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Sample records for mycobacterial interspersed repetitive-unit-variable-number

  1. Implementation of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Loci in Mycobacterium tuberculosis Molecular Epidemiology.

    PubMed

    Trovato, Alberto; Tafaj, Silva; Battaglia, Simone; Alagna, Riccardo; Bardhi, Donika; Kapisyzi, Perlat; Bala, Silvana; Haldeda, Migena; Borroni, Emanuele; Hafizi, Hasan; Cirillo, Daniela Maria

    2016-02-01

    This study shows that the addition of a consensus 4-locus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) loci to the spoligotyping-24-locus MIRU-VNTR typing strategy is a well-standardized approach that can contribute to an improvement of the true cluster definition while retaining high typeability in non-Beijing strains. PMID:26659207

  2. Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis, Variable-Number Tandem-Repeat Analysis, Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing, and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods

    PubMed Central

    Jeon, Semi; Lim, Nara; Kwon, Seungjik; Shim, Taesun; Park, Misun; Kim, Bum-Joon; Kim, Seonghan

    2014-01-01

    Objectives Mycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods. Methods Antibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed. Results All the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR. Conclusion The high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types. PMID:25180144

  3. Drug-resistant tuberculosis can be predicted by Mycobacterial interspersed repetitive unit locus

    PubMed Central

    Yu-feng, Wen; Chao, Jiang; Xian-feng, Cheng

    2015-01-01

    It is unknown whether MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeat) is associated with drug resistance of Mycobacterium tuberculosis. The purpose of this study was to explore the ability of 24 MIRU loci to predict the drug resistance of Isoniazid (INH), Rifampicin (RFP), Streptomycin (SM), Ethambutol (EMB) and Pyrazinamide (PZA). We collected the drug resistance and MIRU loci information of 109 strains of M. tuberculosis from an open database. The results of multivariate logistic regression showed that the VNTR polymorphism of MTUB04 was related to INH resistance [odds ratio (OR) = 2.82, P = 0.00], RFP resistance (OR = 1.91, P = 0.02), SM resistance (OR = 1.98, P = 0.01) and EMB resistance (OR = 1.95, P = 0.03). MIRU40 was associated with INH resistance (OR = 2.22, P = 0.00). MTUB21 was connected with INH resistance (OR = 1.63, P = 0.02) and SM resistance (OR = 1.69, P = 0.01). MIRU26 was correlated with SM resistance (OR = 1.52, P = 0.04). MIRU39 was associated with EMB resistance (OR = 4.07, P = 0.02). The prediction power of MIRU loci were 0.84, 0.70, 0.85, and 0.74 respectively for INH (predicted by MTUB04, MIRU20, and MTUB21), RFP (predicted by MTUB04), SM (predicted by MTUB21 and MIRU26) and EMB (MTUB04 and MIRU39) through ROC analysis. Our results showed that MIRU loci were related to anti-tuberculosis drug and could predict the drug resistance of tuberculosis. PMID:25759689

  4. Determination of Major Lineages of Mycobacterium tuberculosis Complex using Mycobacterial Interspersed Repetitive Units.

    PubMed

    Aminian, Minoo; Shabbeer, Amina; Bennett, Kristin P

    2009-11-01

    We present a novel Bayesian network (BN) to classify strains of Mycobacterium tuberculosis Complex (MTBC) into six major genetic lineages using mycobacterial interspersed repetitive units (MIRUs), a high-throughput biomarker. MTBC is the causative agent of tuberculosis (TB), which remains one of the leading causes of disease and morbidity world-wide. DNA fingerprinting methods such as MIRU are key components of modern TB control and tracking. The BN achieves high accuracy on four large MTBC genotype collections consisting of over 4700 distinct 12-loci MIRU genotypes. The BN captures distinct MIRU signatures associated with each lineage, explaining the excellent performance of the BN. The errors in the BN support the need for additional biomarkers such as the expanded 24-loci MIRU used in CDC genotyping labs since May 2009. The conditional independence assumption of each locus given the lineage makes the BN easily extensible to additional MIRU loci and other biomarkers. PMID:20953280

  5. Characterization of Mycobacterium caprae Isolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping‡

    PubMed Central

    Prodinger, Wolfgang M.; Brandstätter, Anita; Naumann, Ludmila; Pacciarini, Maria; Kubica, Tanja; Boschiroli, Maria Laura; Aranaz, Alicia; Nagy, György; Cvetnic, Zeljko; Ocepek, Matjaz; Skrypnyk, Artem; Erler, Wilfried; Niemann, Stefan; Pavlik, Ivo; Moser, Irmgard

    2005-01-01

    Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex. PMID:16207952

  6. Mycobacterial Interspersed Repetitive Unit Can Predict Drug Resistance of Mycobacterium tuberculosis in China

    PubMed Central

    Cheng, Xian-feng; Jiang, Chao; Zhang, Min; Xia, Dan; Chu, Li-li; Wen, Yu-feng; Zhu, Ming; Jiang, Yue-gen

    2016-01-01

    Background: Recently, Mycobacterial Interspersed Repetitive Unit (MIRU) was supposed to be associated with drug resistance in Mycobacterium tuberculosis (M. tuberculosis), but whether the association exists actually in local strains in China was still unknown. This research was conducted to explore that association and the predictability of MIRU to drug resistance of Tuberculosis (TB). Methods: The clinical isolates were collected and the susceptibility test were conducted with Lowenstein–Jensen (LJ) medium for five anti-TB drug. Based on PCR of MIRU-VNTR (Variable Number of Tandem Repeat) genotyping, we tested the number of the repeat unite of MIRU. Then, we used logistic regression to evaluate the association between 15 MIRU and drug resistance. In addition, we explored the most suitable MIRU locus of identified MIRU loci for drug resistance by multivariate logistic regression. Results: Of the 102 strains, one isolate was resistant to rifampicin and one isolate was resistant to streptomycin. Among these fifteen MIRU, there was a association between MIRU loci polymorphism and anti-tuberculosis drug resistance, ETRB (P = 0.03, OR = 0.19, 95% CI 0.05–0.81) and ETRC (P = 0.01, OR = 0.14, 95% CI 0.03–0.64) were negatively related to isoniazid resistance; MIRU20 (P = 0.05, OR = 2.87, 95% CI 1.01–8.12) was positively associated with ethambutol resistance; and QUB11a (P = 0.02, OR = 0.79, 95% CI 0.65–0.96) was a negative association factor of p-aminosalicylic acid resistance. Conclusion: Our research showed that MIRU loci may predict drug resistance of tuberculosis in China. However, the mechanism still needs further exploration. PMID:27047485

  7. Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus) in South Australia.

    PubMed

    Boardman, Wayne S J; Shephard, Lisa; Bastian, Ivan; Globan, Maria; Fyfe, Janet A M; Cousins, Debby V; Machado, Aaron; Woolford, Lucy

    2014-12-01

    This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed. PMID:25632695

  8. Evaluation of the Epidemiologic Utility of Secondary Typing Methods for Differentiation of Mycobacterium tuberculosis Isolates

    PubMed Central

    Kwara, Awewura; Schiro, Ronald; Cowan, Lauren S.; Hyslop, Newton E.; Wiser, Mark F.; Roahen Harrison, Stephanie; Kissinger, Patricia; Diem, Lois; Crawford, Jack T.

    2003-01-01

    Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64 Mycobacterium tuberculosis isolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping. PMID:12791904

  9. Monstrous Mycobacterial Lipids.

    PubMed

    Seeliger, Jessica; Moody, D Branch

    2016-02-18

    When it comes to lipid diversity, no bacterial genus approaches Mycobacterium. In this issue of Cell Chemical Biology, Burbaud et al. (2016) provide a multi-genic working model for the biosynthesis of trehalose polyphleate (TPP), one of the largest known lipids in mycobacteria. They demonstrate that this lipid is made by diverse mycobacterial species, including those of medical importance. PMID:26971870

  10. Within-Host Heterogeneity of Mycobacterium tuberculosis Infection Is Associated With Poor Early Treatment Response: A Prospective Cohort Study.

    PubMed

    Cohen, Ted; Chindelevitch, Leonid; Misra, Reshma; Kempner, Maria E; Galea, Jerome; Moodley, Prashini; Wilson, Douglas

    2016-06-01

    The clinical management of tuberculosis is a major challenge in southern Africa. The prevalence of within-host genetically heterogeneous Mycobacterium tuberculosis infection and its effect on treatment response are not well understood. We enrolled 500 patients with tuberculosis in KwaZulu-Natal and followed them through 2 months of treatment. Using mycobacterial interspersed repetitive units-variable number of tandem repeats genotyping to identify mycobacterial heterogeneity, we report the prevalence and evaluate the association of heterogeneity with treatment response. Upon initiation of treatment, 21.1% of participants harbored a heterogeneous M. tuberculosis infection; such heterogeneity was independently associated with a nearly 2-fold higher odds of persistent culture positivity after 2 months of treatment (adjusted odds ratio, 1.90; 95% confidence interval, 1.03-3.50). PMID:26768249

  11. Sternal mycobacterial infections

    PubMed Central

    Yuan, Shi-Min

    2016-01-01

    Sternal mycobacterial infections are rare. Due to the rarity, its clinical characteristics, diagnoses, and regular management strategies are still scanty. A total of 76 articles on this topic were obtained by a comprehensive literature collection. The clinical features, diagnosis, management strategies and prognosis were carefully analyzed. There were totally 159 patients including 152 (95%) cases of tuberculosis (TB) and seven (5%) cases of non-TB sternal infections. Sternal mycobacterial infections can be categorized into three types: Primary, secondary, and postoperative, according to the pathogenesis; and categorized into isolated, peristernal, and multifocal, according to the extent of the lesions. Microbiological investigation is more sensitive than medical imaging and Mantoux tuberculin skin test in the diagnosis of sternal infections. Most patients show good responses to the standard four-drug regimen and a surgical intervention was necessary in 28.3% patients. The prognoses of the patients are good with a very low mortality. A delayed diagnosis of sternal mycobacterial infections may bring about recurrent sternal infections and sustained incurability. An early diagnosis and prompt antibiotic regimens may significantly improve the patients' outcomes. PMID:27168857

  12. Nontuberculous mycobacterial osteomyelitis

    PubMed Central

    Bi, Sheng; Hu, Fei-Shu; Yu, Hai-Ying; Xu, Kai-Jin; Zheng, Bei-Wen; Ji, Zhong-Kang; Li, Jun-Jie; Deng, Mei; Hu, Hai-Yang; Sheng, Ji-Fang

    2015-01-01

    Abstract Osteomyelitis caused by nontuberculous mycobacteria (NTM) can have severe consequences and a poor prognosis. Physicians therefore need to be alert to this condition, especially in immunocompromised patients. Although the pathogenesis of NTM osteomyelitis is still unclear, studies in immunodeficient individuals have revealed close relationships between NTM osteomyelitis and defects associated with the interleukin-12–interferon-γ–tumor necrosis factor-α axis, as well as human immunodeficiency virus infection, various immunosuppressive conditions, and diabetes mellitus. Culture and species identification from tissue biopsies or surgical debridement tissue play crucial roles in diagnosing NTM osteomyelitis. Suitable imaging examinations are also important. Adequate surgical debridement and the choice of appropriate, combined antibiotics for long-term anti-mycobacterial chemotherapy, based on in vitro drug susceptibility tests, are the main therapies for these bone infections. Bacillus Calmette–Guerin vaccination might have limited prophylactic value. The use of multiple drugs and long duration of treatment mean that the therapeutic process needs to be monitored closely to detect potential side effects. Adequate duration of anti-mycobacterial chemotherapy together with regular monitoring with blood and imaging tests are key factors determining the recovery outcome in patients with NTM osteomyelitis. PMID:25915177

  13. Species-specific accumulation of interspersed sequences in genus Saccharum.

    PubMed

    Nakayama, Shigeki

    2004-12-01

    The genus Saccharum consists of two wild and four cultivated species. Novel interspersed sequences were isolated from cultivated sugar cane S. officinarum. These sequences were accumulated in all four cultivated species and their wild ancestral species S. robustum, but were not detected in the other wild species S. spontaneum and the relative Erianthus arundinaceus. The species-specific accumulation of interspersed sequences would correlate to the domestication of sugar canes. PMID:15729004

  14. Genetic dissection of mycobacterial biofilms.

    PubMed

    Ojha, Anil K; Jacobs, William R; Hatfull, Graham F

    2015-01-01

    Our understanding of the biological principles of mycobacterial tolerance to antibiotics is crucial for developing shorter anti-tuberculosis regimens. Various in vitro approaches have been developed to identify the conditions that promote mycobacterial persistence against antibiotics. In our laboratories, we have developed a detergent-free in vitro growth model, in which mycobacteria spontaneously grow at the air-medium interface as self-organized multicellular structures, called biofilms. Mycobacterial biofilms harbor a subpopulation of drug tolerant persisters at a greater frequency than their planktonic counterpart. Importantly, development of these structures is genetically programmed, and defective biofilms of isogenic mutants harbor fewer persisters. Thus, genetic analysis of mycobacterial biofilms in vitro could potentially be a powerful tool to unravel the biology of drug tolerance in mycobacteria. In this chapter we describe a method for screening biofilm-defective mutants of mycobacteria in a 96-well format, which readily yields a clonally pure mutant for further studies. PMID:25779318

  15. Molecular typing of Mycobacterium bovis isolates: A review

    PubMed Central

    Ramos, Daniela Fernandes; Tavares, Lucas; da Silva, Pedro Eduardo Almeida; Dellagostin, Odir Antônio

    2014-01-01

    Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications. PMID:25242917

  16. Highly structured genetic diversity of the Mycobacterium tuberculosis population in Djibouti.

    PubMed

    Godreuil, S; Renaud, F; Choisy, M; Depina, J J; Garnotel, E; Morillon, M; Van de Perre, P; Bañuls, A L

    2010-07-01

    Djibouti is an East African country with a high tuberculosis incidence. This study was conducted over a 2-month period in Djibouti, during which 62 consecutive patients with pulmonary tuberculosis (TB) were included. Genetic characterization of Mycobacterium tuberculosis, using mycobacterial interspersed repetitive-unit variable-number tandem-repeat typing and spoligotyping, was performed. The genetic and phylogenetic analysis revealed only three major families (Central Asian, East African Indian and T). The high diversity and linkage disequilibrium within each family suggest a long period of clonal evolution. A Bayesian approach shows that the phylogenetic structure observed in our sample of 62 isolates is very likely to be representative of the phylogenetic structure of the M. tuberculosis population in the total number of TB cases. PMID:19694762

  17. Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

    PubMed

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-08-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. PMID:24865686

  18. Molecular characterization of Mycobacterium orygis isolates from wild animals of Nepal.

    PubMed

    Thapa, Jeewan; Nakajima, Chie; Maharjan, Bhagwan; Poudell, Ajay; Suzuki, Yasuhiko

    2015-08-01

    Mycobacterium orygis, a new member of the Mycobacterium tuberculosis complex, was isolated from a captive spotted deer (Axis axis) and a blue bull (Boselaphus tragocamelus) in Nepal. Analyses by spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing, region of difference and single nucleotide polymorphism of genes gyrB, mmpL6, TbD1, PPE55 and Rv2042c confirmed the isolates as M. orygis. Moreover, analyses by spoligotyping (SIT587) as well as MIRU-VNTR showed that the isolates shared a similar pattern with many reported isolates. From previous and the present studies, it can be inferred that South Asia is one of the endemic regions for M. orygis. Further investigation including a larger sample size and different host interaction will help to understand the ecology and epidemiology of M. orygis in Nepal. PMID:26563034

  19. [Biologics and mycobacterial diseases].

    PubMed

    Tsuyuguchi, Kazunari; Matsumoto, Tomoshige

    2013-03-01

    Various biologics such as TNF-alpha inhibitor or IL-6 inhibitor are now widely used for treatment of rheumatoid arthritis. Many reports suggested that one of the major issues is high risk of developing tuberculosis (TB) associated with using these agents, which is especially important in Japan where tuberculosis still remains endemic. Another concern is the risk of development of nontuberculous mycobacterial (NTM) diseases and we have only scanty information about it. The purpose of this symposium is to elucidate the role of biologics in the development of mycobacterial diseases and to establish the strategy to control them. First, Dr. Tohma showed the epidemiologic data of TB risks associated with using biologics calculated from the clinical database on National Database of Rheumatic Diseases by iR-net in Japan. He estimated TB risks in rheumatoid arthritis (RA) patients to be about four times higher compared with general populations and to become even higher by using biologics. He also pointed out a low rate of implementation of QuantiFERON test (QFT) as screening test for TB infection. Next, Dr. Tokuda discussed the issue of NTM disease associated with using biologics. He suggested the airway disease in RA patients might play some role in the development of NTM disease, which may conversely lead to overdiagnosis of NTM disease in RA patients. He suggested that NTM disease should not be uniformly considered a contraindication to treatment with biologics, considering from the results of recent multicenter study showing relatively favorable outcome of NTM patients receiving biologics. Patients with latent tuberculosis infection (LTBI) should receive LTBI treatment before starting biologics. Dr. Kato, a chairperson of the Prevention Committee of the Japanese Society for Tuberculosis, proposed a new LTBI guideline including active implementation of LTBI treatment, introducing interferon gamma release assay, and appropriate selection of persons at high risk for

  20. [Nontuberculous mycobacterial pulmonary disease].

