analysis of the ITS fragment. One major cluster, comprising six M. fortuitum isolates, and a second cluster of two isolates, were identified by the four methods. RAPD-PCR and ITS genotyping were less discriminative than ERIC-PCR. ERIC-PCR was comparable to PFGE as a valuable complementary tool for investigation of this type of outbreak.

  10. Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain

    PubMed Central

    2014-01-01

    Background Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. Results BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. Conclusions Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions. PMID:24766746

  11. Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

    PubMed Central

    Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

    2013-01-01

    Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

  12. Characterization and growth in human macrophages of Mycobacterium avium complex strains isolated from the blood of patients with acquired immunodeficiency syndrome.

    PubMed Central

    Meylan, P R; Richman, D D; Kornbluth, R S

    1990-01-01

    Strains of the Mycobacterium avium complex (MAC) yield opaque and transparent colonial variants when cultivated in vitro. The transparent variants are more virulent than the opaque for animals, but little is known about the respective roles of these colonial variants in humans. To assess which variant infects humans, various blood fractions from eight patients with MAC bacteremia were plated directly onto 7H10 agar. In cell fractionation studies, all the M. avium complex CFU were associated with leukocytes and none were found free in plasma. All colonies on the primary culture plate exhibited the transparent phenotype. However, during subculture in 7H9 broth or on Lowenstein-Jensen agar, opaque variants appeared in seven of eight strains. Isogenic pairs of transparent and opaque variants were prepared and used to infect in vitro human monocyte-derived macrophages from healthy seronegative individuals. Transparent variants invariably grew inside macrophages, but only one of seven opaque variants did so. These observations indicate that the bacteremia of M. avium complex in acquired immunodeficiency syndrome patients consists exclusively of the transparent variants, perhaps because these variants are able to multiply inside macrophages. In contrast, opaque variants appear after in vitro subculture and are controlled by human macrophages, consistent with their reduced virulence in animals. Images PMID:2370109

  13. Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen.

    PubMed Central

    Thangaraj, H S; Lamb, F I; Davis, E O; Jenner, P J; Jeyakumar, L H; Colston, M J

    1990-01-01

    The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences. The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme. The gene is not expressed from its own promoter in E. coli but is expressed from its own promoter in Mycobacterium smegmatis. The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined. Images PMID:1692812

  14. High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E.

    PubMed

    Homolka, Susanne; Ubben, Tanja; Niemann, Stefan

    2016-01-01

    The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.

  15. Novel vaccine potential of Rv3131, a DosR regulon-encoded putative nitroreductase, against hyper-virulent Mycobacterium tuberculosis strain K.

    PubMed

    Kwon, Kee Woong; Kim, Woo Sik; Kim, Hongmin; Han, Seung Jung; Hahn, Mi-Young; Lee, Jong Seok; Nam, Ki Taek; Cho, Sang-Nae; Shin, Sung Jae

    2017-03-08

    Accumulating evidence indicates that latency-associated Mycobacterium tuberculosis (Mtb)-specific antigens from the dormancy survival regulator regulon (DosR) may be promising novel vaccine target antigens for the development of an improved tuberculosis vaccine. After transcriptional profiling of DosR-related genes in the hyper-virulent Beijing Mtb strain K and the reference Mtb strain H37Rv, we selected Rv3131, a hypothetical nitroreductase, as a vaccine antigen and evaluated its vaccine efficacy against Mtb K. Mtb K exhibited stable and constitutive up-regulation of rv3131 relative to Mtb H37Rv under three different growth conditions (at least 2-fold induction) including exponential growth in normal culture conditions, hypoxia, and inside macrophages. Mice immunised with Rv3131 formulated in GLA-SE, a well-defined TLR4 adjuvant, displayed enhanced Rv3131-specific IFN-γ and serum IgG2c responses along with effector/memory T cell expansion and remarkable generation of Rv3131-specific multifunctional CD4(+) T cells co-producing TNF-α, IFN-γ and IL-2 in both spleen and lung. Following challenge with Mtb K, the Rv3131/GLA-SE-immunised group exhibited a significant reduction in bacterial number and less extensive lung inflammation accompanied by the obvious persistence of Rv3131-specific multifunctional CD4(+) T cells. These results suggest that Rv3131 could be an excellent candidate for potential use in a multi-antigenic Mtb subunit vaccine, especially against Mtb Beijing strains.

  16. Novel vaccine potential of Rv3131, a DosR regulon-encoded putative nitroreductase, against hyper-virulent Mycobacterium tuberculosis strain K

    PubMed Central

    Kwon, Kee Woong; Kim, Woo Sik; Kim, Hongmin; Han, Seung Jung; Hahn, Mi-Young; Lee, Jong Seok; Nam, Ki Taek; Cho, Sang-Nae; Shin, Sung Jae

    2017-01-01

    Accumulating evidence indicates that latency-associated Mycobacterium tuberculosis (Mtb)-specific antigens from the dormancy survival regulator regulon (DosR) may be promising novel vaccine target antigens for the development of an improved tuberculosis vaccine. After transcriptional profiling of DosR-related genes in the hyper-virulent Beijing Mtb strain K and the reference Mtb strain H37Rv, we selected Rv3131, a hypothetical nitroreductase, as a vaccine antigen and evaluated its vaccine efficacy against Mtb K. Mtb K exhibited stable and constitutive up-regulation of rv3131 relative to Mtb H37Rv under three different growth conditions (at least 2-fold induction) including exponential growth in normal culture conditions, hypoxia, and inside macrophages. Mice immunised with Rv3131 formulated in GLA-SE, a well-defined TLR4 adjuvant, displayed enhanced Rv3131-specific IFN-γ and serum IgG2c responses along with effector/memory T cell expansion and remarkable generation of Rv3131-specific multifunctional CD4+ T cells co-producing TNF-α, IFN-γ and IL-2 in both spleen and lung. Following challenge with Mtb K, the Rv3131/GLA-SE-immunised group exhibited a significant reduction in bacterial number and less extensive lung inflammation accompanied by the obvious persistence of Rv3131-specific multifunctional CD4+ T cells. These results suggest that Rv3131 could be an excellent candidate for potential use in a multi-antigenic Mtb subunit vaccine, especially against Mtb Beijing strains. PMID:28272457

  17. Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K.

    PubMed

    Cha, Seung Bin; Kim, Woo Sik; Kim, Jong-Seok; Kim, Hongmin; Kwon, Kee Woong; Han, Seung Jung; Cho, Sang-Nae; Coler, Rhea N; Reed, Steven G; Shin, Sung Jae

    2016-04-27

    The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4(+) T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8(+) central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent.

  18. Immunogenicity and protective efficacy conferred by a novel recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing interleukin-12p70 of human cytokine and Ag85A of Mycobacterium tuberculosis fusion protein.

    PubMed

    Deng, Y H; He, H Y; Zhang, F J

    2013-12-01

    Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG-IA), which could express IL-12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4(+) T and CD8(+) T cells stimulated by specific antigens and levels of IFN-γ production in antigen-stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid-fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG-IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN-γ production among groups and gained improved ratio of CD4(+) T and CD8(+) T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG-IA group were obviously lower than that in rBCG-I group, rBCG-A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG-IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.

  19. Comparison between RFLP and MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Isolated in Stockholm 2009 to 2011

    PubMed Central

    Jonsson, Jerker; Hoffner, Sven; Berggren, Ingela; Bruchfeld, Judith; Ghebremichael, Solomon; Pennhag, Alexandra; Groenheit, Ramona

    2014-01-01

    Our aim was to analyze the difference between methods for genotyping of Mycobacterium tuberculosis complex isolates. We collected genotyping results from Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units - Variable Numbers of Tandem Repeat (MIRU-VNTR) in a geographically limited area (Stockholm) during a period of three years. The number and proportion of isolates belonging to clusters was reduced by 45 and 35% respectively when combining the two methods compared with using RFLP or MIRU-VNTR only. The mean size of the clusters was smaller when combining methods and smaller with RFLP compared to MIRU-VNTR. In clusters with confirmed epidemiological links RFLP coincided slightly better than MIRU-VNTR but where there was a difference, the variation in MIRU-VNTR pattern was only in a single locus. In isolates with few IS6110 bands in RFLP, MIRU-VNTR differentiated the isolates more, dividing the RFLP clusters. Since MIRU-VNTR is faster and less labour-intensive it is the method of choice for routine genotyping. In most cases it will be sufficient for epidemiological purposes but true clustering might still be considered if there are epidemiological links and the MIRU-VNTR results differ in only one of its 24 loci. PMID:24733167

  20. The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis

    PubMed Central

    Sacco, Emmanuelle; Covarrubias, Adrian Suarez; O'Hare, Helen M.; Carroll, Paul; Eynard, Nathalie; Jones, T. Alwyn; Parish, Tanya; Daffé, Mamadou; Bäckbro, Kristina; Quémard, Annaïk

    2007-01-01

    The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C12) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs. PMID:17804795

  1. 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

  2. Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

    PubMed

    Liang, Jinping; Teng, Xindong; Yuan, Xuefeng; Zhang, Ying; Shi, Chunwei; Yue, Tingting; Zhou, Lei; Li, Jianrong; Fan, Xionglin

    2015-08-01

    Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

  3. Effect of milk fermentation by kefir grains and selected single strains of lactic acid bacteria on the survival of Mycobacterium bovis BCG.

    PubMed

    Macuamule, C L S; Wiid, I J; van Helden, P D; Tanner, M; Witthuhn, R C

    2016-01-18

    Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to determine the effect of the fermentation process on the survival of M. bovis BCG in milk. M. bovis BCG at concentrations of 6 log CFU/ml was added to products of kefir fermentation. The survival of M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar plates enriched with 2% BD BACTEC PANTA™. M. bovis BCG was increasingly reduced in sterile kefir that was fermented for a period of 24h and longer. In the milk fermented with kefir grains, Lactobacillus paracasei subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24h and by 2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation. Results from this study show that long term fermentation under certain conditions may have the potential to inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation should be combined with other hurdle technologies such as boiling and milk pasteurisation.

  4. Preliminary structure-activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains.

    PubMed

    Onajole, Oluseye K; Pieroni, Marco; Tipparaju, Suresh K; Lun, Shichun; Stec, Jozef; Chen, Gang; Gunosewoyo, Hendra; Guo, Haidan; Ammerman, Nicole C; Bishai, William R; Kozikowski, Alan P

    2013-05-23

    Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the world's population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

  5. Multiple strain infections and high genotypic diversity among Mycobacterium avium subsp. paratuberculosis field isolates from diseased wild and domestic ruminant species in the eastern Alpine region of Austria.

    PubMed

    Gerritsmann, H; Stalder, G L; Spergser, J; Hoelzl, F; Deutz, A; Kuebber-Heiss, A; Walzer, C; Smith, S

    2014-01-01

    Johne's disease, or paratuberculosis, is a chronic fatal ruminant gastroenteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP) whose foodborne zoonotic potential and association with Crohn's disease are still under debate. The disease is widespread but its epidemiology and epizootiology remains elusive. Wildlife is suspected to play a major role. After a surge in MAP seroprevalence in Austrian cattle, paratuberculosis was declared a notifiable disease in Austria in 2006. At the same time a rise in MAP cases in wild ruminant populations in the Austrian province of Styria was reported. All five autochthonous ruminants were affected. Genetic analysis of isolates, yielded numerous genotypes (>15) and several multiple strain infections (15%) across host species. Identical MIRU-VNTR profiles were identified in different species and sampling locations. On the other hand varying MIRU-VNTR profiles were revealed at the same location and in conspecifics. Our data, taken together with earlier epidemiological studies on MAP and other mycobacteria, raised concerns about the organisms' ecology. Constraints regarding in vitro culture of this highly fastidious organism potentially bias our current understanding of its epidemiology. We suggest that MAP infections could be polyclonal and question the informative value of genotyping a single MAP colony derived from a single specimen for epidemiological analysis of MAP.

  6. A mechanism of virulence: virulent Mycobacterium tuberculosis strain H37Rv, but not attenuated H37Ra, causes significant mitochondrial inner membrane disruption in macrophages leading to necrosis.

    PubMed

    Chen, Minjian; Gan, Huixian; Remold, Heinz G

    2006-03-15

    Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48-72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca(2+) flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Deltapsi(m)) loss caused by mitochondrial permeability transition. Dissipation of Deltapsi(m) also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca(2+) loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Deltapsi(m) 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.

  7. Evaluation of the effect of Pulicaria gnaphalodes and Perovskia abrotanoides essential oil extracts against Mycobacterium tuberculosis strains

    PubMed Central

    Hozoorbakhsh, Fereshte; Esfahani, Bahram Nasr; Moghim, Sharareh; Asghari, Gholamreza

    2016-01-01

    Background: Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), which remains one of the major public health problems in the world. The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) worldwide highlights the urgent need to search for alternative antimycobacterial agents. More and more people in developing countries utilize traditional medicine for their major primary health care needs. It has been determined that the medicinal plants Pulicaria gnaphalodes and Perovskia abrotanoides possess strong antibacterial effect. Materials and Methods: In this study, the antimycobacterial effects of P. gnaphalodes and P. abrotanoides essential oil on MTB were examined. Essential oil was prepared from P. gnaphalodes aerial parts and P. abrotanoides flower. The effects of six different concentrations (20 μg/ml, 40 μg/ml, 80 μg/ml, 160 μg/ml, 320 μg/ml, and 640 μg/ml) were examined against sensitive isolates of MTB and MTB H37Rv (ATCC 27294). Results: The results showed that P. gnaphalodes and P. abrotanoides essential oil extracts have strong inhibitory effects on MTB. This activity for P. gnaphalodes was observed from very low (4%) to good (70.9%) effect; meanwhile, this activity for P. abrotanoides was observed from very low (4%) to strong (86%) effect. Conclusion: The mean of inhibition percentage for P. gnaphalodes and P. abrotanoides in 640 μg/ml was 58.1% and 76.2%, respectively. So, P. abrotanoides plant is more effective against MTB than P. gnaphalodes. Identification of the effective fraction against MTB is a further step to be studied. PMID:27195252

  8. Spoligotyping and polymorphic GC-rich repetitive sequence fingerprinting of mycobacterium tuberculosis strains having few copies of IS6110.

    PubMed

    Yang, Z H; Ijaz, K; Bates, J H; Eisenach, K D; Cave, M D

    2000-10-01

    Several genetic loci have been utilized to genotype isolates of Mycobacterium tuberculosis. A shortcoming of the most commonly used method, IS6110 fingerprinting, is that it does not adequately discriminate between isolates having few copies of IS6110. This study was undertaken to compare pTBN12 fingerprinting of polymorphic GC-rich repetitive sequence genes and spoligotyping of the direct repeat locus as secondary typing procedures for M. tuberculosis isolates having fewer than six copies of IS6110. A total of 88 isolates (100% of the isolates with fewer than six copies of IS6110 isolated in Arkansas during 1996 and 1997) were included in this study. Among the 88 isolates, 34 different IS6110 patterns were observed, 10 of which were shared by more than 1 isolate, involving a total of 64 isolates. The 64 isolates were subdivided into 13 clusters (containing 37 isolates) and 27 unique isolates based on a combination of IS6110 and pTBN12 fingerprinting and into 11 clusters (containing 51 isolates) and 13 unique isolates based on a combination of IS6110 fingerprinting and spoligotyping. Identical spoligotypes were found among isolates having different IS6110 patterns, as well as among isolates showing different pTBN12 patterns. In contrast, all isolates that had different IS6110 patterns were found to be unique by pTBN12 typing. The clustering rate was 73, 58, and 42%, respectively, for IS6110 fingerprinting alone, IS6110 fingerprinting and spoligotyping combined, and IS6110 and pTBN12 combined fingerprinting. The data indicate that the pTBN12 method has greater discriminating power among low-copy-number isolates than does spoligotyping.

  9. Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea.

    PubMed

    Jnawali, Hum Nath; Hwang, Sung Chul; Park, Young Kil; Kim, Hyejin; Lee, Yeong Seon; Chung, Gyung Tae; Choe, Kang Hyeon; Ryoo, Sungweon

    2013-06-01

    In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

  10. In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1.

    PubMed

    Gopal, Pooja; Tasneen, Rokeya; Yee, Michelle; Lanoix, Jean-Philippe; Sarathy, Jansy; Rasic, George; Li, Liping; Dartois, Véronique; Nuermberger, Eric; Dick, Thomas

    2017-03-16

    Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

  11. Molecular Typing of Mycobacterium bovis Strains Isolated in Italy from 2000 to 2006 and Evaluation of Variable-Number Tandem Repeats for Geographically Optimized Genotyping▿

    PubMed Central

    Boniotti, M. Beatrice; Goria, Maria; Loda, Daniela; Garrone, Annalisa; Benedetto, Alessandro; Mondo, Alessandra; Tisato, Ernesto; Zanoni, Mariagrazia; Zoppi, Simona; Dondo, Alessandro; Tagliabue, Silvia; Bonora, Stefano; Zanardi, Giorgio; Pacciarini, M. Lodovica

    2009-01-01

    Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/ETR analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/ETR-A to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/ETR methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of 0.987 that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336. The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains. PMID:19144792

  12. Biodegradation of vinyl chloride, cis-dichloroethene and 1,2-dichloroethane in the alkene/alkane-oxidising Mycobacterium strain NBB4.

    PubMed

    Le, Nga B; Coleman, Nicholas V

    2011-11-01

    Mycobacterium chubuense strain NBB4 can grow on both alkanes and alkenes as carbon sources, and was hypothesised to be an effective bioremediation agent for chlorinated aliphatic pollutants. In this study, the ability of NBB4 to biodegrade vinyl chloride (VC), cis-dichloroethene (cDCE) and 1,2-dichloroethane (DCA) was investigated under pure-culture conditions and in microcosms. Ethene-grown NBB4 cells were capable of biodegrading VC and cDCE, while ethane-grown cells could biodegrade cDCE and DCA. The stoichiometry of inorganic chloride release (1 mol/mol in each case) indicated that VC was completely dechlorinated, while cDCE and DCA were only partially dechlorinated, yielding chloroacetate in the case of DCA, and unknown metabolites in the case of cDCE. The apparent maximum specific activities (k) of whole cells against ethene, cDCE, ethane and DCA were 93 ± 4.6, 89 ± 18, 39 ± 5.5, and 4.8 ± 0.9 nmol/min/mg protein, respectively, while the substrate affinities (K(S)) of whole cells with the same substrates were 2.0 ± 0.15, 46 ± 11, 11 ± 0.33 and 4.0 ± 3.2 μM, respectively. In microcosms containing contaminated aquifer sediments and groundwater, NBB4 cells removed 85-95% of the pollutants (cDCE or DCA at 2 mM) within 24 h, and the cells remained viable for >1 month. Due to its favourable kinetic parameters, and robust survival and biodegradation activities, strain NBB4 is a promising candidate for bioremediation of chlorinated aliphatic pollutants.