    PubMed

    Ringshausen, F C; Rademacher, J

    2016-02-01

    Nontuberculous mycobacteria (NTM) are a group of biologically diverse, ubiquitous and naturally multi-drug resistant bacteria with facultative pathogenicity. Recent data suggest that their clinical significance is increasing worldwide and that susceptible individuals may be at risk for infection via contaminated surfaces and aerosols. These individuals often have a predisposition for chronic respiratory diseases, e. g. bronchiectasis, chronic obstructive pulmonary disease (COPD) and cystic fibrosis and these conditions frequently share the same unspecific signs and symptoms with NTM pulmonary disease (NTM-PD). As a consequence, the diagnosis of NTM-PD, which is established based on clinical, radiological and microbiological criteria, is often delayed. Treating NTM-PD is more demanding than treating pulmonary tuberculosis as therapy is generally more tedious, toxic and expensive as well as being prone to failure. Patient and pathogen-specific factors guide the choice of an appropriate antimicrobial combination regimen, which should comply with national and international recommendations. Adverse events are common, should be anticipated and closely monitored. If infections with infrequently encountered mycobacterial species and severe or refractory disease occur, an interdisciplinary approach should be used, involving infectious disease specialists, experienced thoracic surgeons and referral to an NTM specialist center. PMID:26810111

  1. Interspersal Technique and Behavioral Momentum for Reading Word Lists

    ERIC Educational Resources Information Center

    Burns, Matthew K.; Ardoin, Scott P.; Parker, David C.; Hodgson, Jennifer; Klingbeil, David A.; Scholin, Sarah E.

    2009-01-01

    Academic tasks that include easy responses increase the probability that less preferred and/or more challenging tasks will be performed. The current study applied the process of arranging easier stimuli within reading word lists with behavioral momentum and an interspersal technique. We hypothesized that the behavioral momentum condition, which…

  2. Therapy of nontuberculous mycobacterial infections.

    PubMed

    Jogi, Reena; Tyring, Stephen K

    2004-01-01

    Mycobacterial infections are increasing in incidence worldwide, partly as a result of the increase in immunocompromised individuals. They cause a large number of cutaneous infections with a broad array of manifestations. Because of their diverse manifestations and sometimes fastidious nature, infections with mycobacteria are often misdiagnosed, leading to delay in and sometimes failure of therapy. In addition, many mycobacteria display both in vitro and in vivo drug resistance to antimicrobial agents. Early recognition of affected patients, initiation of appropriate antimicrobial therapy based on current guidelines, and tailoring of therapy after susceptibility testing is available are therefore essential to the successful treatment of mycobacterial infections. PMID:15571497

  3. The cytopathology of mycobacterial infection.

    PubMed

    Michelow, Pamela; Omar, Tanvier; Field, Andrew; Wright, Colleen

    2016-03-01

    Mycobacterial infection, tuberculosis (TB) in particular, remains one of the world's deadliest communicable diseases in adults and particularly in children, in low and middle income countries. The combination of human immunodeficiency virus (HIV) and TB is often lethal with TB accounting for 25% of deaths in the HIV population. One of the cornerstones for reducing the TB epidemic is early case detection using high quality diagnostic techniques. Cytology, especially fine needle aspiration biopsy (FNAB) is able to diagnose mycobacterial infection in a rapid and cost-effective manner without requiring surgery, thus allowing appropriate management to be quickly instituted. Confirmatory ancillary tests can effectively be performed on cytologic material. In this review, the pertinent cytomorphology of mycobacterial infection in various exfoliative and FNAB specimens is presented, in both immunocompetent and immunosuppressed patients. In the immunosuppressed, the typical cytomorphology of caseating granulomatous inflammation may not be seen but suppurative necrotic inflammation, mycobacterial spindle pseudotumour or a specimen comprised entirely of necrosis may be seen instead. This review includes discussion of currently available ancillary tests that can be performed on cytologic specimens. PMID:26800030

  4. The immunology of mycobacterial infections.

    PubMed

    Chaparas, S D

    1982-01-01

    Mycobacteria are endowed with substances that profoundly affect the immune system. Leprosy and tuberculosis exemplify broad spectra of useful and detrimental immune responses of mycobacterial infections that range from intense potentiation to severe specific adn nonspecific suppression of humoral and cellular immune elements. The cellular hypersensitivity induced by mycobacteria serves as a classical model for the analysis of specific and nonspecific immune mechanisms. Mycobacterial disease are prevalent worldwide and rank among the most important bacterial diseases. The kaleidoscope of immunologic events induced by injected mycobacteria and during infections will be reviewed from the standpoint of pathogenesis, pathology, in vitro and in vivo effects on cellular and humoral arms of the immune response, diagnosis, classification, potentiation and suppression. PMID:7042210

  5. Cutaneous mycobacterial spindle cell pseudotumour

    PubMed Central

    Tan, Geok Chin; Yap, Yen Piow; Shiran, Mohd Sidik; Sabariah, Abdul Rahman; Pathmanathan, Rajadurai

    2009-01-01

    Mycobacterial spindle cell pseudotumour (MSCP) has been reported in various sites, including skin, lymph nodes, bone marrow, lung and spleen. Cutaneous lesions are extremely rare and the differential diagnoses include various spindle cell lesions. Literature review shows that this lesion has preponderance for upper limb involvement and occurs largely in immunosuppressed individuals. We report a case of MSCP of the skin due to atypical mycobacterium and discuss the risk of misdiagnosis as a sarcoma. PMID:21686408

  6. Cutaneous mycobacterial spindle cell pseudotumour.

    PubMed

    Tan, Geok Chin; Yap, Yen Piow; Shiran, Mohd Sidik; Sabariah, Abdul Rahman; Pathmanathan, Rajadurai

    2009-01-01

    Mycobacterial spindle cell pseudotumour (MSCP) has been reported in various sites, including skin, lymph nodes, bone marrow, lung and spleen. Cutaneous lesions are extremely rare and the differential diagnoses include various spindle cell lesions. Literature review shows that this lesion has preponderance for upper limb involvement and occurs largely in immunosuppressed individuals. We report a case of MSCP of the skin due to atypical mycobacterium and discuss the risk of misdiagnosis as a sarcoma. PMID:21686408

  7. Mycobacterial truncated hemoglobins: from genes to functions.

    PubMed

    Ascenzi, Paolo; Bolognesi, Martino; Milani, Mario; Guertin, Michel; Visca, Paolo

    2007-08-15

    Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging threats for human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification mechanisms. Mycobacterial truncated hemoglobins (trHbs) have recently been implicated in scavenging of reactive nitrogen species. Individual members from each trHb family (N, O, and P) can be present in the same mycobacterial species. The distinct features of the heme active site structure combined with different ligand binding properties and in vivo expression patterns of mycobacterial trHbs suggest that these globins may accomplish diverse functions. Here, recent genomic, structural and biochemical information on mycobacterial trHbs is reviewed, with the aim of providing further insights into the role of these globins in mycobacterial physiology. PMID:17532149

  8. Tuberculosis Caused by Mycobacterium africanum, United States, 2004–2013

    PubMed Central

    Bloss, Emily; Heilig, Charles M.; Click, Eleanor S.

    2016-01-01

    Mycobacterium africanum is endemic to West Africa and causes tuberculosis (TB). We reviewed reported cases of TB in the United States during 2004–2013 that had lineage assigned by genotype (spoligotype and mycobacterial interspersed repetitive unit variable number tandem repeats). M. africanum caused 315 (0.4%) of 73,290 TB cases with lineage assigned by genotype. TB caused by M. africanum was associated more with persons from West Africa (adjusted odds ratio [aOR] 253.8, 95% CI 59.9–1,076.1) and US-born black persons (aOR 5.7, 95% CI 1.2–25.9) than with US-born white persons. TB caused by M. africanum did not show differences in clinical characteristics when compared with TB caused by M. tuberculosis. Clustered cases defined as >2 cases in a county with identical 24-locus mycobacterial interspersed repetitive unit genotypes, were less likely for M. africanum (aOR 0.1, 95% CI 0.1–0.4), which suggests that M. africanum is not commonly transmitted in the United States. PMID:26886258

  9. Short interspersed elements (SINEs) of the Geomyoidea superfamily rodents.

    PubMed

    Gogolevsky, Konstantin P; Kramerov, Dmitri A

    2006-05-24

    A new short interspersed element (SINE) was isolated from the genome of desert kangaroo rat (Dipodomys deserti) using single-primer PCR. This SINE consists of two monomers: the left monomer (IDL) resembles rodent ID element and other tRNAAla(CGC)-derived SINEs, whereas the right one (Geo) shows no similarity with known SINE sequences. PCR and hybridization analyses demonstrated that IDL-Geo SINE is restricted to the rodent superfamily Geomyoidea (families Geomyidea and Heteromyidea). Isolation and analysis of IDL-Geo from California pocket mouse (Chaetodipus californicus) and Botta's pocket gopher (Thomomys bottae) revealed some species-specific features of this SINE family. The structure and evolution of known dimeric SINEs are discussed. PMID:16517098

  10. Interspersed requests: a nonaversive procedure for reducing aggression and self-injury during instruction.

    PubMed

    Horner, R H; Day, H M; Sprague, J R; O'Brien, M; Heathfield, L T

    1991-01-01

    Interspersed requests are simple commands, with a high likelihood of being followed correctly, that are interspersed among instructional trials to increase the probability that a learner will attempt to perform new or difficult tasks without engaging in aggression or self-injurious behavior. This report presents two assessments of the effect of interspersed requests on aggression and self-injury during instruction. The participants were individuals with severe mental retardation who used aggression and self-injury to avoid difficult instructional situations. Results from both studies indicate that interspersed requests were effective at increasing the responsiveness of the learners to instructions and reducing levels of aggression and self-injury. PMID:1890047

  11. Biosynthesis of mycobacterial phosphatidylinositol mannosides.

    PubMed Central

    Morita, Yasu S; Patterson, John H; Billman-Jacobe, Helen; McConville, Malcolm J

    2004-01-01

    All mycobacterial species, including pathogenic Mycobacterium tuberculosis, synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for normal growth and viability. These glycolipids are important cell-wall and/or plasma-membrane components in their own right and can also be hyperglycosylated to form other wall components, such as lipomannan and lipoarabinomannan. We have investigated the steps involved in the biosynthesis of the major PIM species in a new M. smegmatis cell-free system. A number of apolar and polar PIM intermediates were labelled when this system was continuously labelled or pulse-chase-labelled with GDP-[3H]Man, and the glycan head groups and the acylation states of these species were determined by chemical and enzymic treatments and octyl-Sepharose chromatography respectively. These analyses showed that (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least two pathways that differ in the timing of the first acylation step, (2) early PIM intermediates containing a single mannose residue can be modified with two fatty acid residues, (3) formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicating that polyprenol phosphate-Man, rather than GDP-Man, is the donor for these reactions, (4) modification of acylated PIM4 with alpha1-2- or alpha1-6-linked mannose residues is probably the branch point in the biosyntheses of polar PIM and lipoarabinomannan respectively and (5) GDP strongly inhibits the synthesis of early PIM intermediates and increases the turnover of polyprenol phosphate-Man. These findings are incorporated into a revised pathway for mycobacterial PIM biosynthesis. PMID:14627436

  12. Personalized medicine approach in mycobacterial disease

    PubMed Central

    Mirsaeidi, Mehdi

    2014-01-01

    Mycobacterial diseases are a group of illnesses that cause a considerable number of deaths throughout the world, regardless of years of public health control efforts. Personalized medicine is a new but rapidly advancing field of healthcare. Personalized medicine in the field of mycobacteriology may be applied in the different levels of management such as prevention, diagnosis, treatment and prognosis. A genetic predisposition and a protein dysfunction study are recommended to tailor an individual approach in mycobacterial diseases. PMID:25126491

  13. An Exploratory Analysis of Task-Interspersal Procedures While Teaching Object Labels To Children with Autism

    ERIC Educational Resources Information Center

    Volkert, Valerie M.; Lerman, Dorothea C.; Trosclair, Nicole; Addison, Laura; Kodak, Tiffany

    2008-01-01

    Research has demonstrated that interspersing mastered tasks with new tasks facilitates learning under certain conditions; however, little is known about factors that influence the effectiveness of this treatment strategy. The initial purpose of the current investigation was to evaluate the effects of similar versus dissimilar interspersed tasks…

  14. CD36 deficiency attenuates experimental mycobacterial infection

    PubMed Central

    2010-01-01

    Background Members of the CD36 scavenger receptor family have been implicated as sensors of microbial products that mediate phagocytosis and inflammation in response to a broad range of pathogens. We investigated the role of CD36 in host response to mycobacterial infection. Methods Experimental Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection in Cd36+/+ and Cd36-/- mice, and in vitro co-cultivation of M. tuberculosis, BCG and M. marinum with Cd36+/+ and Cd36-/-murine macrophages. Results Using an in vivo model of BCG infection in Cd36+/+ and Cd36-/- mice, we found that mycobacterial burden in liver and spleen is reduced (83% lower peak splenic colony forming units, p < 0.001), as well as the density of granulomas, and circulating tumor necrosis factor (TNF) levels in Cd36-/- animals. Intracellular growth of all three mycobacterial species was reduced in Cd36-/- relative to wild type Cd36+/+ macrophages in vitro. This difference was not attributable to alterations in mycobacterial uptake, macrophage viability, rate of macrophage apoptosis, production of reactive oxygen and/or nitrogen species, TNF or interleukin-10. Using an in vitro model designed to recapitulate cellular events implicated in mycobacterial infection and dissemination in vivo (i.e., phagocytosis of apoptotic macrophages containing mycobacteria), we demonstrated reduced recovery of viable mycobacteria within Cd36-/- macrophages. Conclusions Together, these data indicate that CD36 deficiency confers resistance to mycobacterial infection. This observation is best explained by reduced intracellular survival of mycobacteria in the Cd36-/- macrophage and a role for CD36 in the cellular events involved in granuloma formation that promote early bacterial expansion and dissemination. PMID:20950462

  15. Targeting Mycobacterial Enzymes with Natural Products.

    PubMed

    Sieniawska, Elwira

    2015-10-22

    Tuberculosis (TB) is a recurring threat to contemporary civilization. It affects not only those within developing countries, but has also appeared again in places where it was once considered eradicated. TB co-infection in patients infected by HIV is, at the time of writing, the most common cause of death. In the field of searching for new antimycobacterial drug leads, compounds of natural origin still remain a promising source. The review is intended to gather information about natural products (metabolites of plants, fungi, bacteria, and marine sponges) that show activity against mycobacterial enzymes. Here, natural metabolites are presented as being inhibitors/activators of the mycobacterial enzymes involved in mycobacterial growth in vitro (ClpC1, ClpP, MurE ligase, mycothiol S-conjugate amidase, β-ketoacyl-ACP synthase, InhA) and in vivo, as regards the host cell (PtpB). Each enzyme is briefly described so as to generate an understanding of its role in mycobacterial growth and engender a perception of the mechanism of action of the studied natural compounds. Furthermore, after the introduction of the enzyme, its inhibitors are listed and exactly characterized. PMID:26441042

  16. Functions and importance of mycobacterial extracellular vesicles.

    PubMed

    Rodriguez, G Marcela; Prados-Rosales, Rafael

    2016-05-01

    The release of cellular factors by means of extracellular vesicles (EVs) is conserved in archaea, bacteria, and eukaryotes. EVs are released by growing bacteria as part of their interaction with their environment and, for pathogenic bacteria, constitute an important component of their interactions with the host. While EVs released by gram-negative bacteria have been extensively studied, the vesicles released by thick cell wall microorganisms like mycobacteria were recognized only recently and are less well understood. Nonetheless, studies of mycobacterial EVs have already suggested roles in pathogenesis, opening exciting new avenues of research aimed at understanding their biogenesis and potential use in antitubercular strategies. In this minireview, we discuss the discovery of mycobacterial vesicles, the current understanding of their nature, content, regulation, and possible functions, as well as their potential therapeutic applications. PMID:27020292

  17. Mycobacterial signaling through toll-like receptors

    PubMed Central

    Basu, Joyoti; Shin, Dong-Min; Jo, Eun-Kyeong

    2012-01-01

    Studies over the past decade have helped to decipher molecular networks dependent on Toll-like receptor (TLR) signaling, in mycobacteria-infected macrophages. Stimulation of TLRs by mycobacteria and their antigenic components rapidly induces intracellular signaling cascades involved in the activation of nuclear factor-κB and mitogen-activated protein kinase pathways, which play important roles in orchestrating proinflammatory responses and innate defense through generation of a variety of antimicrobial effector molecules. Recent studies have provided evidence that mycobacterial TLR-signaling cross talks with other intracellular antimicrobial innate pathways, the autophagy process and functional vitamin D receptor (VDR) signaling. In this article we describe recent advances in the recognition, responses, and regulation of mycobacterial signaling through TLRs. PMID:23189273

  18. Metabolomics: Applications and Promise in Mycobacterial Disease.

    PubMed

    Mirsaeidi, Mehdi; Banoei, Mohammad Mehdi; Winston, Brent W; Schraufnagel, Dean E

    2015-09-01

    Until recently, the study of mycobacterial diseases was trapped in culture-based technology that is more than a century old. The use of nucleic acid amplification is changing this, and powerful new technologies are on the horizon. Metabolomics, which is the study of sets of metabolites of both the bacteria and host, is being used to clarify mechanisms of disease, and can identify changes leading to better diagnosis, treatment, and prognostication of mycobacterial diseases. Metabolomic profiles are arrays of biochemical products of genes in their environment. These complex patterns are biomarkers that can allow a more complete understanding of cell function, dysfunction, and perturbation than genomics or proteomics. Metabolomics could herald sweeping advances in personalized medicine and clinical trial design, but the challenges in metabolomics are also great. Measured metabolite concentrations vary with the timing within a condition, the intrinsic biology, the instruments, and the sample preparation. Metabolism profoundly changes with age, sex, variations in gut microbial flora, and lifestyle. Validation of biomarkers is complicated by measurement accuracy, selectivity, linearity, reproducibility, robustness, and limits of detection. The statistical challenges include analysis, interpretation, and description of the vast amount of data generated. Despite these drawbacks, metabolomics provides great opportunity and the potential to understand and manage mycobacterial diseases. PMID:26196272

  19. Ubiquitous mammalian-wide interspersed repeats (MIRs) are molecular fossils from the mesozoic era.

    PubMed Central

    Jurka, J; Zietkiewicz, E; Labuda, D

    1995-01-01

    Short interspersed elements (SINEs) are ubiquitous in mammalian genomes. Remarkable variety of these repeats among placental orders indicates that most of them amplified in each lineage independently, following mammalian radiation. Here, we present an ancient family of repeats, whose sequence divergence and common occurrence among placental mammals, marsupials and monotremes indicate their amplification during the Mesozoic era. They are called MIRs for abundant Mammalian-wide Interspersed Repeats. With approximately 120,000 copies still detectable in the human genome (0.2-0.3% DNA), MIRs represent a 'fossilized' record of a major genetic event preceding the radiation of placental orders. Images PMID:7870583

  20. Ubiquitous mammalian-wide interspersed repeats (MIRs) are molecular fossils from the mesozoic era.

    PubMed

    Jurka, J; Zietkiewicz, E; Labuda, D

    1995-01-11

    Short interspersed elements (SINEs) are ubiquitous in mammalian genomes. Remarkable variety of these repeats among placental orders indicates that most of them amplified in each lineage independently, following mammalian radiation. Here, we present an ancient family of repeats, whose sequence divergence and common occurrence among placental mammals, marsupials and monotremes indicate their amplification during the Mesozoic era. They are called MIRs for abundant Mammalian-wide Interspersed Repeats. With approximately 120,000 copies still detectable in the human genome (0.2-0.3% DNA), MIRs represent a 'fossilized' record of a major genetic event preceding the radiation of placental orders. PMID:7870583

  1. 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

  2. In vivo storage of XR family interspersed RNA in Xenopus oocytes.

    PubMed

    Liu, C; Smith, L D

    1995-02-01

    Interspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNA (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. In small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentation constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentation constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis. PMID:7542141

  3. Task Interspersal and Performance of Matching Tasks by Preschoolers with Autism

    ERIC Educational Resources Information Center

    Benavides, Christian A.; Poulson, Claire L.

    2009-01-01

    The current study examined the effects of task interspersal on the performance of matching-to-sample tasks by three children with autism. A pre-baseline assessed each child's mastery level of a large body of matching stimuli. These matching tasks included matching identical and non-identical animals, numbers, letters, and shapes. Through this…

  4. Constant Time Delay and Interspersal of Known Items To Teach Sight Words to Students with Mental Retardation and Learning Disabilities.

    ERIC Educational Resources Information Center

    Knight, Melissa G.; Ross, Denise E.; Taylor, Ronald L.; Ramasamy, Rangasamy

    2003-01-01

    This study compared efficacy and efficiency of constant time delay and interspersal of known items to teach sight words to four students with mild mental retardation and learning disabilities. Results support effectiveness of constant time delay and suggest that interspersal of known items was more effective with students with learning…

  5. Genotyping of clinical Mycobacterium tuberculosis isolates based on IS6110 and MIRU-VNTR polymorphisms.

    PubMed

    Żaczek, Anna; Brzostek, Anna; Wojtasik, Arkadiusz; Dziadek, Jarosław; Sajduda, Anna

    2013-01-01

    In this study, 155 clinical Mycobacterium tuberculosis isolates were subject to genotyping with fast ligation-mediated PCR (FLiP). This typing method is a modified mixed-linker PCR, a rapid approach based on the PCR amplification of HhaI restriction fragments of genomic DNA containing the 3' end of IS6110 and resolving the amplicons by polyacrylamide gel electrophoresis. The results were compared with previous data of the more commonly used methods, 15-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and, to verify combined FLiP/MIRU-VNTR clusters, the reference IS6110 restriction fragment length polymorphism (RFLP). FLiP banding patterns were highly reproducible and polymorphic. This method differentiated 119 types among the study set compared to 108 distinct MIRU-VNTR profiles. The discriminatory power of FLiP was slightly higher than that of MIRU-VNTR analysis (Hunter-Gaston Discriminatory Index = 0.991 and 0.990, resp.). Detailed comparison of the clusters defined by each of the methods revealed, however, a more apparent difference in the discriminatory abilities that favored FLiP. Clustering of strains by using combined results of these two PCR-based methods correlated well with IS6110 RFLP-defined clusters, further confirming high discriminatory potential of FLiP typing. These results indicate that FLiP could be an attractive and valuable secondary typing technique for verification of MIRU-VNTR clusters of M. tuberculosis strains. PMID:24455734

  6. Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

    PubMed Central

    Peñuelas-Urquides, Katia; Martínez-Rodríguez, Herminia Guadalupe; Enciso-Moreno, José Antonio; Molina-Salinas, Gloria María; Silva-Ramírez, Beatriz; Padilla-Rivas, Gerardo Raymundo; Vera-Cabrera, Lucio; Torres-de-la-Cruz, Víctor Manuel; Martínez-Martínez, Yazmin Berenice; Ortega-García, Jorge Luis; Garza-Treviño, Elsa Nancy; Enciso-Moreno, Leonor; Saucedo-Cárdenas, Odila; Becerril-Montes, Pola; Said-Fernández/, Salvador

    2014-01-01

    The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB. PMID:25317710

  7. Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current genotyping procedures: a population-based study in northeast Mexico.

    PubMed

    Peñuelas-Urquides, Katia; Martínez-Rodríguez, Herminia Guadalupe; Enciso-Moreno, José Antonio; Molina-Salinas, Gloria María; Silva-Ramírez, Beatriz; Padilla-Rivas, Gerardo Raymundo; Vera-Cabrera, Lucio; Torres-de-la-Cruz, Víctor Manuel; Martínez-Martínez, Yazmin Berenice; Ortega-García, Jorge Luis; Garza-Treviño, Elsa Nancy; Enciso-Moreno, Leonor; Saucedo-Cárdenas, Odila; Becerril-Montes, Pola; Said-Fernández, Salvador

    2014-09-01

    The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman's rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB. PMID:25317710

  8. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.

    PubMed

    Thorne, N; Evans, J T; Smith, E G; Hawkey, P M; Gharbia, S; Arnold, C

    2007-10-01

    A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events. PMID:17803750

  9. A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India

    PubMed Central

    Singhal, Parul; Dixit, Pratima; Singh, Pooja; Jaiswal, Indu; Singh, Mastan; Jain, Amita

    2016-01-01

    Background & objectives: Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB. Methods: A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR). Results: Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. Pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types. Interpretation & conclusions: Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes. PMID:27241648

  10. Occurrence of diverse mutations in isoniazid- and rifampicin-resistant Mycobacterium tuberculosis isolates from autochthonous and immigrant populations of Saudi Arabia.

    PubMed

    Varghese, Bright; Shoukri, Mohammed; Memish, Ziad; Abuljadayel, Naila; Alhakeem, Raafat; Alrabiah, Fahad; Al-Hajoj, Sahal

    2014-12-01

    For the first time in Saudi Arabia, the impact of a patient's ethnic background on mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis isolates was analyzed on a nationwide sample collection. Four hundred fifteen isolates were subjected to drug susceptibility testing, mutation analysis, spoligotyping, and 24 loci-based Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat typing, respectively. Phenotypically, 41 (9.9%) isolates were resistant to RIF, 239 (57.6%) to INH, and 135 (32.5%) to both RIF and INH, respectively. Forty (9.6%), 236 (56.8%), and 133 (32%) isolates were determined as resistant to RIF, INH, and to both by molecular assay. Codon 531 (S531L) mutations (69.4%) in the rpoB gene and codon 315 (S315T) mutations (67.2%) in the katG gene were the most prominent among RIF- and INH-resistant isolates, respectively. The autochthonous population showed a predominance of rpoB codon 516 and 526 mutations, while the inhA promoter position -15 and -8 mutations were prominent among immigrants. A strain cluster ratio of 32% (30 clusters) was observed and 24 clusters displayed identical mutations. Overall, Euro-American lineages were predominant. However, Beijing (56.7%) and EAI (42.7%) were noticed with the highest cluster rate. In Saudi Arabia, the occurrence of mutations responsible for INH and RIF resistance was significantly associated with the ethnic origin of the patient. PMID:25014484

  11. Occupational Tuberculosis in Denmark through 21 Years Analysed by Nationwide Genotyping

    PubMed Central

    Andersen, Aase Bengaard; Andersen, Peter Henrik; Svensson, Erik; Jensen, Sidse Graff; Lillebaek, Troels

    2016-01-01

    Tuberculosis (TB) is a well-known occupational hazard. Based on more than two decades (1992–2012) of centralized nationwide genotyping of all Mycobacterium tuberculosis culture-positive TB patients in Denmark, we compared M. tuberculosis genotypes from all cases notified as presumed occupational (N = 130) with M. tuberculosis genotypes from all TB cases present in the country (N = 7,127). From 1992 through 2006, the IS6110 Restriction Fragment Length Polymorphism (RFLP) method was used for genotyping, whereas from 2005 to present, the 24-locus-based Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeat (MIRU-VNTR) was used. An occupational TB case was classified as clustered if the genotype was 100% identical to at least one other genotype. Subsequently, based on genotype, time period, smear positivity, geography, susceptibility pattern, and any reported epidemiological links between the occupational cases and any potential source cases, the occupational case was categorized as confirmed, likely, possible or unlikely occupationally infected. Among the 130 notified presumed occupational cases, 12 (9.2%) could be classified as confirmed and 46 (35.4%) as unlikely, accounting for nearly half of all cases (44.6%). The remaining 72 cases (55.4%) were categorized as possible. Within this group, 15 cases (11.5%) were assessed to be likely occupational. Our study shows that genotyping can serve as an important tool for disentangle occupational TB in high-income low incidence settings, but still needs to be combined with good epidemiological linkage information. PMID:27082745

  12. Genetic Diversity and Population Structure of Mycobacterium marinum: New Insights into Host and Environmental Specificities

    PubMed Central

    Broutin, Vincent; Bañuls, Anne-Laure; Aubry, Alexandra; Keck, Nicolas; Choisy, Marc; Bernardet, Jean-François; Michel, Christian; Raymond, Jean-Christophe; Libert, Cédric; Barnaud, Antoine; Stragier, Pieter; Portaels, Françoise; Terru, Dominique; Belon, Claudine; Dereure, Olivier; Gutierrez, Cristina; Boschiroli, Maria-Laura; Van De Perre, Philippe; Cambau, Emmanuelle

    2012-01-01

    Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role. PMID:22952269

  13. Genomic epidemiology of multidrug-resistant Mycobacterium tuberculosis during transcontinental spread.

    PubMed

    Coscolla, Mireia; Barry, Pennan M; Oeltmann, John E; Koshinsky, Heather; Shaw, Tambi; Cilnis, Martin; Posey, Jamie; Rose, Jordan; Weber, Terry; Fofanov, Viacheslav Y; Gagneux, Sebastien; Kato-Maeda, Midori; Metcalfe, John Z

    2015-07-15

    The transcontinental spread of multidrug-resistant (MDR) tuberculosis is poorly characterized in molecular epidemiologic studies. We used genomic sequencing to understand the establishment and dispersion of MDR Mycobacterium tuberculosis within a group of immigrants to the United States. We used a genomic epidemiology approach to study a genotypically matched (by spoligotype, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive units-variable number of tandem repeat signature) lineage 2/Beijing MDR strain implicated in an outbreak of tuberculosis among refugees in Thailand and consecutive cases within California. All 46 MDR M. tuberculosis genomes from both Thailand and California were highly related, with a median difference of 10 single-nucleotide polymorphisms (SNPs). The Wat Tham Krabok (WTK) strain is a new sequence type distinguished from all known Beijing strains by 55 SNPs and a genomic deletion (Rv1267c) associated with increased fitness. Sequence data revealed a highly prevalent MDR strain that included several closely related but distinct allelic variants within Thailand, rather than the occurrence of a single outbreak. In California, sequencing data supported multiple independent introductions of WTK with subsequent transmission and reactivation within the state, as well as a potential super spreader with a prolonged infectious period. Twenty-seven drug resistance-conferring mutations and 4 putative compensatory mutations were found within WTK strains. Genomic sequencing has substantial epidemiologic value in both low- and high-burden settings in understanding transmission chains of highly prevalent MDR strains. PMID:25601940

  14. Genetic diversity of the Mycobacterium tuberculosis Beijing family based on multiple genotyping profiles.

    PubMed

    Liu, Y; Wang, S; Lu, H; Chen, W; Wang, W

    2016-06-01

    Among the most prevalent Mycobacterium tuberculosis (Mtb) strains worldwide is the Beijing genotype, which has caused large outbreaks of tuberculosis (TB). Characteristics facilitating the dissemination of Beijing family strains remain unknown, but they are presumed to have been acquired through evolution of the lineage. To explore the genetic diversity of the Beijing family Mtb and explore the discriminatory ability of mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) loci in several regions of East Asia, a cross-sectional study was conducted with a total of 163 Beijing strains collected from registered TB patients between 1 June 2009 and 31 November 2010 in Funing County, China. The isolated strains were analysed by 15-MIRU-VNTR loci typing and compared with published MIRU-VNTR profiles of Beijing strains. Synonymous single nucleotide polymorphisms at 10 chromosomal positions were also analysed. The combination of SNP and MIRU-VNTR typing may be used to assess Mtb genotypes in areas dominated by Beijing strains. The modern subfamily in Shanghai overlapped with strains from other countries, whereas the ancient subfamily was genetically differentiated across several countries. Modern subfamilies, especially ST10, were prevalent. Qub11b and four other loci (MIRU 26, Mtub21, Qub26, Mtub04) could be used to discriminate Beijing strains. PMID:26667080

  15. Whole genome sequence analysis of multidrug-resistant Mycobacterium tuberculosis Beijing isolates from an outbreak in Thailand.

    PubMed

    Regmi, Sanjib Mani; Chaiprasert, Angkana; Kulawonganunchai, Supasak; Tongsima, Sissades; Coker, Olabisi Oluwabukola; Prammananan, Therdsak; Viratyosin, Wasna; Thaipisuttikul, Iyarit

    2015-10-01

    The Mycobacterium tuberculosis Beijing family is often associated with multidrug resistance and large outbreaks. Conventional genotyping study of a community outbreak of multidrug-resistant tuberculosis (MDR-TB) that occurred in Kanchanaburi Province, Thailand was carried out. The study revealed that the outbreak was clonal and the strain was identified as a member of Beijing family. Although, the outbreak isolates showed identical spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeats patterns, a discrepancy regarding ethambutol resistance was observed. In-depth characterization of the isolates through whole genome sequencing of the first and the last three isolates from our culture collection showed them to belong to principal genetic group 1, single nucleotide polymorphism (SNP) cluster group 2, sequence type 10. Compared with the M. tuberculosis H37Rv reference genome, 1242 SNPs were commonly found in all isolates. The genomes of these isolates were shown to be clonal and highly stable over a 5-year period and two or three unique SNPs were identified in each of the last three isolates. Genes known to be associated with drug resistance and their promoter regions, where applicable, were analyzed. The presence of low or no fitness cost mutations for drug resistance and an additional L731P SNP in the rpoB gene was observed in all isolates. These findings might account for the successful transmission of this MDR-TB strain. PMID:25903079

  16. Prospective Genotyping of Mycobacterium tuberculosis from Fresh Clinical Samples

    PubMed Central

    Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

    2014-01-01

    Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample. PMID:25313883

  17. Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia

    PubMed Central

    Fernández-Silva, J. A.; Abdulmawjood, A.; Bülte, M.