  13. 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

  14. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis

    PubMed Central

    Amin, Anita G.; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Araβ(1→2)Araα(1→5)] [Araβ(1→2)Araα(1→3)]Araα(1→5)Araα1→(Ara6) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from PembA(Mtb) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. PembA(Mtb) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on PembA(Mtb) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis. PMID:18174142

  15. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis.

    PubMed

    Amin, Anita G; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.

  16. Asymmetric cell division in Mycobacterium tuberculosis and its unique features.

    PubMed

    Vijay, Srinivasan; Nagaraja, Mukkayyan; Sebastian, Jees; Ajitkumar, Parthasarathi

    2014-03-01

    Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

  17. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    PubMed

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

    2015-03-01

    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.

  18. Mycobacterium avium subspecies paratuberculosis isolates from sheep and goats show reduced persistence in bovine macrophages than cattle, bison, deer and wild boar strains regardless of genotype.

    PubMed

    Abendaño, Naiara; Sevilla, Iker A; Prieto, José Miguel; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2013-05-03

    Assessment of the virulence of isolates of Mycobacterium avium subsp. paratuberculosis (Map) exhibiting distinct genotypes and isolated from different hosts may help to clarify the degree to which clinical manifestations of the disease in cattle can be attributed to bacterial or to host factors. The objective of this study was to test the ability of 10 isolates of Map representing distinct genotypes and isolated from domestic (cattle, sheep, and goat), and wildlife animal species (fallow deer, deer, wild boar, and bison) to enter and grow in bovine macrophages. The isolates were previously typed using IS1311 PCR followed by restriction endonuclease analysis into types C, S or B. Intracellular growth of the isolates in a bovine macrophage-like cell line (BoMac) and in primary bovine monocyte-derived macrophages (MDM) was evaluated by quantification of CFU numbers in the initial inoculum and inside of the host cells at 2h and 7 d p.i. using an automatic liquid culture system (Bactec MGIT 960). Individual data illustrated that growth was less variable in BoMac than in MDM cells. All the isolates from goat and sheep hosts persisted within BoMac cells in lower CFU numbers than the other tested isolates after 7 days of infection regardless of genotype. In addition, BoMac cells exhibited differential inflammatory, apoptotic and destructive responses when infected with a bovine or an ovine isolate; which correlated with the differential survival of these strains within BoMac cells. Our results indicated that the survival of the tested Map isolates within bovine macrophages is associated with the specific host from which the isolates were initially isolated.

  19. Structure of a New Glycolipid from the Mycobacterium avium-Mycobacterium intracellulare Complex

    PubMed Central

    Watanabe, Motoko; Ohta, Akihiro; Sasaki, Shun-ichi; Minnikin, David E.

    1999-01-01

    From the lipid fraction of a freeze-dried cell mass of a strain of the Mycobacterium avium-Mycobacterium intracellulare complex, a new glycolipid was isolated and was characterized as 5-mycoloyl-α-arabinofuranosyl (1→1′)-glycerol, mainly on the basis of nuclear magnetic resonance spectroscopy studies. PMID:10094713

  20. Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

    PubMed Central

    Bouklata, Nada; Supply, Philip; Jaouhari, Sanae; Charof, Reda; Seghrouchni, Fouad; Sadki, Khalid; El Achhab, Youness; Nejjari, Chakib; Filali-Maltouf, Abdelkarim

    2015-01-01

    Background Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community. Methodology During 2010–2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database. Principal Findings Spoligotyping resulted in 137 isolates in 18 clusters (2–50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as “unknown” according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6). Conclusions 24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco

  1. The PE_PGRS Proteins of Mycobacterium tuberculosis Are Ca(2+) Binding Mediators of Host-Pathogen Interaction.

    PubMed

    Yeruva, Veena C; Kulkarni, Apoorva; Khandelwal, Radhika; Sharma, Yogendra; Raghunand, Tirumalai R

    2016-08-23

    The phenomenal success of Mycobacterium tuberculosis (M.tb) as a pathogen is primarily based on its ability to modulate host immune responses. The genome of M.tb encodes multiple immunomodulatory proteins, including several members of the multigenic PE_PPE family of which the PE_PGRS proteins are a subset. Curiously, 56 of the 61 PE_PGRS proteins contain multiple copies of the glycine-rich sequence motif GGXGXD/NXUX, a nonapeptide sequence predicted to bind Ca(2+), but the functional significance of these motifs remains a mystery. Here we provide evidence via isothermal titration calorimetry, (45)Ca blotting, fluorescence, and circular dichroism spectroscopy that Ca(2+) binds to the PE_PGRS proteins, PE_PGRS33 (Rv1818c) (10 motifs) and PE_PGRS61 (Rv3653) (one motif). Ca(2+) was observed not to bind to PE_PGRS8 (Rv0742), which lacks nonapeptide motifs. Using recombinant Mycobacterium smegmatis strains expressing Rv1818c and Rv3653 and the THP-1 macrophage model of infection, we show that the two proteins mediate Ca(2+)-dependent upregulation of the anti-inflammatory cytokine IL-10, events critical to the pathogenesis of M.tb. Both Rv1818c and Rv3653 interact with TLR2 in a Ca(2+)-dependent manner, providing a novel mechanistic basis for their immunomodulatory effects. Mutations in the nonapeptide motif of Rv3653 led to compromised Ca(2+) binding, validating the functional criticality of this motif. This study demonstrates for the first time not only their Ca(2+) binding properties but also an essential role for Ca(2+) in the functioning of the M.tb PE_PGRS proteins, opening up the possibility of developing novel anti-tuberculosis therapeutics that inhibit Ca(2+)-PE_PGRS binding.

  2. Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.

    PubMed

    Mohanty, Soumitra; Dal Molin, Michael; Ganguli, Geetanjali; Padhi, Avinash; Jena, Prajna; Selchow, Petra; Sengupta, Srabasti; Meuli, Michael; Sander, Peter; Sonawane, Avinash

    2016-01-01

    Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

  3. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

    PubMed Central

    Gey van Pittius, Nico C; Gamieldien, Junaid; Hide, Winston; Brown, Gordon D; Siezen, Roland J; Beyers, Albert D

    2001-01-01

    Background The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. Results Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. Conclusions Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium. PMID:11597336

  4. Mycobacterium bovis in Panama, 2013

    PubMed Central

    Acosta, Fermín; Chernyaeva, Ekatherina; Mendoza, Libardo; Sambrano, Dilcia; Correa, Ricardo; Rotkevich, Mikhail; Tarté, Miroslava; Hernández, Humberto; Velazco, Bredio; de Escobar, Cecilia; de Waard, Jacobus H.

    2015-01-01

    Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak indicated minimal homology with strains previously circulating in Panama. M. bovis dispersion into Panama highlights the need for enhanced genotype testing to track zoonotic infections. PMID:25988479

  5. Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

    PubMed Central

    Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor

    2016-01-01

    More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB. In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis. PMID:27245412

  6. Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis.

    PubMed

    Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor; Philips, Jennifer A

    2016-08-01

    More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis.

  7. Mycobacterium massilipolynesiensis” sp. nov., a rapidly-growing mycobacterium of medical interest related to Mycobacterium phlei

    PubMed Central

    Phelippeau, M.; Asmar, S.; Osman, D. Aboubaker; Sassi, M.; Robert, C.; Michelle, C.; Musso, D.; Drancourt, M.

    2017-01-01

    In French Polynesia, respiratory tract clinical isolate M26, displayed unusual phenotype and contradictory phylogenetic affiliations, suggesting a hitherto unidentified rapidly-growing Mycobacterium species. The phenotype of strain M26 was further characterized and its genome sequenced. Strain M26 genome consists in a 5,732,017-bp circular chromosome with a G + C% of 67.54%, comprising 5,500 protein-coding genes and 52 RNA genes (including two copies of the 16 S rRNA gene). One region coding for a putative prophage was also predicted. An intriguing characteristic of strain M26’s genome is the large number of genes encoding polyketide synthases and nonribosomal peptide synthases. Phylogenomic analysis showed that strain M26’s genome is closest to the Mycobacterium phlei genome with a 76.6% average nucleotide identity. Comparative genomics of 33 Mycobacterium genomes yielded 361 genes unique to M26 strain which functional annotation revealed 84.21% of unknown function and 3.88% encoding lipid transport and metabolism; while 48.87% of genes absent in M26 strain have unknown function, 9.5% are implicated in transcription and 19% are implicated in transport and metabolism. Strain M26’s unique phenotypic and genomic characteristics indicate it is representative of a new species named “Mycobacterium massilipolynesiensis”. Looking for mycobacteria in remote areas allows for the discovery of new Mycobacterium species. PMID:28074866

  8. Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae–Mycobacterium abscessus group

    PubMed Central

    Nogueira, Christiane Lourenço; Whipps, Christopher M.; Matsumoto, Cristianne Kayoko; Chimara, Erica; Droz, Sara; Tortoli, Enrico; de Freitas, Denise; Cnockaert, Margo; Palomino, Juan Carlos; Martin, Anandi; Vandamme, Peter

    2015-01-01

    Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and, in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae–Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M. abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1–2 nt differences in rpoB and internal transcribed spacer (ITS) sequences. Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values >70 % confirmed that the five isolates belong to the same species, while values < 70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results, demonstrated that they share characteristics with M. chelonae–M. abscessus members, but constitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM 10906T ( = CCUG 66554T = LMG 28586T = INCQS 0733T). PMID:26358475

  9. Mycobacterium marseillense sp. nov., Mycobacterium timonense sp. nov. and Mycobacterium bouchedurhonense sp. nov., members of the Mycobacterium avium complex.