    2011-01-01

    The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme–linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis. PMID:21785685

  18. Diverse Molecular Genotypes of Mycobacterium tuberculosis Complex Isolates Circulating in the Free State, South Africa.

    PubMed

    Van der Spoel van Dijk, Anneke; Makhoahle, Pakiso M; Rigouts, Leen; Baba, Kamaldeen

    2016-01-01

    Tuberculosis is a serious public health concern especially in Africa and Asia. Studies describing strain diversity are lacking in the Free State region of South Africa. The aim of the study was to describe the diversity of Mycobacterium tuberculosis (M. tuberculosis) strain families in the Free State province of South Africa. A total of 86 M. tuberculosis isolates were genotyped using spoligotyping. A 12-locus mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTRs) typing was used to further characterize the resulting spoligotyping clusters. SITVITWEB identified 49 different patterns with allocation to six lineages including Latin-American-Mediterranean (LAM) (18 isolates), T (14 isolates), Beijing (five isolates), S (six isolates), Haarlem (one isolate), and X (five isolates), while 37 (43.0%) orphans were identified. Eight clusters included 37 isolates with identical spoligotypes (2 to 13/cluster). MIRU-VNTR typing further differentiated three spoligotyping clusters: SIT1/Beijing/MIT17, SIT33/LAM3/MIT213, and confirmed one SIT34/S/MIT311. In addition, SpolDB3/RIM assignment of the orphan strains resulted in a further 10 LAM and 13 T families. In total, LAM (28 isolates) and T (27 isolates) cause 63% of the individual cases of MTB in our study. The Free State has a highly diverse TB population with LAM being predominant. Further studies with inclusion of multidrug-resistant strains with larger sample size are warranted. PMID:27073397

  19. Diverse Molecular Genotypes of Mycobacterium tuberculosis Complex Isolates Circulating in the Free State, South Africa

    PubMed Central

    Rigouts, Leen

    2016-01-01

    Tuberculosis is a serious public health concern especially in Africa and Asia. Studies describing strain diversity are lacking in the Free State region of South Africa. The aim of the study was to describe the diversity of Mycobacterium tuberculosis (M. tuberculosis) strain families in the Free State province of South Africa. A total of 86 M. tuberculosis isolates were genotyped using spoligotyping. A 12-locus mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTRs) typing was used to further characterize the resulting spoligotyping clusters. SITVITWEB identified 49 different patterns with allocation to six lineages including Latin-American-Mediterranean (LAM) (18 isolates), T (14 isolates), Beijing (five isolates), S (six isolates), Haarlem (one isolate), and X (five isolates), while 37 (43.0%) orphans were identified. Eight clusters included 37 isolates with identical spoligotypes (2 to 13/cluster). MIRU-VNTR typing further differentiated three spoligotyping clusters: SIT1/Beijing/MIT17, SIT33/LAM3/MIT213, and confirmed one SIT34/S/MIT311. In addition, SpolDB3/RIM assignment of the orphan strains resulted in a further 10 LAM and 13 T families. In total, LAM (28 isolates) and T (27 isolates) cause 63% of the individual cases of MTB in our study. The Free State has a highly diverse TB population with LAM being predominant. Further studies with inclusion of multidrug-resistant strains with larger sample size are warranted. PMID:27073397

  20. Boromycin Kills Mycobacterial Persisters without Detectable Resistance.

    PubMed

    Moreira, Wilfried; Aziz, Dinah B; Dick, Thomas

    2016-01-01

    Boromycin is a boron-containing polyether macrolide antibiotic isolated from Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative bacteria where the outer membrane appears to block access of the molecule to the cytoplasmic membrane. Here we asked whether boromycin is active against Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer membrane. The results show that boromycin is a potent inhibitor of mycobacterial growth (MIC50 = 80 nM) with strong bactericidal activity against growing and non-growing drug tolerant persister bacilli. Exposure to boromycin resulted in a rapid loss of membrane potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein. Consistent with boromycin acting as a potassium ionophore, addition of KCl to the medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50 = 30 μM, HC50 = 40 μM) with a selectivity index of more than 300. Spontaneous resistant mutants could not be isolated suggesting a mutation frequency of less than 10(-9)/CFU. Taken together, the results suggests that targeting mycobacterial transmembrane ion gradients may be an attractive chemotherapeutic intervention level to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance. PMID:26941723

  1. Boromycin Kills Mycobacterial Persisters without Detectable Resistance

    PubMed Central

    Moreira, Wilfried; Aziz, Dinah B.; Dick, Thomas

    2016-01-01

    Boromycin is a boron-containing polyether macrolide antibiotic isolated from Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative bacteria where the outer membrane appears to block access of the molecule to the cytoplasmic membrane. Here we asked whether boromycin is active against Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer membrane. The results show that boromycin is a potent inhibitor of mycobacterial growth (MIC50 = 80 nM) with strong bactericidal activity against growing and non-growing drug tolerant persister bacilli. Exposure to boromycin resulted in a rapid loss of membrane potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein. Consistent with boromycin acting as a potassium ionophore, addition of KCl to the medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50 = 30 μM, HC50 = 40 μM) with a selectivity index of more than 300. Spontaneous resistant mutants could not be isolated suggesting a mutation frequency of less than 10-9/CFU. Taken together, the results suggests that targeting mycobacterial transmembrane ion gradients may be an attractive chemotherapeutic intervention level to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance. PMID:26941723

  2. Nontuberculous mycobacterial otomastoiditis: a case report.

    PubMed

    Tsai, Li-Tai; Wang, Ching-Yuan; Lin, Chia-Der; Tsai, Ming-Hsui

    2013-01-01

    Nontuberculous mycobacterial otomastoiditis is rare and can be easily confused with various different forms of otitis media. We describe the case of a 50-year-old woman who presented with left-sided chronic otitis media that had persisted for more than 1 year. It was not eradicated by standard antimicrobial therapy and surgical debridement. After appropriate antibiotic therapy for nontuberculous mycobacteria was added to the therapeutic regimen, the patient improved significantly and the lesion had healed by 6 months. Based on our experience with this case, we conclude that early bacterial culture and staining for acid-fast bacilli in ear drainage material or granulation tissue should be performed when standard antimicrobial therapy fails to eradicate chronic otitis media of an undetermined origin that is accompanied by granulation tissue over the external auditory canal or middle ear. Polymerase chain reaction testing is also effective for rapid diagnosis. Surgical debridement and removal of the foreign body can successfully treat nontuberculous mycobacterial otomastoiditis only when effective antimicrobial therapy is also administered. PMID:23354889

  3. Mycobacterial Lung Disease Complicating HIV Infection.

    PubMed

    Haas, Michelle K; Daley, Charles L

    2016-04-01

    Mycobacterial infections have caused enormous morbidity and mortality in people living with human immunodeficiency virus (HIV) infection. Of these, the most devastating has been tuberculosis (TB), the leading cause of death among HIV-positive persons globally. TB has killed more people living with HIV than any other infection. Diagnosis of latent TB infection (LTBI) is critical as treatment can prevent emergence of TB disease. Bacteriologic confirmation of TB disease should be sought whenever possible as well as drug susceptibility testing. When detected early, drug susceptible TB is curable. Similar to TB, nontuberculous mycobacteria (NTM) can also produce pulmonary and extrapulmonary infections including disseminated disease that can be fatal. Diagnosis through accurate identification of the pathogenic organism will greatly inform treatment. Depending on the NTM identified, treatment may not be curable. Ultimately, preventive strategies such as initiation of antiretroviral drugs and treatment of LTBI are interventions expected to have significant impacts on control of TB and NTM in the setting of HIV. This chapter will review the impact of pulmonary mycobacterial infections on HIV-positive individuals. PMID:26974300

  4. Network Analysis of Human Genes Influencing Susceptibility to Mycobacterial Infections

    PubMed Central

    Lipner, Ettie M.; Garcia, Benjamin J.; Strong, Michael

    2016-01-01

    Tuberculosis and nontuberculous mycobacterial infections constitute a high burden of pulmonary disease in humans, resulting in over 1.5 million deaths per year. Building on the premise that genetic factors influence the instance, progression, and defense of infectious disease, we undertook a systems biology approach to investigate relationships among genetic factors that may play a role in increased susceptibility or control of mycobacterial infections. We combined literature and database mining with network analysis and pathway enrichment analysis to examine genes, pathways, and networks, involved in the human response to Mycobacterium tuberculosis and nontuberculous mycobacterial infections. This approach allowed us to examine functional relationships among reported genes, and to identify novel genes and enriched pathways that may play a role in mycobacterial susceptibility or control. Our findings suggest that the primary pathways and genes influencing mycobacterial infection control involve an interplay between innate and adaptive immune proteins and pathways. Signaling pathways involved in autoimmune disease were significantly enriched as revealed in our networks. Mycobacterial disease susceptibility networks were also examined within the context of gene-chemical relationships, in order to identify putative drugs and nutrients with potential beneficial immunomodulatory or anti-mycobacterial effects. PMID:26751573

  5. PRESENCE OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN ALPACAS (LAMA PACOS) INHABITING THE CHILEAN ALTIPLANO.

    PubMed

    Salgado, Miguel; Sevilla, Iker; Rios, Carolina; Crossley, Jorge; Tejeda, Carlos; Manning, Elizabeth

    2016-03-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis. The organism causes disease in both domestically managed and wild ruminant species. South American camelids have a long, shared history with indigenous people in the Andes. Over the last few decades, increasing numbers of alpacas were exported to numerous countries outside South America. No paratuberculosis surveillance has been reported for these source herds. In this study, individual fecal samples from 85 adult alpacas were collected from six separate herds in the Chilean Altiplano. A ParaTB mycobacterial growth indicator tube (MGIT) liquid culture of each individual fecal sample, followed by real-time polymerase chain reaction (PCR) protocol was used for confirmation. DNA extracts from a subset of confirmed MAP isolates were subjected to mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Fifteen alpaca were fecal culture test-positive. Five false-positive culture samples were negative on PCR analysis for Mycobacterium avium subsp. avium (MAA), Mycobacterium bovis (M. bovis), and the 16 S rDNA gene. Three MAP isolates subset-tested belonged to the same MIRU-VNTR type, showing four repeats for TR292 (locus 1) in contrast to the three repeats typical of the MAP reference strain K10. The number of repeats found in the remaining loci was identical to that of the K10 strain. It is not known how nor when MAP was introduced into the alpaca population in the Chilean Altiplano. The most plausible hypothesis to explain the presence of MAP in these indigenous populations is transmission by contact with infected domestic small ruminant species that may on occasion share pastures or range with alpacas. Isolation of this mycobacterial pathogen from such a remote region suggests that MAP has found its way beyond the confines of intensively managed domestic agriculture premises. PMID:27010259

  6. Drug Targets in Mycobacterial Sulfur Metabolism

    PubMed Central

    Bhave, Devayani P.; Muse, Wilson B.; Carroll, Kate S.

    2011-01-01

    The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes. PMID:17970225

  7. High mycobacterial diversity in recreational lakes.

    PubMed

    Roguet, A; Therial, C; Saad, M; Boudahmane, L; Moulin, L; Lucas, F S

    2016-05-01

    Although nontuberculous mycobacteria (NTM) are natural inhabitants of freshwater ecosystems, few studies have focused on their distribution in these habitats. Thus, the knowledge about the abundance as well as the composition of NTM remains limited and patchy in these environments. In this context, a prospective study was performed to identify favourable habitats for mycobacteria in two recreational lakes. Mycobacterial density and diversity were measured using quantitative real-time PCR and the MiSeq Illumina platform. For both lakes, five compartments were investigated, i.e. water column, air-water interface, sediment, epilithon and epiphyton biofilms. Nontuberculous mycobacteria were detected in all compartments in large densities and displayed a remarkable diversity. NTM were dominated by fast-growing species. Lakes and compartments appeared to shape mycobacteria assemblage composition as well as their densities. In both lakes, some OTUs assigned to the species level were identified as related to known opportunistic pathogens. PMID:26873594

  8. Nontuberculous mycobacterial pulmonary disease mimicking lung cancer

    PubMed Central

    Hong, Su Jin; Kim, Tae Jung; Lee, Jae-Ho; Park, Jeong-Soo

    2016-01-01

    Abstract To describe the features and clinical implications of computed tomography (CT), positron emission tomography (PET), and percutaneous needle aspiration biopsy (PCNB) in pulmonary nontuberculous mycobacterial (NTM) disease manifesting as a solitary nodule, mass, or mass-like consolidation mimicking malignancy. Among a cohort of 388 patients with NTM pulmonary disease, 14 patients with clinically and radiologically suspected lung cancer were included in our study. Two chest radiologists evaluated CT features, including lesion type (nodule, mass, or mass-like consolidation), morphologic features (margin, degree of enhancement, calcification), and presence of accompanying findings suggestive of NTM pulmonary disease (bronchiectasis with clustered centrilobular nodules or upper-lobe cavitary lesions) by consensus. Diagnostic procedures for microbiologic diagnosis of NTM disease and clinical outcome were reviewed. Incidence of NTM pulmonary disease presenting as solitary nodule/mass (n = 8) or mass-like consolidation (n = 6) was 3.6% (14 of 388). Most lesions were detected incidentally during routine health check-up or evaluation of other disease (11 of 14, 79%). Lesions typically showed poor contrast-enhancement (9 of 12) and internal calcification (6 of 14). No lesions had CT features suggestive of NTM pulmonary disease. All 4 lesions for which PET/CT imaging was performed showed strong fluorodeoxyglucose uptake simulating malignant lesions (mean, 4.9; range, 3.6–7.8). PCNB revealed mycobacterial histology in 6 of 11 specimens and positive culture results were obtained for 7 of 7 specimens. NTM pulmonary disease may present as a solitary nodule, mass, or mass-like consolidation mimicking malignancy. CT features and PCNB are important to diagnose NTM disease mimicking lung cancer to avoid unnecessary surgery. PMID:27367996

  9. [Nontuberculous mycobacterial infections of the lung].

    PubMed

    Latshang, Tsogyal D; Lo Cascio, Christian M; Russi, Erich W

    2011-07-01

    Nontuberculous mycobacterium (NTM) species are mycobacterial species other than those belonging to the Mycobacterium tuberculosis complex and M. leprae. NTM are generally free-living organisms that are ubiquitous in the environment. Pulmonary disease, especially in older persons with and without underlying lung disease, is caused primarily by M. avium complex (MAC) and M. kansasii. The symptoms and signs of MAC lung disease are variable and not specific, but include cough, malaise, weakness, dyspnoea, chest discomfort and occasionally hemoptoe. Two major clinical presentations include disease in those with underlying lung disease, primarily white, middle-aged or elderly men - often alcoholics and/or smokers with underlying chronic obstructive lung disease, patients in whom MAC develops in areas of prior bronchiectasis, and patients with cystic fibrosis; and those without known underlying lung disease, including non-smoking women over age 50 who have interstitial patterns on chest radiography. M. kansasii infections are endemic in cities with infected tap water. Symptoms of the M. kansasii lung disease resemble to tuberculosis. M. abszessus is the most pathogenic rapid growing Mycobacterium which causes pulmonary infection. The American Thoracic Society and Infectious Disease Society of America's diagnostic criteria for nontuberculous mycobacterial pulmonary infections include both imaging studies consistent with pulmonary disease and recurrent isolation of mycobacteria from sputum or isolated from at least one bronchial wash in a symptomatic patient. For treatment of MAC lung disease we recommend depending on severity and susceptibility testing a three to four drug treatment with a macrolide, rifampicin and ethambutol and for M. kansasii a treatment with Isoniazid, rifampicin and ethambutol. Surgical management only plays a role in rare and special cases. Treatment should be continued until sputum cultures are consecutively negative for at least one year. PMID

  10. Purification of a mycobacterial adhesin for fibronectin.

    PubMed Central

    Ratliff, T L; McCarthy, R; Telle, W B; Brown, E J

    1993-01-01

    Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria. Images PMID:8478078

  11. Methylation Status of Alu and LINE-1 Interspersed Repetitive Sequences in Behcet's Disease Patients.

    PubMed

    Yüksel, Şahru; Kucukazman, Selma Ozbek; Karataş, Gülten Sungur; Ozturk, Mehmet Akif; Prombhul, Sasiprapa; Hirankarn, Nattiya

    2016-01-01

    Behcet's Disease (BD) is a multisystem chronic inflammatory disease. The pathology is believed to involve both genetic susceptibility and environmental factors. Hypomethylation leading to activation of interspersed repetitive sequences (IRSs) such as LINE-1 and Alu contributes to the pathologies of autoimmune diseases and cancer. Herein, the epigenetic changes of IRSs in BD were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences (COBRA-IRS). DNA from neutrophils and peripheral blood mononuclear cells (PBMCs) of BD patients with ocular involvement that were in active or inactive states and healthy controls were used to analyze LINE-1 and Alu methylation levels. For Alu sequences, significant differences were observed in the frequency of (u)C(u)C alleles between PBMCs of patients and controls (p = 0.03), and between inactive patients and controls (p = 0.03). For neutrophils, the frequency of (u)C(u)C was significantly higher between patients and controls (p = 0.006) and between inactive patients and controls (p = 0.002). The partial methylation ((u)C(m)C + (m)C(u)C) frequencies of Alu between inactive patients and control samples also differed (p = 0.02). No statistically significant differences for LINE-1 were detected. Thus, changes in the methylation level of IRS elements might contribute to the pathogenesis of BD. The role of Alu transcripts in BD should be investigated further. PMID:27123441

  12. Methylation Status of Alu and LINE-1 Interspersed Repetitive Sequences in Behcet's Disease Patients

    PubMed Central

    Yüksel, Şahru; Kucukazman, Selma Ozbek; Karataş, Gülten Sungur; Ozturk, Mehmet Akif; Prombhul, Sasiprapa; Hirankarn, Nattiya

    2016-01-01

    Behcet's Disease (BD) is a multisystem chronic inflammatory disease. The pathology is believed to involve both genetic susceptibility and environmental factors. Hypomethylation leading to activation of interspersed repetitive sequences (IRSs) such as LINE-1 and Alu contributes to the pathologies of autoimmune diseases and cancer. Herein, the epigenetic changes of IRSs in BD were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences (COBRA-IRS). DNA from neutrophils and peripheral blood mononuclear cells (PBMCs) of BD patients with ocular involvement that were in active or inactive states and healthy controls were used to analyze LINE-1 and Alu methylation levels. For Alu sequences, significant differences were observed in the frequency of uCuC alleles between PBMCs of patients and controls (p = 0.03), and between inactive patients and controls (p = 0.03). For neutrophils, the frequency of uCuC was significantly higher between patients and controls (p = 0.006) and between inactive patients and controls (p = 0.002). The partial methylation (uCmC + mCuC) frequencies of Alu between inactive patients and control samples also differed (p = 0.02). No statistically significant differences for LINE-1 were detected. Thus, changes in the methylation level of IRS elements might contribute to the pathogenesis of BD. The role of Alu transcripts in BD should be investigated further. PMID:27123441

  13. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  14. Anti-mycobacterial peptides: from human to phage.

    PubMed

    Teng, Tieshan; Liu, Jiafa; Wei, Hongping

    2015-01-01

    Mycobacterium tuberculosis is the major pathogen of tuberculosis (TB). With the growing problem of M. tuberculosis resistant to conventional antibiotics, especially multi-drug resistant tuberculosis (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB), the need for new TB drugs is now more prominent than ever. Among the promising candidates for anti-TB drugs, anti-mycobacterial peptides have a few advantages, such as low immunogenicity, selective affinity to prokaryotic negatively charged cell envelopes, and diverse modes of action. In this review, we summarize the recent progress in the anti-mycobacterial peptides, highlighting the sources, effectiveness and bactericidal mechanisms of these antimicrobial peptides. Most of the current anti-mycobacterial peptides are derived either from host immune cells, bacterial extraction, or mycobacteriophages. Besides trans-membrane pore formation, which is considered to be the common bactericidal mechanism, many of the anti-mycobacterial peptides have the second non-membrane targets within mycobacteria. Additionally, some antimicrobial peptides play critical roles in innate immunity. However, a few obstacles, such as short half-life in vivo and resistance to antimicrobial peptides, need overcoming before clinical applications. Nevertheless, the multiple functions of anti-mycobacterial peptides, especially direct killing of pathogens and immune-modulators in infectious and inflammatory conditions, indicate that they are promising candidates for future drug development. PMID:25613372

  15. Sequencing instructional tasks. A comparison of contingent and noncontingent interspersal of preferred academic tasks.

    PubMed

    Noell, George H; Whitmarsh, Ernest L; VanDerHeyden, Amanda M; Gatti, Susan L; Slider, Natalie J

    2003-04-01

    This study compared two strategies for increasing accurate responding on a low-preference academic task by interspersing presentations of a preferred academic task. Five children attending a preschool program for children with delayed language development participated in this study. Preferred and nonpreferred tasks were identified through a multiple-stimulus, free-operant preference assessment. Contingent access to a preferred academic task was associated with improved response accuracy when compared to noncontingent access to that activity for 3 students. For 1 student, noncontingent access to the preferred activity led to improved response accuracy, and 1 student's analysis suggested the importance of procedural variety. The implications of these findings for use of preference assessments to devise instructional sequences that improve student responding are discussed. PMID:12705105

  16. Characterization of the Plasmodium Interspersed Repeats (PIR) proteins of Plasmodium chabaudi indicates functional diversity.

    PubMed

    Yam, Xue Yan; Brugat, Thibaut; Siau, Anthony; Lawton, Jennifer; Wong, Daniel S; Farah, Abdirahman; Twang, Jing Shun; Gao, Xiaohong; Langhorne, Jean; Preiser, Peter R

    2016-01-01

    Plasmodium multigene families play a central role in the pathogenesis of malaria. The Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium spp. However their function(s) remains unknown. Using the rodent model of malaria, Plasmodium chabaudi, we show that individual CIR proteins have differential localizations within infected red cell (iRBC), suggesting different functional roles in a blood-stage infection. Some CIRs appear to be located on the surface of iRBC and merozoites and are therefore well placed to interact with host molecules. In line with this hypothesis, we show for the first time that a subset of recombinant CIRs bind mouse RBCs suggesting a role for CIR in rosette formation and/or invasion. Together, our results unravel differences in subcellular localization and ability to bind mouse erythrocytes between the members of the cir family, which strongly suggest different functional roles in a blood-stage infection. PMID:26996203

  17. Characterization of the Plasmodium Interspersed Repeats (PIR) proteins of Plasmodium chabaudi indicates functional diversity

    PubMed Central

    Yam, Xue Yan; Brugat, Thibaut; Siau, Anthony; Lawton, Jennifer; Wong, Daniel S.; Farah, Abdirahman; Twang, Jing Shun; Gao, Xiaohong; Langhorne, Jean; Preiser, Peter R.

    2016-01-01

    Plasmodium multigene families play a central role in the pathogenesis of malaria. The Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium spp. However their function(s) remains unknown. Using the rodent model of malaria, Plasmodium chabaudi, we show that individual CIR proteins have differential localizations within infected red cell (iRBC), suggesting different functional roles in a blood-stage infection. Some CIRs appear to be located on the surface of iRBC and merozoites and are therefore well placed to interact with host molecules. In line with this hypothesis, we show for the first time that a subset of recombinant CIRs bind mouse RBCs suggesting a role for CIR in rosette formation and/or invasion. Together, our results unravel differences in subcellular localization and ability to bind mouse erythrocytes between the members of the cir family, which strongly suggest different functional roles in a blood-stage infection. PMID:26996203

  18. Role of immunity to mycobacterial stress proteins in rheumatoid arthritis.

    PubMed Central

    McLean, L.; Winrow, V.; Blake, D.

    1990-01-01

    'Stress Proteins in Inflammation' provided a forum for the discussion of topical issues in this rapidly moving field. The mycobacterial 65 kDa stress proteins play a key role in certain animal models of inflammatory arthritis. However, the impression emerging is that the mechanism probably involves more than a simple cross-reaction between mycobacterial SP65 and either the host SP65 or a cartilage antigen, and that evidence for a primary role in human rheumatoid arthritis is lacking. A realistic role for immune responses against stress proteins might be the amplification or perpetuation of inflammation. If so, this is unlikely to be limited to arthritis. PMID:2184873

  19. Phosphorylation Modulates Catalytic Activity of Mycobacterial Sirtuins

    PubMed Central

    Yadav, Ghanshyam S.; Ravala, Sandeep K.; Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Sirtuins are NAD+-dependent deacetylases involved in the regulation of diverse cellular processes and are conserved throughout phylogeny. Here we report about in vitro transphosphorylation of the only NAD+-dependent deacetylase (mDAC) present in the genome of Mycobacterium tuberculosis by eukaryotic-type Ser/Thr kinases, particularly PknA. The phosphorylated mDAC displayed decreased deacetylase activity compared to its unphosphorylated counterpart. Mass-spectrometric study identified seven phosphosites in mDAC; however, mutational analysis highlighted major contribution of Thr-214 for phosphorylation of the protein. In concordance to this observation, variants of mDAC substituting Thr-214 with either Ala (phospho-ablated) or Glu (phosphomimic) exhibited significantly reduced deacetylase activity suggesting phosphorylation mediated control of enzymatic activity. To assess the role of phosphorylation towards functionality of mDAC, we opted for a sirtuin knock-out strain of Escherichia coli (Δdac), where interference of endogenous mycobacterial kinases could be excluded. The Δdac strain in nutrient deprived acetate medium exhibited compromised growth and complementation with mDAC reversed this phenotype. The phospho-ablated or phosphomimic variant, on the other hand, was unable to restore the functionality of mDAC indicating the role of phosphorylation per se in the process. We further over-expressed mDAC or mDAC-T214A as His-tagged protein in M. smegmatis, where endogenous eukaryotic-type Ser/Thr kinases are present. Anti-phosphothreonine antibody recognized both mDAC and mDAC-T214A proteins in western blotting. However, the extent of phosphorylation as adjudged by scanning the band intensity, was significantly low in the mutant protein (mDAC-T214A) compared to that of the wild-type (mDAC). Furthermore, expression of PknA in the mDAC complemented Δdac strain was able to phosphorylate M. tuberculosis sirtuin. The growth profile of this culture in acetate medium was

  20. Risk for Mycobacterial Disease among Patients with Rheumatoid Arthritis, Taiwan, 2001–2011

    PubMed Central

    Liao, Tsai-Ling; Lin, Ching-Heng; Shen, Gwan-Han; Chang, Chia-Li; Lin, Chin-Fu

    2015-01-01

    Increasing evidence indicates that the risk of acquiring tuberculosis (TB) and nontuberculous mycobacterial disease is elevated among patients with rheumatoid arthritis (RA). To determine the epidemiology of mycobacterial diseases among RA patients in Asia, we conducted a retrospective cohort study. We used a nationwide database to investigate the association of RA with mycobacterial diseases. The risk for development of TB or nontuberculous mycobacterial disease was 2.28-fold and 6.24-fold higher among RA patients than among the general population, respectively. Among RA patients, risk for development of mycobacterial disease was higher among those who were older, male, or both. Furthermore, among RA patients with mycobacterial infections, the risk for death was increased. Therefore, RA patients, especially those with other risk factors, should be closely monitored for development of mycobacterial disease. PMID:26196158

  1. Prevalence of Nontuberculous Mycobacterial Pulmonary Disease, Germany, 2009–2014

    PubMed Central

    Wagner, Dirk; de Roux, Andrés; Diel, Roland; Hohmann, David; Hickstein, Lennart; Welte, Tobias; Rademacher, Jessica

    2016-01-01

    We analyzed routine statutory health insurance claim data to determine prevalence of nontuberculous mycobacterial pulmonary disease in Germany. Documented prevalence rates of this nonnotifiable disease increased from 2.3 to 3.3 cases/100,000 population from 2009 to 2014. Prevalence showed a strong association with advanced age and chronic obstructive pulmonary disease. PMID:27191473

  2. Prevalence of Nontuberculous Mycobacterial Pulmonary Disease, Germany, 2009-2014.

    PubMed

    Ringshausen, Felix C; Wagner, Dirk; de Roux, Andrés; Diel, Roland; Hohmann, David; Hickstein, Lennart; Welte, Tobias; Rademacher, Jessica

    2016-06-01

    We analyzed routine statutory health insurance claim data to determine prevalence of nontuberculous mycobacterial pulmonary disease in Germany. Documented prevalence rates of this nonnotifiable disease increased from 2.3 to 3.3 cases/100,000 population from 2009 to 2014. Prevalence showed a strong association with advanced age and chronic obstructive pulmonary disease. PMID:27191473

  3. Age-dependent humoral responses of children to mycobacterial antigens.

    PubMed Central

    Fairchok, M P; Rouse, J H; Morris, S L

    1995-01-01

    In the United States, disseminated infection with environmental mycobacteria, including the Mycobacterium avium complex, is the most common opportunistic bacterial infection seen in AIDS patients. However, the source and relative degree of exposure to environmental mycobacteria during childhood are unknown. To examine the age-related exposure to mycobacteria, we obtained serum samples from 150 children ranging in age from 6 months to 18 years. Each sample was tested against both M. avium (serovar 1) sonic extracts and mycobacterial lipoarabinomannan, using an enzyme-linked immunosorbent assay (ELISA). All serum samples were also subjected to immunoblot analysis with the sonic extract antigen. These studies established that elevated ELISA values (P < 0.0001) and increased immunoblot reactivity (P < 0.0001) against mycobacterial antigens were both associated with increasing age. The seroreactivity differences were most striking when comparing the age groups of children below the age of 6 with the older age groups. Our results suggest that the development of humoral immune responses to mycobacterial antigens in children correlates with increasing age and that there may be an environmental factor predisposing to mycobacterial exposure which is related to advancing age. PMID:7583921

  4. Comparison of the Explicit Timing and Interspersal Interventions: Analysis of Problem Completion Rates, Student Preference, and Teacher Acceptability

    ERIC Educational Resources Information Center

    Rhymer, Katrina N.; Morgan, Sandra K.

    2005-01-01

    Explicit timing and interspersal interventions were investigated using a within-subjects design with 45 third-grade students. A control assignment consisted of subtraction of a two digit number from a two digit number (i.e., target problem) and served as a baseline. An explicit timing assignment consisted of similar problems as those for the…

  5. Mycobacterial infections in striped bass from Delaware Bay

    USGS Publications Warehouse

    Ottinger, C.A.; Brown, J.J.; Densmore, Christine L.; Starliper, C.E.; Blazer, V.S.; Weyers, H.S.; Beauchamp, K.A.; Rhodes, M.W.; Kator, H.; Gauthier, David T.; Vogelbein, W.K.

    2007-01-01

    Eighty striped bass Morone saxatilis were obtained from Delaware Bay using commercial gill nets set adjacent to Woodland Beach (n = 70) and Bowers Beach (n = 10) in December 2003. Fish were examined for gross lesions. Total lengths (TLs) and eviscerated weights were determined to calculate condition factors (K). Portions of spleens were aseptically harvested for bacterial culture, and portions of spleens, kidneys (anterior and posterior), livers, and gonads were obtained for histological examination. The size distribution of the striped bass was relatively homogeneous; the mean TL was about 600 mm for all samples. Mean K exceeded 0.95 in all samples and was not significantly different (P > 0.05) among samples. Significant differences in mycobacterial infection prevalence (P ??? 0.05) were observed among samples; samples obtained at Woodland Beach (WB) on December 10 (53.8%, n = 13) and December 17 (7.1%, n = 42) exhibited the most striking differences in prevalence. Mycobacterial infection intensity ranged from 1 ?? 102 to 1 ?? 107 colony-forming units per gram of spleen. Acanthocephalan infection prevalence and intensity, non-acid-fast bacterial infection prevalence, and fish sex ratio were also significantly different among the samples (P ??? 0.05). Similar to the mycobacterial infections, differences in sex ratio, acanthocephalan infection, and non-acid-fast bacterial infection were observed between the WB samples taken on December 10 and 17. However, no significant associations (P > 0.05) were observed between sex ratio or these infections and mycobacterial infection. The differences in bacterial and parasite infection prevalence and intensity and fish sex ratio in some samples indicate that these fish had a different history and that the epizootiology of mycobacterial infection in striped bass from Delaware Bay may be relatively complex. ?? Copyright by the American Fisheries Society 2007.