    PubMed

    Ben Salah, Iskandar; Cayrou, Caroline; Raoult, Didier; Drancourt, Michel

    2009-11-01

    An rpoB sequence-based evaluation of 100 Mycobacterium avium complex (MAC) clinical isolates led to the identification of five respiratory tract isolates that were potential representatives of three novel MAC species. Distinctive phenotypic features of isolates 62863 and 5356591(T) included a pseudomycelium morphology and both esterase and acid phosphatase activities. These two isolates exhibited sequence similarities of 99.8 % for the 16S rRNA gene, 86.3 and 86.1 % for 16S-23S rRNA gene internal transcribed spacer (ITS-1) sequence, 96.7 and 97.8 % for rpoB and 97.6 and 97.4 % for hsp65, respectively, with the type strain of Mycobacterium chimaera, the most closely related species. Isolates 3256799 and 5351974(T) lacked alpha-mannosidase and beta-glucosidase activities. They exhibited sequence similarities of 99.6 % for the 16S rRNA gene, 90.1 and 90.4 % for ITS-1, 97.8 % for rpoB and 98.0 and 98.1 % for hsp65, respectively, with the type strain of M. chimaera, the most closely related species. Isolate 4355387(T) lacked urease and alpha-glucosidase activities, but it exhibited valine arylamidase, cystine arylamidase and acid phosphatase activities. It had sequence similarities of 99.3 % for the 16S rRNA gene, 51.8 % for ITS-1, 97.1 % for rpoB and 97.8 % for hsp65 with the type strain of Mycobacterium colombiense, the most closely related species. A phylogenetic tree based on concatenated 16S rRNA gene, ITS-1, rpoB and hsp65 sequences showed the uniqueness of these five isolates as representatives of three novel species, with bootstrap values >/=95 % in all nodes. On the basis of these phenotypic and genetic characteristics, these five isolates are proposed as representatives of three novel MAC species: Mycobacterium marseillense sp. nov., with strain 5356591(T) (=CCUG 56325(T) =CIP 109828(T) =CSUR P30(T)) as the type strain; Mycobacterium timonense sp. nov., with strain 5351974(T) (=CCUG 56329(T) =CIP 109830(T) =CSUR P32(T)) as the type strain; and Mycobacterium

  10. Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates.

    PubMed

    Schinsky, Mark F; Morey, Roger E; Steigerwalt, Arnold G; Douglas, Michael P; Wilson, Rebecca W; Floyd, Margaret M; Butler, W Ray; Daneshvar, Maryam I; Brown-Elliott, Barbara A; Wallace, Richard J; McNeil, Michael M; Brenner, Don J; Brown, June M

    2004-09-01

    The Mycobacterium fortuitum third biovariant complex (sorbitol-negative and sorbitol-positive) contains unnamed taxa first characterized in 1991. These organisms can cause respiratory infections, a spectrum of soft tissue and skeletal infections, bacteraemia and disseminated disease. To evaluate this group of organisms, clinical reference isolates and the type strains of M. fortuitum third biovariant complex sorbitol-negative (n = 21), M. fortuitum third biovariant complex sorbitol-positive (n = 3), M. fortuitum (n = 3), Mycobacterium peregrinum (pipemidic acid-susceptible) (n = 1), Mycobacterium porcinum (n = 1), Mycobacterium senegalense (n = 2) and Mycobacterium septicum (n = 1) were characterized by using conventional phenotypic (morphological, physiological and antimicrobial susceptibilities), chemotaxonomic (HPLC and cellular fatty acids) and genotypic [RFLP of the rRNA gene (ribotyping), PCR-RFLP of a 439 bp segment of the 65 kDa hsp gene (PCR restriction analysis) and 16S rRNA gene sequence] analysis, DNA G + C content and DNA-DNA relatedness analyses. The results of these studies indicated that the strains comprised M. porcinum (n = 13), M. septicum (n = 1) and four novel closely related genetic groups within the M. fortuitum third biovariant complex: Mycobacterium boenickei sp. nov. (n = 6), Mycobacterium houstonense sp. nov. (n = 2), Mycobacterium neworleansense sp. nov. (n = 1) and Mycobacterium brisbanense sp. nov. (n = 1), with type strains ATCC 49935T (= W5998T = DSM 44677T), ATCC 49403T (= W5198T = DSM 44676T) ATCC 49404T (= W6705T = DSM 44679T) and ATCC 49938T (= W6743T = DSM 44680T), respectively.

  11. Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts

    PubMed Central

    Devasundaram, Santhi; Gopalan, Akilandeswari; Das, Sulochana D.; Raja, Alamelu

    2016-01-01

    In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms. PMID:27667981

  12. The Global Reciprocal Reprogramming between Mycobacteriophage SWU1 and Mycobacterium Reveals the Molecular Strategy of Subversion and Promotion of Phage Infection.

    PubMed

    Fan, Xiangyu; Duan, Xiangke; Tong, Yan; Huang, Qinqin; Zhou, Mingliang; Wang, Huan; Zeng, Lanying; Young, Ry F; Xie, Jianping

    2016-01-01

    Bacteriophages are the viruses of bacteria, which have contributed extensively to our understanding of life and modern biology. The phage-mediated bacterial growth inhibition represents immense untapped source for novel antimicrobials. Insights into the interaction between mycobacteriophage and Mycobacterium host will inform better utilizing of mycobacteriophage. In this study, RNA sequencing technology (RNA-seq) was used to explore the global response of Mycobacterium smegmatis mc(2)155 at an early phase of infection with mycobacteriophage SWU1, key host metabolic processes of M. smegmatis mc(2)155 shut off by SWU1, and the responsible phage proteins. The results of RNA-seq were confirmed by Real-time PCR and functional assay. 1174 genes of M. smegmatis mc(2)155 (16.9% of the entire encoding capacity) were differentially regulated by phage infection. These genes belong to six functional categories: (i) signal transduction, (ii) cell energetics, (iii) cell wall biosynthesis, (iv) DNA, RNA, and protein biosynthesis, (v) iron uptake, (vi) central metabolism. The transcription patterns of phage SWU1 were also characterized. This study provided the first global glimpse of the reciprocal reprogramming between the mycobacteriophage and Mycobacterium host.

  13. Identification of the Mycobacterium tuberculosis protein PE-PGRS62 as a novel effector that functions to block phagosome maturation and inhibit iNOS expression.

    PubMed

    Thi, Emily P; Hong, Chris Joon Ho; Sanghera, Gaganjit; Reiner, Neil E

    2013-05-01

    Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.

  14. The Global Reciprocal Reprogramming between Mycobacteriophage SWU1 and Mycobacterium Reveals the Molecular Strategy of Subversion and Promotion of Phage Infection

    PubMed Central

    Fan, Xiangyu; Duan, Xiangke; Tong, Yan; Huang, Qinqin; Zhou, Mingliang; Wang, Huan; Zeng, Lanying; Young, Ry F.; Xie, Jianping

    2016-01-01

    Bacteriophages are the viruses of bacteria, which have contributed extensively to our understanding of life and modern biology. The phage-mediated bacterial growth inhibition represents immense untapped source for novel antimicrobials. Insights into the interaction between mycobacteriophage and Mycobacterium host will inform better utilizing of mycobacteriophage. In this study, RNA sequencing technology (RNA-seq) was used to explore the global response of Mycobacterium smegmatis mc2155 at an early phase of infection with mycobacteriophage SWU1, key host metabolic processes of M. smegmatis mc2155 shut off by SWU1, and the responsible phage proteins. The results of RNA-seq were confirmed by Real-time PCR and functional assay. 1174 genes of M. smegmatis mc2155 (16.9% of the entire encoding capacity) were differentially regulated by phage infection. These genes belong to six functional categories: (i) signal transduction, (ii) cell energetics, (iii) cell wall biosynthesis, (iv) DNA, RNA, and protein biosynthesis, (v) iron uptake, (vi) central metabolism. The transcription patterns of phage SWU1 were also characterized. This study provided the first global glimpse of the reciprocal reprogramming between the mycobacteriophage and Mycobacterium host. PMID:26858712

  15. Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) to Identify environmental Strains of Mycobacterium Complex

    EPA Science Inventory

    Species within the Mycobacterium avium Complex (MAC) group are found to be both prevalent and persistent in drinking water distribution systems. The MAC is composed of two predominant species: M. avium and M. intracellulare. These species have the ability to survive drinking ...

  16. [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

  17. Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Vila, Joaquim; López, Zaira; Sabaté, Jordi; Minguillón, Cristina; Solanas, Anna M.; Grifoll, Magdalena

    2001-01-01

    Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures. PMID:11722898

  18. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  19. Genotyping of Mycobacterium avium complex organisms using multispacer sequence typing.