  6. Inhaled Amikacin for Treatment of Refractory Pulmonary Nontuberculous Mycobacterial Disease

    PubMed Central

    Shaw, Pamela A.; Glaser, Tanya S.; Bhattacharyya, Darshana; Fleshner, Michelle; Brewer, Carmen C.; Zalewski, Christopher K.; Folio, Les R.; Siegelman, Jenifer R.; Shallom, Shamira; Park, In Kwon; Sampaio, Elizabeth P.; Zelazny, Adrian M.; Holland, Steven M.; Prevots, D. Rebecca

    2014-01-01

    Rationale: Treatment of pulmonary nontuberculous mycobacteria, especially Mycobacterium abscessus, requires prolonged, multidrug regimens with high toxicity and suboptimal efficacy. Options for refractory disease are limited. Objectives: We reviewed the efficacy and toxicity of inhaled amikacin in patients with treatment-refractory nontuberculous mycobacterial lung disease. Methods: Records were queried to identify patients who had inhaled amikacin added to failing regimens. Lower airway microbiology, symptoms, and computed tomography scan changes were assessed together with reported toxicity. Measurements and Main Results: The majority (80%) of the 20 patients who met entry criteria were women; all had bronchiectasis, two had cystic fibrosis and one had primary ciliary dyskinesia. At initiation of inhaled amikacin, 15 were culture positive for M. abscessus and 5 for Mycobacterium avium complex and had received a median (range) of 60 (6, 190) months of mycobacterial treatment. Patients were followed for a median of 19 (1, 50) months. Eight (40%) patients had at least one negative culture and 5 (25%) had persistently negative cultures. A decrease in smear quantity was noted in 9 of 20 (45%) and in mycobacterial culture growth for 10 of 19 (53%). Symptom scores improved in nine (45%), were unchanged in seven (35%), and worsened in four (20%). Improvement on computed tomography scans was noted in 6 (30%), unchanged in 3 (15%), and worsened in 11 (55%). Seven (35%) stopped amikacin due to: ototoxicity in two (10%), hemoptysis in two (10%), and nephrotoxicity, persistent dysphonia, and vertigo in one each. Conclusions: In some patients with treatment-refractory pulmonary nontuberculous mycobacterial disease, the addition of inhaled amikacin was associated with microbiologic and/or symptomatic improvement; however, toxicity was common. Prospective evaluation of inhaled amikacin for mycobacterial disease is warranted. PMID:24460437

  7. The Effect of an Interspersed Refuge on Aphis glycines (Hemiptera: Aphididae), Their Natural Enemies, and Biological Control

    PubMed Central

    O’Neal, M. E.

    2016-01-01

    Soybean production in the north central United States has relied heavily on the use of foliar and seed applied insecticides to manage Aphis glycines (Hemiptera: Aphididae). An additional management strategy is the use soybean cultivars containing A. glycines resistance genes (Rag). Previous research has demonstrated that Rag cultivars are capable of preventing yield loss equivalent to the use of foliar and seed-applied insecticides. However, the presence of virulent biotypes in North America has raised concern for the durability of Rag genes. A resistance management program that includes a refuge for avirulent biotypes could limit the frequency at which virulent biotypes increase within North America. To what extent such a refuge reduces the effectiveness of aphid-resistant soybean is not clear. We conducted an experiment to determine whether a susceptible refuge mixed into resistant soybean (i.e., interspersed refuge or refuge-in-a-bag) affects the seasonal exposure of aphids, their natural enemies, biological control, and yield protection provided by aphid resistance. We compared three ratios of interspersed refuges (resistant: susceptible; 95:5, 90:10, 75:25) to plots grown with 100% susceptible or resistant soybean. We determined that an interspersed refuge of at least 25% susceptible seed would be necessary to effectively produce avirulent individuals. Interspersed refuges had negligible effects on yield and the natural enemy community. However, there was evidence that they increased the amount of biological control that occurred within a plot. We discuss the compatibility of interspersed refuges for A. glycines management and whether resistance management can prolong the durability of Rag genes. PMID:26476557

  8. The Effect of an Interspersed Refuge on Aphis glycines (Hemiptera: Aphididae), Their Natural Enemies, and Biological Control.

    PubMed

    Varenhorst, A J; O'Neal, M E

    2016-02-01

    Soybean production in the north central United States has relied heavily on the use of foliar and seed applied insecticides to manage Aphis glycines (Hemiptera: Aphididae). An additional management strategy is the use soybean cultivars containing A. glycines resistance genes (Rag). Previous research has demonstrated that Rag cultivars are capable of preventing yield loss equivalent to the use of foliar and seed-applied insecticides.However, the presence of virulent biotypes in North America has raised concern for the durability of Rag genes. A resistance management program that includes a refuge for avirulent biotypes could limit the frequency at which virulent biotypes increase within North America. To what extent such a refuge reduces the effectiveness of aphid-resistant soybean is not clear. We conducted an experiment to determine whether a susceptible refuge mixed into resistant soybean (i.e., interspersed refuge or refuge-in-a-bag) affects the seasonal exposure of aphids, their natural enemies, biological control, and yield protection provided by aphid resistance. We compared three ratios of interspersed refuges (resistant: susceptible; 95:5, 90:10, 75:25) to plots grown with 100%susceptible or resistant soybean. We determined that an interspersed refuge of at least 25% susceptible seed would be necessary to effectively produce avirulent individuals. Interspersed refuges had negligible effects onyield and the natural enemy community. However, there was evidence that they increased the amount of biological control that occurred within a plot. We discuss the compatibility of interspersed refuges for A. glycines management and whether resistance management can prolong the durability of Rag genes. PMID:26476557

  9. Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution

    SciTech Connect

    Kass, D.H.; Batzer, M.A.; Deininger, P.L. |

    1995-01-01

    The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolutions. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.

  10. Monitoring Long Interspersed Nuclear Element 1 Expression During Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Bodak, Maxime; Ciaudo, Constance

    2016-01-01

    Long Interspersed Elements-1 (LINE-1 or L1) are a class of transposable elements which account for almost 19 % of the mouse genome. This represents around 600,000 L1 fragments, among which it is estimated that 3000 intact copies still remain capable to retrotranspose and to generate deleterious mutation by insertion into genomic coding region. In differentiated cells, full length L1 are transcriptionally repressed by DNA methylation. However at the blastocyst stage, L1 elements are subject to a demethylation wave and able to be expressed and to be inserted into new genomic locations. Mouse Embryonic Stem Cells (mESCs) are pluripotent stem cells derived from the inner cell mass of blastocysts. Mouse ESCs can be maintained undifferentiated under controlled culture conditions or induced into the three primary germ layers, therefore they represent a suitable model to follow mechanisms involved in L1 repression during the process of differentiation of mESCs. This protocol presents how to maintain culture of undifferentiated mESCs, induce their differentiation, and monitor L1 expression at the transcriptional and translational levels. L1 transcriptional levels are assessed by real-time qRT-PCR performed on total RNA extracts using specific L1 primers and translation levels are measured by Western blot analysis of L1 protein ORF1 using a specific L1 antibody. PMID:26895058

  11. Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor

    PubMed Central

    Chang, Hui-Bo; Zou, Ji-Zhen; He, Cai; Zeng, Rui; Li, Yuan-Yuan; Ma, Fei-Fei; Liu, Zhuo; Ye, Hui; Wu, Jian-Xin

    2015-01-01

    Background: DNA hypomethylation of long interspersed nuclear elements-1 (LINEs-1) occurs during carcinogenesis, whereas information addressing LINE-1 methylation in Wilms tumor (WT) is limited. The main purpose of our study was to quantify LINE-1 methylation levels and evaluate their relationship with relative telomere length (TL) in WT. Methods: We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction (PCR) pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line. Significant changes were analyzed by t-tests. Results: LINE-1 methylation levels were significantly lower (P < 0.05) and relative TLs were significantly shorter (P < 0.05) in WT compared with normal kidney. There was a significant positive relationship between LINE-1 methylation and relative TL in WT (r = 0.671, P = 0.001). LINE-1 Methylation levels were significantly associated with global DNA methylation (r = 0.332, P < 0.01). In addition, relative TL was shortened and LINE-1 methylation was decreased in a WT cell line treated with the hypomethylating agent 5-aza-2′-deoxycytidine compared with untreated WT cell line. Conclusion: These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT. PMID:26608986

  12. Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes.

    PubMed

    Kordis, D; Gubensek, F

    1998-09-01

    We have shown previously by Southern blot analysis that Bov-B long interspersed nuclear elements (LINEs) are present in different Viperidae snake species. To address the question as to whether Bov-B LINEs really have been transmitted horizontally between vertebrate classes, the analysis has been extended to a larger number of vertebrate, invertebrate, and plant species. In this paper, the evolutionary origin of Bov-B LINEs is shown unequivocally to be in Squamata. The previously proposed horizontal transfer of Bov-B LINEs in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The horizontal transfer of Bov-B LINEs from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution. The ancestor of Colubroidea snakes is a possible donor of Bov-B LINEs to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINEs in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINEs from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINEs have been maintained stably by vertical transmission since the origin of Squamata in the Mesozoic era. PMID:9724768

  13. Long Interspersed Element-1 Protein Expression Is a Hallmark of Many Human Cancers

    PubMed Central

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S.; Hruban, Ralph H.; Iacobuzio-Donahue, Christine A.; Maitra, Anirban; Torbenson, Michael S.; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S.; Montgomery, Elizabeth A.; Gabrielson, Edward; Netto, George J.; Lotan, Tamara L.; De Marzo, Angelo M.; Westra, William; Binder, Zev A.; Orr, Brent A.; Gallia, Gary L.; Eberhart, Charles G.; Boeke, Jef D.; Harris, Chris R.; Burns, Kathleen H.

    2014-01-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1–encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1–encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. PMID:24607009

  14. RUDI, a short interspersed element of the V-SINE superfamily widespread in molluscan genomes.

    PubMed

    Luchetti, Andrea; Šatović, Eva; Mantovani, Barbara; Plohl, Miroslav

    2016-06-01

    Short interspersed elements (SINEs) are non-autonomous retrotransposons that are widespread in eukaryotic genomes. They exhibit a chimeric sequence structure consisting of a small RNA-related head, an anonymous body and an AT-rich tail. Although their turnover and de novo emergence is rapid, some SINE elements found in distantly related species retain similarity in certain core segments (or highly conserved domains, HCD). We have characterized a new SINE element named RUDI in the bivalve molluscs Ruditapes decussatus and R. philippinarum and found this element to be widely distributed in the genomes of a number of mollusc species. An unexpected structural feature of RUDI is the HCD domain type V, which was first found in non-amniote vertebrate SINEs and in the SINE from one cnidarian species. In addition to the V domain, the overall sequence conservation pattern of RUDI elements resembles that found in ancient AmnSINE (~310 Myr old) and Au SINE (~320 Myr old) families, suggesting that RUDI might be among the most ancient SINE families. Sequence conservation suggests a monophyletic origin of RUDI. Nucleotide variability and phylogenetic analyses suggest long-term vertical inheritance combined with at least one horizontal transfer event as the most parsimonious explanation for the observed taxonomic distribution. PMID:26987730

  15. Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

    PubMed Central

    Zhu, Wei; Kuo, Dwight; Nathanson, Jason; Satoh, Akira; Pao, Gerald M.; Yeo, Gene W.; Bryant, Susan V.; Voss, S. Randal; Gardiner, David M.; Hunter, Tony

    2012-01-01

    Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration. PMID:22913491

  16. Polycaprolactone nanofiber interspersed collagen type-I scaffold for bone regeneration: a unique injectable osteogenic scaffold.

    PubMed

    Baylan, Nuray; Bhat, Samerna; Ditto, Maggie; Lawrence, Joseph G; Lecka-Czernik, Beata; Yildirim-Ayan, Eda

    2013-08-01

    There is an increasing demand for an injectable cell coupled three-dimensional (3D) scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed using cells, a natural polymer (collagen type-I), and a synthetic polymer (polycaprolactone (PCL)). The injectable nanofibrous PN-COL is created by interspersing PCL nanofibers within pre-osteoblast cell embedded collagen type-I. This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen was coupled with the structural support of PCL nanofibers, to create cell encapsulated injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity. The effects of PCL nanofiber inclusion within the cell encapsulated collagen matrix has been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity. The structural analysis of novel bioactive material proved that the material is chemically stable enough in an aqueous solution for an extended period of time without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation data suggests that novel PN-COL scaffolds promote the osteoblast proliferation, phenotype expression, and formation of mineralized matrix. This study demonstrates for the first time the feasibility of creating a structurally competent, injectable, cell embedded bone tissue scaffold. Furthermore, the results demonstrate the advantages of mimicking the hierarchical architecture of native bone with nano- and micro-size formation through introducing PCL nanofibers within macron-size collagen fibers and in

  17. Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling.

    PubMed

    Warncke, Jan D; Vakonakis, Ioannis; Beck, Hans-Peter

    2016-12-01

    During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family. PMID:27582258

  18. The spatial arrangement of reefs alters the ecological patterns of fauna between interspersed algal habitats

    NASA Astrophysics Data System (ADS)

    Tuya, F.; Wernberg, T.; Thomsen, M. S.

    2008-07-01

    Reef landscapes dominated by canopy-forming species are often irregular mosaics of habitats, with important influences on associated fauna. This study tested if differences in the ecological patterns of mobile fauna inhabiting interspersed (morphologically distinct) algal habitats were altered by the spatial arrangement of reefs of varying proximity to the shoreline. Specifically, prosobranch gastropods were used as models to test that: (1) there were differences in the ecological patterns (species composition and abundances) between three algal habitats (the kelp Ecklonia radiata, fucalean macroalgae, and erect red algae); (2) the magnitude of these differences depended on the position of reef lines ('in-shore' vs. 'off-shore'); and (3) these effects were regionally consistent across a ˜4° latitudinal gradient (˜600 km of coastline) in Western Australia. The ecological patterns of algal-associated gastropods responded strongly to the presence of algal habitats with different physical structure at small spatial scales. Importantly, differences in assemblage structure (e.g. differences in total abundances) between habitats across the latitudinal gradient were especially accentuated on the in-shore reefs compared with the off-shore reefs, where a general amelioration of differences between habitats was observed, probably associated with a more widespread effect of stronger wave forces across habitats. Overall, red algae supported higher total abundances and species richness (per algal weight) compared to the other algal habitats, particularly on in-shore reefs. Patterns for individual species were considerably location-dependent, reflecting the natural variability of species across geographical gradients. In contrast, patterns at the assemblage-level were consistent, providing evidence for the existence of general rules underlying the assemblage-level organization of mobile invertebrates on subtidal reefs across this geographical gradient.

  19. Viral protein R of human immunodeficiency virus type-1 induces retrotransposition of long interspersed element-1

    PubMed Central

    2013-01-01

    Background Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients’ blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). Results We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein β. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. Conclusions Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients’ blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases. PMID:23915234

  20. Twenty Years of Mycobacterial Glycans: Furanosides and Beyond.

    PubMed

    Lowary, Todd L

    2016-07-19

    The cell surface (or cell wall) of bacteria is coated with carbohydrate (or glycan) structures that play a number of important roles. These include providing structural integrity, serving as a permeability barrier to extracellular compounds (e.g., drugs) and modulating the immune system of the host. Of interest to this Account is the cell wall structure of mycobacteria. There are a host of different mycobacterial species, some of which cause human disease. The most well-known is Mycobacterium tuberculosis, the causative agent of tuberculosis. The mycobacterial cell wall is characterized by the presence of unusual carbohydrate structures that fulfill the roles described above. However, in many cases, a molecular-level understanding of how mycobacterial cell wall glycans mediate these processes is lacking. Inspired by a seminar he heard as a postdoctoral fellow, the author began his independent research program with a focus on the chemical synthesis of mycobacterial glycans. The goals were not only to develop synthetic approaches to these unique structures but also to provide molecules that could be used to probe their biological function. Initial work addressed the preparation of fragments of two key polysaccharides, arabinogalactan and lipoarabinomannan, which contain large numbers of sugar residues in the furanose (five-membered) ring form. At the time these investigations began, there were few methods reported for the synthesis of oligosaccharides containing furanose rings. Thus, early in the program, a major area of interest was methodology development, particularly for the preparation of 1,2-cis-furanosides. To solve this challenge, a range of conformationally restricted donors have been developed, both in the author's group and others, which provide 1,2-cis-furanosidic linkages with high stereoselectivity. These investigations were followed by application of the developed methods to the synthesis of a range of target molecules containing arabinofuranose and

  1. Nontuberculous Mycobacterial Infection after Fractionated CO2 Laser Resurfacing

    PubMed Central

    Culton, Donna A.; Miller, Becky A.; Miller, Melissa B.; MacKuen, Courteney; Groben, Pamela; White, Becky; Cox, Gary M.; Stout, Jason E.

    2013-01-01

    Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection. PMID:23628077

  2. Highlight on Advances in Nontuberculous Mycobacterial Disease in North America

    PubMed Central

    Mirsaeidi, Mehdi; Farshidpour, Maham; Allen, Mary Beth; Ebrahimi, Golnaz; Falkinham, Joseph O.

    2014-01-01

    Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and exist as an important cause of pulmonary infections in humans. Pulmonary involvement is the most common disease manifestation of NTM and the incidence of NTM is growing in North America. Susceptibility to NTM infection is incompletely understood; therefore preventative tools are not well defined. Treatment of pulmonary nontuberculous mycobacterial (NTM) infection is difficult and entails multiple antibiotics and an extended treatment course. Also, there is a considerable variation in treatment management that should be considered before initiating treatment. We highlight the new findings in the epidemiology diagnosis and treatment of mycobacterial infections. We debate new advances regarding NTM infection in cystic fibrosis patients and solid organ transplant recipients. Finally, we introduce a new epidemiologic model for NTM disease based on virulence-exposure-host factors. PMID:25574470

  3. Targeting the mycobacterial envelope for tuberculosis drug development

    PubMed Central

    Favrot, Lorenza; Ronning, Donald R

    2013-01-01

    The bacterium that causes tuberculosis, Mycobacterium tuberculosis, possesses a rather unique outer membrane composed largely of lipids that possess long-chain and branched fatty acids, called mycolic acids. These lipids form a permeability barrier that prevents entry of many environmental solutes, thereby making these bacteria acid-fast and able to survive extremely hostile surroundings. Antitubercular drugs must penetrate this layer to reach their target. This review highlights drug development efforts that have added to the slowly growing tuberculosis drug pipeline, identified new enzyme activities to target with drugs and increased the understanding of important biosynthetic pathways for mycobacterial outer membrane and cell wall core assembly. In addition, a portion of this review looks at discovery efforts aimed at weakening this barrier to decrease mycobacterial virulence, decrease fitness in the host or enhance the efficacy of the current drug repertoire by disrupting the permeability barrier. PMID:23106277

  4. Nontuberculous Mycobacterial Ocular Infections: A Systematic Review of the Literature

    PubMed Central

    Kheir, Wajiha J.; Sheheitli, Huda; Abdul Fattah, Maamoun; Hamam, Rola N.

    2015-01-01

    Nontuberculous or atypical mycobacterial ocular infections have been increasing in prevalence over the past few decades. They are known to cause periocular, adnexal, ocular surface and intraocular infections and are often recalcitrant to medical therapy. These infections can potentially cause detrimental outcomes, in part due to a delay in diagnosis. We review 174 case reports and series on nontuberculous mycobacterial (NTM) ocular infections and discuss etiology, microbiology, risk factors, diagnosis, clinical presentation, and treatment of these infections. History of interventions, trauma, foreign bodies, implants, contact lenses, and steroids are linked to NTM ocular infections. Steroid use may prolong the duration of the infection and cause poorer visual outcomes. Early diagnosis and initiation of treatment with multiple antibiotics are necessary to achieve the best visual outcome. PMID:26106601

  5. ‘Black bronchoscopy’: a case of active mycobacterial tuberculosis

    PubMed Central

    Inaty, Hanine; Arora, Ayush; Diacovo, Julia M.; Mehta, Atul

    2016-01-01

    A 63-year-old male presents with chronic cough and hemoptysis. Computed tomography of the chest revealed a left lower lobe (LLL) area of consolidation with prominent ipsilateral hilar lymphadenopathy. Bronchoscopic airway examination revealed black mucosal discoloration and airway narrowing at the superior segment of the LLL. Bronchoalveolar lavage from the corresponding site grew mycobacterial tuberculosis. The patient's symptoms subsided with anti-tuberculous therapy with a significant decrease in the size of the LLL mass. PMID:27471594

  6. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

    PubMed

    Vemula, Mani H; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  7. Vaccination Against Tuberculosis With Whole-Cell Mycobacterial Vaccines.

    PubMed

    Scriba, Thomas J; Kaufmann, Stefan H E; Henri Lambert, Paul; Sanicas, Melvin; Martin, Carlos; Neyrolles, Olivier

    2016-09-01

    Live attenuated and killed whole-cell vaccines (WCVs) offer promising vaccination strategies against tuberculosis. A number of WCV candidates, based on recombinant bacillus Calmette-Guerin (BCG), attenuated Mycobacterium tuberculosis, or related mycobacterial species are in various stages of preclinical or clinical development. In this review, we discuss the vaccine candidates and key factors shaping the development pathway for live and killed WCVs and provide an update on progress. PMID:27247343

  8. Genetic characterization of mycobacterial l,d-transpeptidases

    PubMed Central

    Sanders, Akeisha N.; Wright, Lori F.

    2014-01-01

    l,d-Transpeptidases (Ldts) catalyse the formation of 3–3 cross-links in peptidoglycans (PGs); however, the role of these enzymes in cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage of 3–3 cross-links (~30–80 %) than the PG in most other bacteria, suggesting that they are particularly important to mycobacterial cell wall biology. The genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis encode multiple Ldt genes, but it is not clear if they are redundant. We compared the sequences of the Ldt proteins from 18 mycobacterial genomes and found that they can be grouped into six classes. We then constructed M. smegmatis strains lacking single or multiple Ldt genes to determine the physiological consequence of the loss of these enzymes. We report that of the single mutants, only one, ΔldtC (MSMEG_0929, class 5), displayed an increased susceptibility to imipenem – a carbapenem antibiotic that inhibits the Ldt enzymes. The invariant cysteine in the active site of LdtC was required for function, consistent with its role as an Ldt. A triple mutant missing ldtC and both of the class 2 genes displayed hypersusceptibility to antibiotics, lysozyme and d-methionine, and had an altered cellular morphology. These data demonstrated that the distinct classes of mycobacterial Ldts may reflect different, non-redundant functions and that the class 5 Ldt was peculiar in that its loss, alone and with the class 2 proteins, had the most profound effect on phenotype. PMID:24855140

  9. Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases

    PubMed Central

    2004-01-01

    PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an α(1→6)LM intermediate through a PPM-dependent α(1→6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a ‘natural’ C75 polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial α(1→6)mannosyltransferase, to a synthetic Manp(1→6)-Manp-O-C10:1 disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis α(1→6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases. PMID:15202931

  10. A hoarse voice: atypical mycobacterial infection of the larynx.

    PubMed

    McEwan, J A; Mohsen, A H; Schmid, M L; McKendrick, M W

    2001-11-01

    Myobacterium malmoense is a non-tuberculous mycobacterium that most commonly causes pulmonary infection, particularly in patients with underlying pulmonary disease or immunodeficiency. We describe a case of Mycobacterium malmoense infection of the larynx in a previously well middle-aged woman, which has previously not been reported. The case highlights the importance of considering atypical mycobacterial infection in the differential diagnosis of laryngeal lesions. PMID:11779312

  11. 'Black bronchoscopy': a case of active mycobacterial tuberculosis.

    PubMed

    Inaty, Hanine; Arora, Ayush; Diacovo, Julia M; Mehta, Atul

    2016-07-01

    A 63-year-old male presents with chronic cough and hemoptysis. Computed tomography of the chest revealed a left lower lobe (LLL) area of consolidation with prominent ipsilateral hilar lymphadenopathy. Bronchoscopic airway examination revealed black mucosal discoloration and airway narrowing at the superior segment of the LLL. Bronchoalveolar lavage from the corresponding site grew mycobacterial tuberculosis. The patient's symptoms subsided with anti-tuberculous therapy with a significant decrease in the size of the LLL mass. PMID:27471594

  12. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish

    PubMed Central

    Vemula, Mani H.; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  13. Mycobacterial Prevalence and Antibiotic Resistance Frequency Trends in Taiwan of Mycobacterial Clinical Isolates From 2002 to 2014.

    PubMed

    Shiau, Ming-Yuh; Lee, Ming-Shih; Huang, Tian-Lin; Tsai, Jen-Ning; Chang, Yih-Hsin

    2016-03-01

    Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC) infections, is one of the most widespread infectious diseases worldwide. Nontuberculous mycobacteria (NTM) also cause chronic pulmonary infections, however, NTM infection is generally overlooked.This study analyzed the frequencies of MTBC and NTM clinical isolates from 181,132 specimens obtained from patients in Taiwan suspected of having a pulmonary mycobacterial infection from 2002 to 2014. The resistant rates to 4 first-line antibiotics (isoniazid, ethambutol, rifampicin, and streptomycin) of 9079 clinical MTBC isolates were also examined by the modified agar proportion method.Overall, the mycobacterial isolation rate was 8.65%, and this consisted of MTBC isolation rate of 5.01% and NTM isolation rate of 3.63%. The prevalence of MTBC isolates among the identified mycobacterial strains could be seen to decrease significantly from 82.5% in 2002 to 41.18% in 2014. Notably, the corresponding NTM prevalence increased 3.36 fold from 17.54% in 2002 to 58.82% in 2014. The frequencies of MTBC and NTM isolates showed a reciprocal trend with the crossing over occurring in the years 2010 and 2011. Although the resistance rates of the MTBC isolates to isoniazid and streptomycin were relatively stable over the study period, resistance rates of the MTBC isolates against rifampicin and ethambutol fluctuated across the study period. Overall, the incidence of multidrug resistance was relatively consistent at about 1.74%.The diagnosis, identification, and susceptibility tests for NTM should be standardized and integrated into appropriate clinical settings to cope with the increase in NTM infections. In addition, the documentation of the antibiotic resistance rates of MTBC clinical isolates to the antibiotic treatments most often clinically prescribed over a decade provides valuable clues and reference points for effective mycobacterial control. PMID:27015168

  14. 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

  15. Identification and Genotyping of Mycobacterium tuberculosis Isolated From Water and Soil Samples of a Metropolitan City

    PubMed Central

    Velayati, Ali Akbar; Farnia, Parissa; Mozafari, Mohadese; Malekshahian, Donya; Farahbod, Amir Masoud; Seif, Shima; Rahideh, Snaz

    2015-01-01

    BACKGROUND: The potential role of environmental Mycobacterium tuberculosis in the epidemiology of TB remains unknown. We investigated the transmission of M tuberculosis from humans to the environment and the possible transmission of M tuberculosis from the environment to humans. METHODS: A total of 1,500 samples were collected from three counties of the Tehran, Iran metropolitan area from February 2012 to January 2014. A total of 700 water samples (47%) and 800 soil samples (53%) were collected. Spoligotyping and the mycobacterial interspersed repetitive units-variable number of tandem repeats typing method were performed on DNA extracted from single colonies. Genotypes of M tuberculosis strains isolated from the environment were compared with the genotypes obtained from 55 patients with confirmed pulmonary TB diagnosed during the study period in the same three counties. RESULTS: M tuberculosis was isolated from 11 of 800 soil samples (1%) and 71 of 700 water samples (10%). T family (56 of 82, 68%) followed by Delhi/CAS (11 of 82, 13.4%) were the most frequent M tuberculosis superfamilies in both water and soil samples. Overall, 27.7% of isolates in clusters were related. No related typing patterns were detected between soil, water, and clinical isolates. The most frequent superfamily of M tuberculosis in clinical isolates was Delhi/CAS (142, 30.3%) followed by NEW-1 (127, 27%). The bacilli in contaminated soil (36%) and damp water (8.4%) remained reculturable in some samples up to 9 months. CONCLUSIONS: Although the dominant M tuberculosis superfamilies in soil and water did not correspond to the dominant M tuberculosis family in patients, the presence of circulating genotypes of M tuberculosis in soil and water highlight the risk of transmission. PMID:25340935

  16. Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China.

    PubMed

    Li, Qin-Jing; Jiao, Wei-Wei; Yin, Qing-Qin; Xu, Fang; Li, Jie-Qiong; Sun, Lin; Xiao, Jing; Li, Ying-Jia; Mokrousov, Igor; Huang, Hai-Rong; Shen, A-Dong

    2016-05-01

    Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains. PMID:26902762

  17. Protocol for a population-based molecular epidemiology study of tuberculosis transmission in a high HIV-burden setting: the Botswana Kopanyo study

    PubMed Central

    Zetola, N M; Modongo, C; Moonan, P K; Click, E; Oeltmann, J E; Shepherd, J; Finlay, A

    2016-01-01

    Introduction Mycobacterium tuberculosis (Mtb) is transmitted from person to person via airborne droplet nuclei. At the community level, Mtb transmission depends on the exposure venue, infectiousness of the tuberculosis (TB) index case and the susceptibility of the index case's social network. People living with HIV infection are at high risk of TB, yet the factors associated with TB transmission within communities with high rates of TB and HIV are largely undocumented. The primary aim of the Kopanyo study is to better understand the demographic, clinical, social and geospatial factors associated with TB and multidrug-resistant TB transmission in 2 communities in Botswana, a country where 60% of all patients with TB are also infected with HIV. This manuscript describes the methods used in the Kopanyo study. Methods and analysis The study will be conducted in greater Gaborone, which has high rates of HIV and a mobile population; and in Ghanzi, a rural community with lower prevalence of HIV infection and home to the native San population. Kopanyo aims to enrol all persons diagnosed with TB during a 4-year study period. From each participant, sputum will be cultured, and for all Mtb isolates, molecular genotyping (24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats) will be performed. Patients with matching genotype results will be considered members of a genotype cluster, a proxy for recent transmission. Demographic, behavioural, clinical and social information will be collected by interview. Participant residence, work place, healthcare facilities visited and social gathering venues will be geocoded. We will assess relationships between these factors and cluster involvement to better plan interventions for reducing TB transmission. Ethics Ethical approval from the Independent Review Boards at the University of Pennsylvania, US Centers for Disease Control and Prevention, Botswana Ministry of Health and University of Botswana has been

  18. Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

    PubMed

    Fritsch, Isabel; Luyven, Gabriele; Köhler, Heike; Lutz, Walburga; Möbius, Petra

    2012-02-01

    Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable

  19. Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

    PubMed

    Pérez-Lago, L; Palacios, J J; Herranz, M; Ruiz Serrano, M J; Bouza, E; García-de-Viedma, D

    2015-02-01

    The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure. PMID:25658553

  20. Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

    PubMed Central

    Sohal, Jagdip Singh; Arsenault, Julie; Labrecque, Olivia; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Fecteau, Gilles

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the “cattle type,” or type II, although 3 strains were of the “bison type.” A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns. PMID:24829229

  1. Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

    PubMed

    Stucki, David; Ballif, Marie; Egger, Matthias; Furrer, Hansjakob; Altpeter, Ekkehardt; Battegay, Manuel; Droz, Sara; Bruderer, Thomas; Coscolla, Mireia; Borrell, Sonia; Zürcher, Kathrin; Janssens, Jean-Paul; Calmy, Alexandra; Mazza Stalder, Jesica; Jaton, Katia; Rieder, Hans L; Pfyffer, Gaby E; Siegrist, Hans H; Hoffmann, Matthias; Fehr, Jan; Dolina, Marisa; Frei, Reno; Schrenzel, Jacques; Böttger, Erik C; Gagneux, Sebastien; Fenner, Lukas

    2016-07-01

    Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB. PMID:27194683

  2. Molecular analysis and MIRU-VNTR typing of Mycobacterium avium subsp. avium, 'hominissuis' and silvaticum strains of veterinary origin.

    PubMed

    Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám; Gyuranecz, Miklós

    2016-06-01

    Besides Mycobacterium avium subsp. paratuberculosis (MAP), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), and 'M. avium subsp. hominissuis' (MAH) are equally important members of M. avium complex, with worldwide distribution and zoonotic potential. Genotypic discrimination is a prerequisite to epidemiological studies which can facilitate disease prevention through revealing infection sources and transmission routes. The primary aim of this study was to identify the genetic diversity within 135 MAA, 62 MAS, and 84 MAH strains isolated from wild and domestic mammals, reptiles and birds. Strains were tested for the presence of large sequence polymorphism LSP(A)17 and were submitted to Mycobacterial interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) analysis at 8 loci, including MIRU1, 2, 3, and 4, VNTR25, 32, and 259, and MATR9. In 12 strains hsp65 sequence code type was also determined. LSP(A)17 was present only in 19.9% of the strains. All LSP(A)17 positive strains belonged to subspecies MAH. The discriminatory power of the MIRU-VNTR loci set used reached 0.9228. Altogether 54 different genotypes were detected. Within MAH, MAA, and MAS strains 33, 16, and 5 different genotypes were observed. The described genotypes were not restricted to geographic regions or host species, but proved to be subspecies specific. Our knowledge about MAS is limited due to isolation and identification difficulties. This is the first study including a large number of MAS field strains. Our results demonstrate the high diversity of MAH and MAA strains and the relative uniformity of MAS strains. PMID:26964909

  3. Mycobacterial species as case-study of comparative genome analysis.

    PubMed

    Zakham, F; Belayachi, L; Ussery, D; Akrim, M; Benjouad, A; El Aouad, R; Ennaji, M M

    2011-01-01

    The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis K—10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYR—1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species. PMID:21396338

  4. Production of matrix metalloproteinases in response to mycobacterial infection.

    PubMed

    Quiding-Järbrink, M; Smith, D A; Bancroft, G J

    2001-09-01

    Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M. tuberculosis in vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-alpha), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-alpha or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-gamma), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-gamma treatment increased macrophage secretion of TNF-alpha but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-alpha available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M. tuberculosis induced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-alpha, IL-18, and IFN-gamma. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections. PMID:11500442

  5. Mycobacterial Infection after Cosmetic Procedure with Botulinum Toxin A

    PubMed Central

    Saeb-Lima, Marcela; Solis-Arreola, Gerardo-Victor

    2015-01-01

    We report a case of mycobacterial infection at the sites of previous injections of botulinum toxin A in a 45-year-old woman. She presented with erythematous, swollen, warm, and tender plaques and nodules at the points of injection from which a biopsy was taken, demonstrating a deep dermal and hypodermal abscessified epithelioid granulomatous inflammatory infiltrate in which some acid-fast bacilli were identified with Ziehl-Neelsen and Fite-Faraco stains. The lesion was first treated with clarithromycin plus azithromycin, to which rifampicin was later added. A good therapeutic response was obtained. PMID:26023629

  6. Outbreak of nontuberculous mycobacterial disease in the central Pacific.

    PubMed

    Lillis, Joseph V; Ansdell, David

    2011-01-01

    Approximately 10% of the island population of Satowan (population, 650 persons), a small, remote coral island in the central Pacific, suffers from an acquired, chronic, disfiguring skin condition known locally as "spam." This skin disease has affected the island population since shortly after World War II. An investigation in 2007 revealed that this skin disease is caused by a nontuberculous mycobacterial infection closely related to Mycobacterium marinum. This article reviews the fascinating history of this skin disease on Satowan, its distinctive clinical presentation, and recommendations for diagnosis and treatment of clinically similar skin lesions in Pacific Islanders. PMID:21095522

  7. Studies of transmission of mycobacterial infections in Chinook salmon

    USGS Publications Warehouse

    Ross, A.J.; Johnson, H.E.