    PubMed

    Cayrou, Caroline; Turenne, Christine; Behr, Marcel A; Drancourt, Michel

    2010-03-01

    Mycobacterium avium complex (MAC) currently comprises eight species of environmental and animal-associated, slowly-growing mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense , Mycobacterium bouchedurhonense, Mycobacterium marseillense and Mycobacterium timonense. In humans, MAC organisms are responsible for opportunistic infections whose unique epidemiology remains poorly understood, in part due to the lack of a genotyping method applicable to all eight MAC species. In this study we developed multispacer sequence typing (MST), a sequencing-based method, for the genotyping of MAC organisms. An alignment of the genome sequence of M. avium subsp. hominissuis strain 104 and M. avium subsp. paratuberculosis strain K-10 revealed 621 intergenic spacers <1000 bp. From these, 16 spacers were selected that ranged from 300 to 800 bp and contained a number of variable bases, <50 within each of the 16 spacers. Four spacers were successfully PCR-amplified and sequenced in 11 reference strains. Combining the sequence of these four spacers in 106 MAC organisms, including 83 M. avium, 11 M. intracellulare , six M. chimaera, two M. colombiense and one each of M. arosiense, M. bouchedurhonense, M. marseillense and M. timonense, yielded a total of 45 spacer types, with an index of discrimination of 0.94. Each spacer type was specific for a species and certain spacer types were specific for subspecies of M. avium. MST is a new method for genotyping of organisms belonging to any one of the eight MAC species tested in this study.

  20. Intra- and Interpatient Variability of the hsp65 and 16S-23S Intergenic Gene Region in Mycobacterium abscessus Strains from Patients with Cystic Fibrosis

    PubMed Central

    König, Brigitte; Tammer, Ina; Sollich, Veronika; König, Wolfgang

    2005-01-01

    To establish the exact pathogenic role of Mycobacterium abscessus in cystic fibrosis (CF), molecular tests are required for accurate identification. Forty-eight M. abscessus isolates from seven patients with CF were analyzed by sequence analysis for sequence variants within the hsp65 gene and the 16S-23S intergenic sequence (ITS). We detected two different hsp65 genes and correspondingly two ITS sequevars belonging to M. abscessus type I and type II. PMID:16000490

  1. The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization

    PubMed Central

    Gupta, Manish; Nayyar, Nishtha; Chawla, Meenakshi; Sitaraman, Ramakrishnan; Bhatnagar, Rakesh; Banerjee, Nirupama

    2016-01-01

    Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5′-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the PparDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis. PMID:27379032

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

    PubMed Central

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

    2016-01-01

    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. bovis BCG. 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. PMID:26850295

  3. 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.

  4. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

    PubMed Central

    2010-01-01

    Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea). M. excelsa (Pohutukawa) was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity. PMID:20537175

  5. Occurrence and Clinical Relevance of Mycobacterium chimaera sp. nov., Germany

    PubMed Central

    Goldenberg, Oliver; Richter, Elvira; Göbel, Ulf B.; Petrich, Annette; Buchholz, Petra; Moter, Annette

    2008-01-01

    Retrospective molecular genetic analysis of 166 Mycobacterium intracellulare isolates showed that 143 (86%) strains could be assigned to Mycobacterium chimaera sp. nov. Of 97 patients from whom M. chimaera sp. nov. was isolated, only 3.3% exhibited mycobacterial lung disease, whereas all M. intracellulare isolates caused severe pulmonary infections. PMID:18760016

  6. Occurrence and clinical relevance of Mycobacterium chimaera sp. nov., Germany.

    PubMed

    Schweickert, Birgitta; Goldenberg, Oliver; Richter, Elvira; Göbel, Ulf B; Petrich, Annette; Buchholz, Petra; Moter, Annette

    2008-09-01

    Retrospective molecular genetic analysis of 166 Mycobacterium intracellulare isolates showed that 143 (86%) strains could be assigned to Mycobacterium chimaera sp. nov. Of 97 patients from whom M. chimaera sp. nov. was isolated, only 3.3% exhibited mycobacterial lung disease, whereas all M. intracellulare isolates caused severe pulmonary infections.

  7. In-vitro evaluation of Perasafe compared with 2% alkaline glutaraldehyde against Mycobacterium spp.

    PubMed

    Hernández, A; Martró, E; Matas, L; Ausina, V

    2003-05-01

    Quantitative suspension and carrier tests were used to compare the activity of Perasafe and Cidex against Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Mycobacterium fortuitum, and Mycobacterium chelonae. The interference of an organic load, and of hard water was also considered. Both agents achieved reductions exceeding 10(5)-fold within 20 and 30 min for all the strains tested. Perasafe is thus mycobactericidal and a viable alternative to Cidex for intermediate or high-level disinfection.

  8. Proposal to elevate Mycobacterium avium complex ITS sequevar MAC-Q to Mycobacterium vulneris sp. nov.

    PubMed

    van Ingen, J; Boeree, M J; Kösters, K; Wieland, A; Tortoli, E; Dekhuijzen, P N R; van Soolingen, D

    2009-09-01

    The Mycobacterium avium complex (MAC) consists of four recognized species, Mycobacterium avium, Mycobacterium colombiense, Mycobacterium intracellulare and Mycobacterium chimaera, and a variety of other strains that may be members of undescribed taxa. We report on two isolates of a scotochromogenic, slowly growing, non-tuberculous Mycobacterium species within the M. avium complex from a lymph node and an infected wound after a dogbite of separate patients in The Netherlands. The extrapulmonary infections in immunocompetent patients suggested a high level of virulence. These isolates were characterized by a unique nucleotide sequence in the 16S rRNA gene, 99% similar to Mycobacterium colombiense, and the MAC-Q 16S-23S internal transcribed spacer (ITS) sequence. Sequence analyses of the hsp65 gene revealed 97% similarity to M. avium. The rpoB gene sequence was 98% similar to M. colombiense. Phenotypically, the scotochromogenicity, positive semi-quantitative catalase and heat-stable catalase tests, negative tellurite reductase and urease tests and susceptibility to hydroxylamine and oleic acid set these isolates apart from related species. High-performance liquid chromatography analysis of cell-wall mycolic acid content revealed a unique pattern, related to that of M. avium and M. colombiense. Together, these findings supported a separate species status within the Mycobacterium avium complex. We propose elevation of scotochromogenic M. avium complex strains sharing this 16S gene and MAC-Q ITS sequence to separate species status, for which the name Mycobacterium vulneris sp. nov. is proposed. The type strain is NLA000700772T (=DSM 45247T=CIP 109859T).

  9. Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

    PubMed Central

    Shepard, C C; Youmans, A Y; Youmans, G P

    1977-01-01

    Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge. PMID:404242

  10. Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

    PubMed

    Lee, So-Young; Kim, Byoung-Jun; Kim, Hong; Won, Yu-Seop; Jeon, Che Ok; Jeong, Joseph; Lee, Seon Ho; Lim, Ji-Hun; Lee, Seung-Heon; Kim, Chang Ki; Kook, Yoon-Hoh; Kim, Bum-Joon

    2016-08-01

    Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T).

  11. Mycobacterium pyrenivorans sp. nov., a novel polycyclic-aromatic-hydrocarbon-degrading species.

    PubMed

    Derz, Kerstin; Klinner, Ulrich; Schuphan, Ingolf; Stackebrandt, Erko; Kroppenstedt, Reiner M

    2004-11-01

    The taxonomic position of a polycyclic-aromatic-hydrocarbon-degrading bacterium, strain 17A3(T), isolated from contaminated soil was determined using a combination of phenotypic and genotypic properties. The isolate showed phenotypic properties that were diagnostic for species of the genus Mycobacterium. Comparative 16S rRNA gene sequence analysis assigned 17A3(T) to the 16S rRNA gene subgroup that contains Mycobacterium aurum, Mycobacterium austroafricanum, Mycobacterium vaccae and Mycobacterium vanbaalenii, but it could clearly be distinguished from these species using a combination of physiological, chemotaxonomic markers and internal rRNA gene spacer analyses. The data showed that strain 17A3(T) (=DSM 44605(T)=NRRL B-24244(T)) merits recognition as the type strain of a novel species of the genus Mycobacterium. The name Mycobacterium pyrenivorans sp. nov. is proposed for the species because of its ability to use pyrene as a sole source of carbon and energy.

  12. 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

  13. Mycobacterium yongonense sp. nov., a slow-growing non-chromogenic species closely related to Mycobacterium intracellulare.

    PubMed

    Kim, Byoung-Jun; Math, Renukaradhya K; Jeon, Che Ok; Yu, Hee-Kyung; Park, Young-Gil; Kook, Yoon-Hoh; Kim, Bum-Joon

    2013-01-01

    A slow-growing non-chromogenic mycobacterium was isolated from a patient with pulmonary disease. Phenotypically, strain 05-1390(T) was similar to Mycobacterium intracellulare ATCC 13950(T). The 16S rRNA gene sequence (1385 bp) of strain 05-1390(T) showed a high degree of similarity to those of the M. intracellulare complex, namely Mycobacterium marseillense 5351974(T) (100 %), M. intracellulare ATCC 13950(T) (99.8 %) and Mycobacterium chimaera DSM 44623(T) (99.9 %). Phylogenetic analysis based on internal transcribed spacer 1 (ITS1) and the hsp65 gene indicated that strain 05-1390(T) was closely related to M. intracellulare ATCC 13950(T), but that it was a distinct phylogenetic entity. Of particular interest, an analysis based on the rpoB gene (701 bp) showed that it is closely related to Mycobacterium parascrofulaceum ATCC BAA-614(T) (99.4 %), a scotochromogenic strain, rather than to the M. intracellulare-related strains. Unique MALDI-TOF MS profiles also supported the taxonomic status of this strain as a distinct species. These data support the conclusion that strain 05-1390(T) represents a novel mycobacterial species, for which the name Mycobacterium yongonense sp. nov. is proposed; the type strain is 05-1390(T) ( = DSM 45126(T) = KCTC 19555(T)).

  14. Draft Genome Sequences of Two Extensively Drug-Resistant Strains of Mycobacterium tuberculosis Belonging to the Euro-American S Lineage.

    PubMed

    Malinga, Lesibana A; Abeel, Thomas; Desjardins, Christopher A; Dlamini, Talent C; Cassell, Gail; Chapman, Sinéad B; Birren, Bruce W; Earl, Ashlee M; van der Walt, Martie

    2016-03-03

    We report the whole-genome sequencing of two extensively drug-resistant tuberculosis strains belonging to the Euro-American S lineage. The RSA 114 strain showed single-nucleotide polymorphisms predicted to have drug efflux activity.

  15. Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., isolated from the pitcher plant Sarracenia purpurea.

    PubMed

    Tran, Phuong M; Dahl, John L

    2016-11-01

    Several fast- to intermediate-growing, acid-fast, scotochromogenic bacteria were isolated from Sarracenia purpurea pitcher waters in Minnesota sphagnum peat bogs. Two strains (DL734T and DL739T) were among these isolates. On the basis of 16S rRNA gene sequences, the phylogenetic positions of both strains is in the genus Mycobacterium with no obvious relation to any characterized type strains of mycobacteria. Phenotypic characterization revealed that neither strain was similar to the type strains of known species of the genus Mycobacterium in the collective properties of growth, pigmentation or fatty acid composition. Strain DL734T grew at temperatures between 28 and 32 °C, was positive for 3-day arylsulfatase production, and was negative for Tween 80 hydrolysis, urease and nitrate reduction. Strain DL739T grew at temperatures between 28 and 37 °C, and was positive for Tween 80 hydrolysis, urea, nitrate reduction and 3-day arylsulfatase production. Both strains were catalase-negative while only DL739T grew with 5 % NaCl. Fatty acid methyl ester profiles were unique for each strain. DL739T showed an ability to survive at 8 °C with little to no cellular replication and is thus considered to be psychrotolerant. Therefore, strains DL734T and DL739T represent two novel species of the genus Mycobacterium with the proposed names Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., respectively. The type strains are DL734T (=JCM 30395T=NCCB 100519T) and DL739T (=JCM 30396T=NCCB 100520T), respectively.

  16. Draft Genome Sequences of Three Mycobacterium chimaera Respiratory Isolates

    PubMed Central

    Roycroft, Emma; Raftery, Philomena; Mok, Simone; Fitzgibbon, Margaret; Rogers, Thomas R.

    2015-01-01

    Mycobacterium chimaera is an opportunistic human pathogen implicated in both pulmonary and cardiovascular infections. Here, we report the draft genome sequences of three strains isolated from human respiratory specimens. PMID:26634757

  17. Draft Genome Sequences of Three Mycobacterium chimaera Respiratory Isolates.

    PubMed

    Mac Aogáin, Micheál; Roycroft, Emma; Raftery, Philomena; Mok, Simone; Fitzgibbon, Margaret; Rogers, Thomas R

    2015-12-03

    Mycobacterium chimaera is an opportunistic human pathogen implicated in both pulmonary and cardiovascular infections. Here, we report the draft genome sequences of three strains isolated from human respiratory specimens.

  18. 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

  19. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

    PubMed Central

    Mavrici, Daniela; Marakalala, Mohlopheni J.; Holton, James M.; Prigozhin, Daniil M.; Gee, Christine L.; Zhang, Yanjia J.; Rubin, Eric J.; Alber, Tom

    2014-01-01

    Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis. PMID:24843173

  20. A standardized production pipeline for high profile targets from Mycobacterium tuberculosis

    PubMed Central

    Milewski, Morlin C.; Broger, Tobias; Kirkpatrick, Joanna; Filomena, Angela; Komadina, Dana; Schneiderhan‐Marra, Nicole; Wilmanns, Matthias

    2016-01-01

    1 Purpose Tuberculosis is still a major threat to global health. New tools and strategies to produce disease‐related proteins are quintessential for the development of novel vaccines and diagnostic markers. 2 Experimental design To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. 3 Results Twelve soluble proteins from Mtb, including one protein complex, were purified to near‐homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. 4 Conclusion and clinical relevance The established workflow comprises a simple, cost‐effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers. PMID:27400835

  1. Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis.

    PubMed

    Stec, Jozef; Vilchèze, Catherine; Lun, Shichun; Perryman, Alexander L; Wang, Xin; Freundlich, Joel S; Bishai, William; Jacobs, William R; Kozikowski, Alan P

    2014-11-01

    New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.

  2. Biological Evaluation of Potent Triclosan-Derived Inhibitors of the Enoyl-Acyl Carrier Protein Reductase InhA in Drug-sensitive and Drug-resistant Strains of Mycobacterium tuberculosis

    PubMed Central

    Vilchèze, Catherine; Lun, Shichun; Perryman, Alexander L.; Wang, Xin; Freundlich, Joel S.; Bishai, William; Jacobs, William R.

    2014-01-01

    New triclosan (TRC) analogs were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA and specific modifications to its positions 5 and 4′ afforded twenty-seven derivatives; of these compounds seven derivatives showed an improved potency in comparison to TRC. These analogs were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 3, had an MIC value of 0.6 μg/mL (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this molecule inhibited the purified InhA enzyme to the extent of 98%, and it showed an IC50 value of 90 nM. Compounds 3 and 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc24914, an Mtb strain overexpressing inhA, was resistant to the compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein. PMID:25165007

  3. Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17(+) IFNγ(-) CD4(+) T cell expansion through an IL-23 and TGF-β-dependent mechanism in patients with MDR-TB tuberculosis.

    PubMed

    Basile, J I; Kviatcovsky, D; Romero, M M; Balboa, L; Monteserin, J; Ritacco, V; Lopez, B; Sabio y García, C; García, A; Vescovo, M; Montaner, P G; Palmero, D; Del Carmen Sasiain, M; de la Barrera, S

    2017-01-01

    We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD(+) HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1β and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17(+) interferon (IFN)-γ(-) and IL-17(+) IFN-γ(+) in CD4(+) T cells from MDR-TB and PPD(+) HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4(+) T cells from PPD(+) HD whereas, together with transforming growth factor (TGF-β), it promotes IL-17(+) IFN-γ(-) expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-β secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17(+) IFN-γ(-) cells and the enhancement of latency-associated protein (LAP) expression in CD14(+) and CD4(+) T cells from MDR-TB, which suggests that the M strain promotes IL-17(+) IFN-γ(-) T cells through a strong TLR-2-dependent TGF-β production by antigen-presenting cells and CD4(+) T cells. Finally, CD4(+) T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17(+) IFN-γ(-) and lower IL-17(+) IFN-γ(+) levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.

  4. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  5. New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

    PubMed

    Nagy, I; Schoofs, G; De Schrijver, A; Vanderleyden, J; De Mot, R

    1997-07-01

    A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

  6. Polycyclic aromatic hydrocarbon-degrading species isolated from Hawaiian soils: Mycobacterium crocinum sp. nov., Mycobacterium pallens sp. nov., Mycobacterium rutilum sp. nov., Mycobacterium rufum sp. nov. and Mycobacterium aromaticivorans sp. nov.

    PubMed

    Hennessee, Christiane T; Seo, Jong-Su; Alvarez, Anne M; Li, Qing X

    2009-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants. In this study, both pristine and contaminated soils were sampled as a source of PAH-degrading organisms. Nine strains isolated from these soils were identified as rapidly growing members of the genus Mycobacterium through basic phenotypic characteristics and through sequence similarity of three genes. Because the sequence similarity of the 16S rRNA gene is relatively high among members of this genus, additional conserved genes encoding the beta subunit of RNA polymerase (rpoB) and a heat-shock protein (hsp65) were sequenced. Several analyses were completed to differentiate the strains from one another and to determine their species-level taxonomy, including fatty acid methyl ester analysis, biochemical tests and substrate-utilization profiling. A phylogenetic tree incorporating sequences for all three genes was constructed with the isolates and their close described relatives. Results for biochemical tests, substrate-utilization tests and DNA sequencing were compared with those of the phylogenetically similar organisms to establish the isolated strains as representatives of novel species with characteristics unlike those of previously described species of Mycobacterium. Finally, DNA-DNA hybridization was performed between strains and their close relatives to confirm their position within novel species. Our results demonstrated that the isolates represent five novel species, which were named Mycobacterium crocinum sp. nov. (type strain czh-42(T) =ATCC BAA-1373(T) =CIP 109262(T); reference strains czh-1A =ATCC BAA-1370 =CIP 109266 and czh-3 =ATCC BAA-1371=CIP 109267), Mycobacterium pallens sp. nov. (type strain czh-8(T) =ATCC BAA-1372(T) =CIP 109268(T)), Mycobacterium rutilum sp. nov. (type strain czh-117(T) =ATCC BAA-1375(T) =CIP 109271(T); reference strains czh-107 =ATCC BAA-1374 =CIP 109270 and czh-132 =ATCC BAA-1376 =CIP 109272), Mycobacterium rufum sp. nov. (type strain JS14(T

  7. Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666.

    PubMed

    Scheps, Daniel; Malca, Sumire Honda; Hoffmann, Helen; Nestl, Bettina M; Hauer, Bernhard

    2011-10-07

    The oxofunctionalization of saturated hydrocarbons is an important goal in basic and applied chemistry. Biocatalysts like cytochrome P450 enzymes can introduce oxygen into a wide variety of molecules in a very selective manner, which can be used for the synthesis of fine and bulk chemicals. Cytochrome P450 enzymes from the CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. Here we report the product yields resulting from C(5)-C(12) alkane and alcohol oxidation catalyzed by CYP153A enzymes from Mycobacterium marinum (CYP153A16) and Polaromonas sp. (CYP153A P. sp.). For all reactions, byproduct formation is described in detail. Following cloning and expression in Escherichia coli, the activity of the purified monooxygenases was reconstituted with putidaredoxin (CamA) and putidaredoxin reductase (CamB). Although both enzyme systems yielded primary alcohols and α,ω-alkanediols, each one displayed a different oxidation pattern towards alkanes. For CYP153A P. sp. a predominant ω-hydroxylation activity was observed, while CYP153A16 possessed the ability to catalyze both ω-hydroxylation and α,ω-dihydroxylation reactions.

  8. Beta-lactamases of Mycobacterium tuberculosis and Mycobacterium kansasii.

    PubMed

    Segura, C; Salvadó, M

    1997-09-01

    Re-emergence of infectious diseases caused by mycobacteria as well as the emergence of multiresistant strains of Mycobacterium has promoted the research on the use of beta-lactames in the treatment of such diseases. Mycobacteria produce beta-lactamases: M. tuberculosis produces a wide-spectrum beta-lactamase whose behaviour mimicks those of Gram-negative bacteria. M. kansasii produces also beta-lactamase which can be inhibited by clavulanic acid. An overview on beta-lactamases from both species is reported.

  9. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  10. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  11. Environmental effects on protection against Mycobacterium tuberculosis after immunization with Ad85A.

    PubMed

    Beverley, Peter; Ronan, Edward; Lee, Lianni; Arnold, Isabelle; Bolinger, Beatrice; Powrie, Fiona; Tchilian, Elma

    2013-02-04

    Previously we have shown that intradermal (i.d.) immunization with a recombinant adenovirus expressing antigen 85A (Ad85A) induced a strong splenic CD8T cell response in BALB/c mice but a weak lung immune response and did not protect mice against challenge with Mycobacterium tuberculosis (Mtb). After moving to a new animal house, the same i.d. immunization induced a strong lung immune response and the mice were protected against Mtb challenge. Increased numbers of antigen 85A-specific CD8 cells were present in lung tissue but were not recoverable by bronchoalveolar lavage (BAL). Mycobacterial growth was inhibited 21 days after Mtb challenge. In contrast, the effects of intranasal (i.n.) immunization did not change between the animal houses; 85A-specific T cells were recovered by BAL and were able to inhibit Mtb growth early after challenge. The effect of alterations to the environment was investigated by administering BCG or Mycobacterium abscessus in the drinking water, which induced protection against Mtb challenge, while Mycobacterium smegmatis did not. However, when Ad85A was given i.d. at the same time as BCG or M. abscessus, but not M. smegmatis, the protection induced by Ad85A was abolished. Treatment of mice with a CD25 antibody during the challenge period, abolished the suppressive effect of oral mycobacterial administration, suggesting that regulatory T cells (T regs) were involved. These results showed that exposure to environmental microorganisms can alter the protective immune response to a parenterally administered subunit vaccine, a result with important implications for the use of such vaccines in humans.

  12. Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property.

    PubMed

    Baghel, Anil S; Tandon, Rashmi; Gupta, Garima; Kumar, Ajit; Sharma, Raman K; Aggarwal, Neha; Kathuria, Abha; Saini, Neeraj K; Bose, Mridula; Prasad, Ashok K; Sharma, Sunil K; Nath, Mahendra; Parmar, Virinder S; Raj, Hanumantharao G

    2011-12-20

    The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.

  13. Multidrug-Resistant Tuberculosis in Panama Is Driven by Clonal Expansion of a Multidrug-Resistant Mycobacterium tuberculosis Strain Related to the KZN Extensively Drug-Resistant M. tuberculosis Strain from South Africa

    PubMed Central

    Lanzas, Fedora; Karakousis, Petros C.; Sacchettini, James C.

    2013-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is a significant health problem in Panama. The extent to which such cases are the result of primary or acquired resistance and the strain families involved are unknown. We performed whole-genome sequencing of a collection of 66 clinical MDR isolates, along with 31 drug-susceptible isolates, that were isolated in Panama between 2001 and 2010; 78% of the MDR isolates belong to the Latin American-Mediterranean (LAM) family. Drug resistance mutations correlated well with drug susceptibility profiles. To determine the relationships among these strains and to better understand the acquisition of resistance mutations, a phylogenetic tree was constructed based on a genome-wide single-nucleotide polymorphism analysis. The phylogenetic tree shows that the isolates are highly clustered, with a single strain (LAM9-c1) accounting for nearly one-half of the MDR isolates (29/66 isolates). The LAM9-c1 strain was most prevalent among male patients of working age and was associated with high mortality rates. Members of this cluster all share identical mutations conferring resistance to isoniazid (KatG S315T mutation), rifampin (RpoB S531L mutation), and streptomycin (rrs C517T mutation). This evidence of primary resistance supports a model in which MDR-TB in Panama is driven by clonal expansion and ongoing transmission of several strains in the LAM family, including the highly successful MDR strain LAM9-c1. The phylogenetic analysis also shows that the LAM9-c1 strain is closely related to the KwaZulu-Natal (KZN) extensively drug-resistant TB strain identified in KwaZulu-Natal, South Africa. The LAM9-c1 and KZN strains likely arose from a recent common ancestor that was transmitted between Panama and South Africa and had the capacity to tolerate an accumulation of multiple resistance mutations. PMID:23884993

  14. Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum.

    PubMed Central

    Erardi, F X; Failla, M L; Falkinham, J O

    1987-01-01

    A copper-tolerant Mycobacterium scrofulaceum strain was able to remove copper from culture medium by sulfate-dependent precipitation as copper sulfide. Such precipitation of copper sulfide was not observed in a derivative that lacks a 173-kilobase plasmid. In addition, the plasmid-carrying strain has a sulfate-independent copper resistance mechanism. PMID:3662522

  15. [A case of pulmonary multiresistant Mycobacterium bovis tuberculosis in Madagascar].

    PubMed

    Ramarokoto, H; Andrianasolo, D; Rasolonavalona, T; Ramaroson, F; Razafitsiarovana, I; Vincent, V; Ratsimba, L; Rasolofo Razanamparany, V

    2003-01-01

    We report a chronic case of pulmonary tuberculosis in a Malagasy citizen from Antsohihy (West of Madagascar), who was infected with a multi-drug resistant Mycobacterium bovis strain. This is the first case reported of the isolation of such a strain in Madagascar.

  16. Collagen degrading activity associated with Mycobacterium species

    PubMed Central

    Masso, F; Paez, A; Varela, E; d Diaz; Zenteno, E; Montano, L

    1999-01-01

    BACKGROUND—The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis.
METHODS—Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme.
RESULTS—CFPE from Mycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity.
CONCLUSIONS—Mycobacterium species possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.

 PMID:10212111

  17. Genetic Structure and Drug Susceptibility Patterns of Mycobacterium tuberculosis Complex Strains Responsible of Human Pulmonary Tuberculosis in the Major Rearing Region in Cameroon.

    PubMed

    Koro Koro, Francioli; Um Boock, Alphonse; Kaiyven, Afi Leslie; Noeske, Juergen; Gutierrez, Cristina; Kuaban, Christopher; Etoa, François-Xavier; Eyangoh, Sara Irène

    2016-01-01

    Background. Cameroon this last decade continues to present a low contribution of M. africanum and M. bovis in human tuberculosis (TB), while M. bovis was prevalent in cattle but all these pieces of information only concerned West and Center regions. Methods. We carried out the first study in Adamaoua, one of the most rearing regions of Cameroon, on the genetic structure and drug susceptibility of the MTBC strains isolated from newly diagnosed sputum smear-positive patients aged 15 years and above. For that purpose, spoligotyping, a modified 15 standard MIRU/VNTR loci typing, and the proportion method were used. Results. Four hundred and thirty-seven MTBC isolates were analyzed by spoligotyping. Of these, 423 were identified as M. tuberculosis, within the Cameroon family being dominant with 278 (65.7%) isolates; twelve (2.75%) isolates were classified as M. africanum and two as M. bovis. MIRU/VNTR typing of the most prevalent sublineage (SIT 61) suggested that this lineage is not a unique clone as thought earlier but could constitute a group of strains implicated to different pocket of TB transmission. Only M. tuberculosis sublineages were associated with antituberculosis drug resistance. Conclusion. These results showed the weak contribution of M. africanum and M. bovis to human active pulmonary tuberculosis in Cameroon even in the rearing region.

  18. Genetic Structure and Drug Susceptibility Patterns of Mycobacterium tuberculosis Complex Strains Responsible of Human Pulmonary Tuberculosis in the Major Rearing Region in Cameroon

    PubMed Central

    Um Boock, Alphonse; Noeske, Juergen; Gutierrez, Cristina; Kuaban, Christopher; Etoa, François-Xavier; Eyangoh, Sara Irène

    2016-01-01

    Background. Cameroon this last decade continues to present a low contribution of M. africanum and M. bovis in human tuberculosis (TB), while M. bovis was prevalent in cattle but all these pieces of information only concerned West and Center regions. Methods. We carried out the first study in Adamaoua, one of the most rearing regions of Cameroon, on the genetic structure and drug susceptibility of the MTBC strains isolated from newly diagnosed sputum smear-positive patients aged 15 years and above. For that purpose, spoligotyping, a modified 15 standard MIRU/VNTR loci typing, and the proportion method were used. Results. Four hundred and thirty-seven MTBC isolates were analyzed by spoligotyping. Of these, 423 were identified as M. tuberculosis, within the Cameroon family being dominant with 278 (65.7%) isolates; twelve (2.75%) isolates were classified as M. africanum and two as M. bovis. MIRU/VNTR typing of the most prevalent sublineage (SIT 61) suggested that this lineage is not a unique clone as thought earlier but could constitute a group of strains implicated to different pocket of TB transmission. Only M. tuberculosis sublineages were associated with antituberculosis drug resistance. Conclusion. These results showed the weak contribution of M. africanum and M. bovis to human active pulmonary tuberculosis in Cameroon even in the rearing region. PMID:28119925

  19. Assessing the effectiveness of low-pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro-organisms

    EPA Science Inventory

    Aims: To assess low-pressure ultraviolet light (LP-UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed t...

  20. Evaluation of MGIT 960-Based Antimicrobial Testing and Determination of Critical Concentrations of First- and Second-Line Antimicrobial Drugs with Drug-Resistant Clinical Strains of Mycobacterium tuberculosis

    PubMed Central

    Krüüner, Annika; Yates, Malcolm D.; Drobniewski, Francis A.

    2006-01-01

    The objectives of this study were to (i) compare agreement of the MGIT 960 system for first-line drugs with a methodology (the resistance ratio method [RRM]) that had been used in clinical trials, relating drug susceptibility to clinical outcome; (ii) compare the performance of the MGIT 960, RRM, and microtiter plate assay (MPA) methodologies for second-line drug testing; and (iii) define critical concentrations for ciprofloxacin and moxifloxacin for liquid-culture-based testing. The large collection of clinical isolates of Mycobacterium tuberculosis (n = 247) used included 176 (71%) multidrug-resistant isolates. The results for MGIT 960 and the RRM for rifampin and isoniazid (n = 200) were in excellent (99 to 100%) agreement for all strains. For streptomycin, 97% of the results at the critical concentration and 92% at high concentration, and for pyrazinamide 92% of results overall, were concordant, but for ethambutol, fewer than 85% (65% for the critical concentration and 84% for the high concentration) of the MGIT-based results were concordant with those for the RRM. The MGIT 960, RRM, and MPA assays (n = 133) correlated well for most second-line drugs tested. For susceptibility to ofloxacin, the MGIT 960 and MPA results were in full agreement. The amikacin and rifabutin results obtained by MGIT 960 agreed with the RRM results in 131 (99%) cases, and for capreomycin, they agreed for 129 of 133 isolates tested (97%). For prothionamide testing, only a limited number of drug-resistant isolates were available for testing and drawing definitive conclusions. We propose critical concentrations of 1.0 μg/ml and 0.125 μg/ml for ciprofloxacin and moxifloxacin, respectively, for liquid-culture-based testing. PMID:16517859

  1. Usefulness of the GenoType MTBC Assay for Differentiating Species of the Mycobacterium tuberculosis Complex in Cultures Obtained from Clinical Specimens

    PubMed Central

    Richter, Elvira; Weizenegger, Michael; Fahr, Anne-Marie; Rüsch-Gerdes, Sabine

    2004-01-01

    A novel DNA strip assay, GenoType MTBC, was evaluated for differentiation of Mycobacterium tuberculosis complex species from 77 positive liquid cultures in clinical practice. Species identification (M. tuberculosis [71 strains], Mycobacterium bovis subsp. bovis [5 strains], and Mycobacterium africanum subtype I [1 strain]) results were identical to conventional results. The sensitivity was slightly higher for this test than for the AccuProbe assay. PMID:15365028

  2. Comparison of the Pathogenicity for Mice of Mycobacterium fortuitum and Mycobacterium abscessus

    PubMed Central

    Saito, Hajime; Tasaka, Hiromichi

    1969-01-01

    The pathogenicity for mice of 12 strains of Mycobacterium abscessus was compared with that for 8 strains of M. fortuitum. Both species caused lesions in kidneys and produced “spinning disease” resulting from inner ear infections. No major differences in pathogenicity of these two species were demonstrated. Strain to strain variation was marked, especially with M. abscessus. For example, 1.6 × 106 organisms of strain 11188 of M. abscessus produced death in four of five animals within 42 days, whereas strain 380 of M. abscessus failed to produce any deaths within 42 days. In the case of M. fortuitum, the greatest mortality observed was one of five animals, yet the incidence of spinning disease and kidney disease occurred earlier postinfection than in mice infected with M. abscessus. Histologically, abscess formation by a strain of M. abscessus was greater than by a strain of M. fortuitum, but this difference cannot be interpreted as a species difference. Images PMID:5370281

  3. Draft genome sequence of a Mycobacterium avium complex isolate from a broadbill bird

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the draft genome sequences of ten Mycobacterium avium complex isolates obtained from diverse hosts. This collection includes isolates obtained from deer, pig, elephant, ruddy duck and Red-tailed hawk species. The type strain of Mycobacterium avium subspecies silvaticum (ATCC 49884) is also...

  4. The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2.

    PubMed

    George, K M; Yuan, Y; Sherman, D R; Barry, C E

    1995-11-10

    The major mycolic acid produced by Mycobacterium tuberculosis contains two cis-cyclopropanes in the meromycolate chain. The gene whose product cyclopropanates the proximal double bond was cloned by homology to a putative cyclopropane synthase identified from the Mycobacterium leprae genome sequencing project. This gene, named cma2, was sequenced and found to be 52% identical to cma1 (which cyclopropanates the distal double bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of the proximal double bond in the alpha 1 series of mycolic acids. Coexpression of both cyclopropane synthases resulted in cyclopropanation of both centers, producing a molecule structurally similar to the M. tuberculosis alpha-dicyclopropyl mycolates. Differential scanning calorimetry of purified cell walls and mycolic acids demonstrated that cyclopropanation of the proximal position raised the observed transition temperature by 3 degrees C. These results suggest that cyclopropanation contributes to the structural integrity of the cell wall complex.

  5. Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry.

    PubMed

    Rabodoarivelo, Marie Sylvianne; Aerts, Maarten; Vandamme, Peter; Palomino, Juan Carlos; Rasolofo, Voahangy; Martin, Anandi

    2016-10-27

    A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.

  6. The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    Serafini, Agnese; Pisu, Davide; Palù, Giorgio; Rodriguez, G. Marcela; Manganelli, Riccardo

    2013-01-01

    ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions. PMID:24155985

  7. Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth.

    PubMed

    Al Zayer, Maha; Stankowska, Dorota; Dziedzic, Renata; Sarva, Krishna; Madiraju, Murty V; Rajagopalan, Malini

    2011-05-01

    The genetic and biochemical aspects of the essential Mycobacteriumtuberculosis MtrAB two-component regulatory signal transduction (2CRS) system have not been extensively investigated. We show by bacterial two-hybrid assay that the response regulator (RR) MtrA and the sensor kinase MtrB interact. We further demonstrate that divalent metal ions [Mg²+, Ca²+ or both] promote MtrB kinase autophosphorylation activity, but only Mg²+ promotes phosphotransfer to MtrA. Replacement of the conserved aspartic acid residues at positions 13 and 56 with alanine (D13A), glutamine (D56E) or asparagine (D56N) prevented MtrA phosphorylation, indicating that these residues are important for phosphorylation. The MtrA(D56E) and MtrA(D13A) proteins bound to the promoter of fbpB, the gene encoding antigen 85B protein, efficiently in the absence of phosphorylation, whereas MtrA(D56N) did not. We also show that M.tuberculosismtrA merodiploids overproducing MtrA(D13A), unlike cells overproducing wild-type MtrA, grow poorly in nutrient broth and show reduced expression of fbpB. These latter findings are reminiscent of a phenotype associated with MtrA overproduction during intramacrophage growth. Our results suggest that MtrA(D13A) behaves like a constitutively active response regulator and that further characterization of mtrA merodiploid strains will provide valuable clues to the MtrAB system.

  8. In vitro susceptibilities of Mycobacterium tuberculosis to 10 antimicrobial agents.

    PubMed Central

    Byrne, S K; Crawford, C E; Geddes, G L; Black, W A

    1988-01-01

    After preliminary in vitro screening of 10 antimicrobial agents against Mycobacterium tuberculosis, the MICs of the 6 most promising agents against 27 clinical isolates were determined by agar dilution. The two quinolone compounds tested (difloxacin and A-56620) were the most active, each inhibiting 50% of the strains at concentrations of 4 micrograms/ml. M. tuberculosis strains previously shown to be resistant to isoniazid, streptomycin, rifampin, or ethambutol were as susceptible to these quinolone compounds as susceptible strains. PMID:3143305

  9. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...