    1962-01-01

    THE INCLUSION OF VISCERA AND CARCASSES OF TUBERCULOUS ADULT SALMON IN THE DIET OF JUVENILE SALMONIDS is considered to be the major source of mycobacterial infections in hatchery-reared fish (Wood and Ordal, 1958; Ross, Earp, and Wood, 1959). In considering additional modes of infection, we speculated about transovarian transmission or a mechanical process arising from contamination of the ova at the egg-taking stage with subsequent entry of the bacteria into the egg at the time of fertilization. This paper is a report on observations made during an experiment designed to test the latter theories.

  8. Activation of human neutrophils by mycobacterial phenolic glycolipids

    PubMed Central

    Fäldt, J; Dahlgren, C; Karlsson, A; Ahmed, A M S; Minnikin, D E; Ridell, M

    1999-01-01

    The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells. PMID:10540187

  9. Ultrathin Carbon with Interspersed Graphene/Fullerene-like Nanostructures: A Durable Protective Overcoat for High Density Magnetic Storage

    PubMed Central

    Dwivedi, Neeraj; Satyanarayana, Nalam; Yeo, Reuben J.; Xu, Hai; Ping Loh, Kian; Tripathy, Sudhiranjan; Bhatia, Charanjit S.

    2015-01-01

    One of the key issues for future hard disk drive technology is to design and develop ultrathin (<2 nm) overcoats with excellent wear- and corrosion protection and high thermal stability. Forming carbon overcoats (COCs) having interspersed nanostructures by the filtered cathodic vacuum arc (FCVA) process can be an effective approach to achieve the desired target. In this work, by employing a novel bi-level surface modification approach using FCVA, the formation of a high sp3 bonded ultrathin (~1.7 nm) amorphous carbon overcoat with interspersed graphene/fullerene-like nanostructures, grown on magnetic hard disk media, is reported. The in-depth spectroscopic and microscopic analyses by high resolution transmission electron microscopy, scanning tunneling microscopy, time-of-flight secondary ion mass spectrometry, and Raman spectroscopy support the observed findings. Despite a reduction of ~37 % in COC thickness, the FCVA-processed thinner COC (~1.7 nm) shows promising functional performance in terms of lower coefficient of friction (~0.25), higher wear resistance, lower surface energy, excellent hydrophobicity and similar/better oxidation corrosion resistance than current commercial COCs of thickness ~2.7 nm. The surface and tribological properties of FCVA-deposited COC was further improved after deposition of lubricant layer. PMID:26109208

  10. Ultrathin Carbon with Interspersed Graphene/Fullerene-like Nanostructures: A Durable Protective Overcoat for High Density Magnetic Storage

    NASA Astrophysics Data System (ADS)

    Dwivedi, Neeraj; Satyanarayana, Nalam; Yeo, Reuben J.; Xu, Hai; Ping Loh, Kian; Tripathy, Sudhiranjan; Bhatia, Charanjit S.

    2015-06-01

    One of the key issues for future hard disk drive technology is to design and develop ultrathin (<2 nm) overcoats with excellent wear- and corrosion protection and high thermal stability. Forming carbon overcoats (COCs) having interspersed nanostructures by the filtered cathodic vacuum arc (FCVA) process can be an effective approach to achieve the desired target. In this work, by employing a novel bi-level surface modification approach using FCVA, the formation of a high sp3 bonded ultrathin (~1.7 nm) amorphous carbon overcoat with interspersed graphene/fullerene-like nanostructures, grown on magnetic hard disk media, is reported. The in-depth spectroscopic and microscopic analyses by high resolution transmission electron microscopy, scanning tunneling microscopy, time-of-flight secondary ion mass spectrometry, and Raman spectroscopy support the observed findings. Despite a reduction of ~37 % in COC thickness, the FCVA-processed thinner COC (~1.7 nm) shows promising functional performance in terms of lower coefficient of friction (~0.25), higher wear resistance, lower surface energy, excellent hydrophobicity and similar/better oxidation corrosion resistance than current commercial COCs of thickness ~2.7 nm. The surface and tribological properties of FCVA-deposited COC was further improved after deposition of lubricant layer.

  11. Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin

    PubMed Central

    Marques, André; Ribeiro, Tiago; Neumann, Pavel; Macas, Jiří; Novák, Petr; Schubert, Veit; Pellino, Marco; Fuchs, Jörg; Ma, Wei; Kuhlmann, Markus; Brandt, Ronny; Vanzela, André L. L.; Beseda, Tomáš; Šimková, Hana; Pedrosa-Harand, Andrea; Houben, Andreas

    2015-01-01

    Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated “Tyba” and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle–dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences. PMID:26489653

  12. Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin.

    PubMed

    Marques, André; Ribeiro, Tiago; Neumann, Pavel; Macas, Jiří; Novák, Petr; Schubert, Veit; Pellino, Marco; Fuchs, Jörg; Ma, Wei; Kuhlmann, Markus; Brandt, Ronny; Vanzela, André L L; Beseda, Tomáš; Šimková, Hana; Pedrosa-Harand, Andrea; Houben, Andreas

    2015-11-01

    Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences. PMID:26489653

  13. Ultrathin Carbon with Interspersed Graphene/Fullerene-like Nanostructures: A Durable Protective Overcoat for High Density Magnetic Storage.

    PubMed

    Dwivedi, Neeraj; Satyanarayana, Nalam; Yeo, Reuben J; Xu, Hai; Ping Loh, Kian; Tripathy, Sudhiranjan; Bhatia, Charanjit S

    2015-01-01

    One of the key issues for future hard disk drive technology is to design and develop ultrathin (<2 nm) overcoats with excellent wear- and corrosion protection and high thermal stability. Forming carbon overcoats (COCs) having interspersed nanostructures by the filtered cathodic vacuum arc (FCVA) process can be an effective approach to achieve the desired target. In this work, by employing a novel bi-level surface modification approach using FCVA, the formation of a high sp(3) bonded ultrathin (~1.7 nm) amorphous carbon overcoat with interspersed graphene/fullerene-like nanostructures, grown on magnetic hard disk media, is reported. The in-depth spectroscopic and microscopic analyses by high resolution transmission electron microscopy, scanning tunneling microscopy, time-of-flight secondary ion mass spectrometry, and Raman spectroscopy support the observed findings. Despite a reduction of ~37% in COC thickness, the FCVA-processed thinner COC (~1.7 nm) shows promising functional performance in terms of lower coefficient of friction (~0.25), higher wear resistance, lower surface energy, excellent hydrophobicity and similar/better oxidation corrosion resistance than current commercial COCs of thickness ~2.7 nm. The surface and tribological properties of FCVA-deposited COC was further improved after deposition of lubricant layer. PMID:26109208

  14. Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

    PubMed

    Vasconcellos, Sidra E G; Acosta, Chyntia Carolina; Gomes, Lia Lima; Conceição, Emilyn Costa; Lima, Karla Valéria; de Araujo, Marcelo Ivens; Leite, Maria de Lourdes; Tannure, Flávio; Caldas, Paulo Cesar de Souza; Gomes, Harrison M; Santos, Adalberto Rezende; Gomgnimbou, Michel K; Sola, Christophe; Couvin, David; Rastogi, Nalin; Boechat, Neio; Suffys, Philip Noel

    2014-01-01

    Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB. PMID:25314118

  15. Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

    PubMed Central

    Vasconcellos, Sidra E. G.; Acosta, Chyntia Carolina; Gomes, Lia Lima; Conceição, Emilyn Costa; Lima, Karla Valéria; de Araujo, Marcelo Ivens; Leite, Maria de Lourdes; Tannure, Flávio; Caldas, Paulo Cesar de Souza; Gomes, Harrison M.; Santos, Adalberto Rezende; Gomgnimbou, Michel K.; Sola, Christophe; Couvin, David; Rastogi, Nalin; Boechat, Neio; Suffys, Philip Noel

    2014-01-01

    Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB. PMID:25314118

  16. Hyper-interspersed NANO/MEMS - Architecture design for new concepts in miniature robotics for space exploration

    NASA Astrophysics Data System (ADS)

    Santoli, Salvatore

    1999-05-01

    Launch weight and volume requirements are substantially decreased by reduction of probe size in exploration mission systems, as mass and volume both scale as the third power of system size. Accordingly, the already quite developed MEMS (Micro Electro Mechanical System) technology, that offers low cost, small, light weight, and increasingly reliable devices through durability and redundancy, is strongly attractive as a near-term technology for significantly reducing the cost to launch and operate space systems. It is shown that the final goal of MEMS technology, i.e. the merging through solid state microdcvices of the functions of sensing, computation, communication and actuation, can lead to a new, biomimetic kind of miniature robotics, particularly suitable for planetary exploration, through molecular mono- electronics/MEMS integration jointly with a hyper-interspersed architecture made up of autonomous units embodying sensors, information processors and actuators. The problem tackled here concerns the basic design of such miniature robots, from some μm to insect size, featuring finely structured intelligent autonomous parts as smart skins, sensory and manipulating members working on the analogue external reality and communicating with their inner molecular level nondiscrete pseudo-analogue information processing networks. The (mesoscopic network)/MEMS units are shown to embody a quantum mechanical/macroscopic world connection, in which the nondiscrete molecular devices allow the automaton parts to perform very complex, fast information processing operations as metaphores of bionic functions like learning, attention, and decision making under uncertain conditions, this last due to the stochasticity inherent in the quantum network. Flexible architectures instead of von Neumann type rigid architectures in addition to hyper-interspersion of autonomous units can be realized through such nano/MEMS devices, and the μm — cm size of the whole robots and their organs

  17. microRNAs in mycobacterial disease: friend or foe?

    PubMed Central

    Mehta, Manali D.; Liu, Philip T.

    2014-01-01

    As the role of microRNA in all aspects of biology continues to be unraveled, the interplay between microRNAs and human disease is becoming clearer. It should come of no surprise that microRNAs play a major part in the outcome of infectious diseases, since early work has implicated microRNAs as regulators of the immune response. Here, we provide a review on how microRNAs influence the course of mycobacterial infections, which cause two of humanity’s most ancient infectious diseases: tuberculosis and leprosy. Evidence derived from profiling and functional experiments suggests that regulation of specific microRNAs during infection can either enhance the immune response or facilitate pathogen immune evasion. Now, it remains to be seen if the manipulation of host cell microRNA profiles can be an opportunity for therapeutic intervention for these difficult-to-treat diseases. PMID:25076967

  18. New Targets and Inhibitors of Mycobacterial Sulfur Metabolism§

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2015-01-01

    The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes. PMID:23808874

  19. A novel quinoline derivative that inhibits mycobacterial FtsZ.

    PubMed

    Mathew, Bini; Ross, Larry; Reynolds, Robert C

    2013-07-01

    High throughput phenotypic screening of large commercially available libraries through two NIH programs has produced thousands of potentially interesting hits for further development as antitubercular agents. Unfortunately, these screens do not supply target information, and further follow up target identification is required to allow optimal rational design and development of highly active and selective clinical candidates. Cheminformatic analysis of the quinoline and quinazoline hits from these HTS screens suggested a hypothesis that certain compounds in these two classes may target the mycobacterial tubulin homolog, FtsZ. In this brief communication, activity of a lead quinoline against the target FtsZ from Mycobacterium tuberculosis (Mtb) is confirmed as well as good in vitro whole cell antibacterial activity against Mtb H37Rv. The identification of a putative target of this highly tractable pharmacophore should help medicinal chemists interested in targeting FtsZ and cell division develop a rational design program to optimize this activity toward a novel drug candidate. PMID:23647650

  20. Management of Nontuberculous Mycobacterial Infection in The Elderly

    PubMed Central

    Mirsaeidi, Mehdi; Farshidpour, Maham; Ebrahimi, Golnaz; Aliberti, Stefano; Falkinham, Joseph O

    2014-01-01

    The incidence of nontuberculous mycobacteria (NTM) has increased over the last decades. Elderly people are more susceptible to NTM and experience increased morbidities. NTM incidence is expected to rise due to an increasing elderly population at least up to 2050. Given the importance of NTM infection in the elderly, an increasing interest exists in studying NTM characteristics in aged population. In this review, we summarize the characteristics of NTM infection among elderly patients. We focus on epidemiology, clinical presentation, and treatment options of NTM in this age group. We highlight the differences in the diagnosis and treatment between rapid and slow growing mycobacterial infections. The current recommendation for treatment of NTM is discussed. We debate if in vitro susceptibility testing has a role in treatment of NTM. Drug-drug interaction between antibiotics used to treat NTM and other medications, particularly warfarin, is another important issue that we discuss. Finally, we review the prognosis of NTM disease in elderly patients. PMID:24685313

  1. Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides

    PubMed Central

    Maranha, Ana; Moynihan, Patrick J.; Miranda, Vanessa; Correia Lourenço, Eva; Nunes-Costa, Daniela; Fraga, Joana S.; José Barbosa Pereira, Pedro; Macedo-Ribeiro, Sandra; Ventura, M. Rita; Clarke, Anthony J.; Empadinhas, Nuno

    2015-01-01

    Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. Enzymatic studies, synthetic chemistry, NMR spectroscopy and mass spectrometry approaches suggest that, in contrast to the prevailing consensus, octanoate is not esterified to the primary hydroxyl group of glycerate but instead to the C6 OH of the second glucose in DGG. These observations raise important new questions about the MGLP reducing end architecture and about subsequent biosynthetic steps. Functional characterization of this unique octanoyltransferase, whose gene has been proposed to be essential for M. tuberculosis growth, adds new insights into a vital mycobacterial pathway, which may inspire new drug discovery strategies. PMID:26324178

  2. Immune biology of macaque lymphocyte populations during mycobacterial infection

    PubMed Central

    LAI, X; SHEN, Y; ZHOU, D; SEHGAL, P; SHEN, L; SIMON, M; QIU, L; LETVIN, N L; CHEN, Z W

    2003-01-01

    Immune responses of lymphocyte populations during early phases of mycobacterial infection and reinfection have not been well characterized in humans. A non-human primate model of Mycobacterium bovis bacille Calmette–Guerin (BCG) infection was employed to characterize optimally the immune responses of mycobacteria-specific T cells. Primary BCG infection induced biphasic immune responses, characterized by initial lymphocytopenia and subsequent expansion of CD4+, CD8+ and γδ T cell populations in the blood, lymph nodes and the pulmonary compartment. The potency of detectable T cell immune responses appears to be influenced by the timing and route of infection as well as challenge doses of BCG organisms. Systemic BCG infection introduced by intravenous challenge induced a dose-dependent expansion of circulating CD4+, CD8+ and γδ T cells whereas, in the pulmonary compartment, the systemic infection resulted in a predominant increase in numbers of γδ T cells. In contrast, pulmonary exposure to BCG through the bronchial route induced detectable expansions of CD4+, CD8+ and γδ T cell populations in only the lung but not in the blood. A rapid recall expansion of these T cell populations was seen in the macaques reinfected intravenously and bronchially with BCG. The expanded αβ and γδ T cell populations exhibited their antigen specificity for mycobacterial peptides and non-peptide phospholigands, respectively. Finally, the major expansion of T cells was associated with a resolution of active BCG infection and reinfection. The patterns and kinetics of CD4+, CD8+ and γδ T cell immune responses during BCG infection might contribute to characterizing immune protection against tuberculosis and testing new tuberculosis vaccines in primates. PMID:12869023

  3. Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle

    PubMed Central

    Janagama, Harish K.; Widdel, Andrea; Vulchanova, Lucy; Stabel, Judith R.; Waters, W. Ray; Palmer, Mitchell V.; Sreevatsan, Srinand

    2009-01-01

    Background Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. Methodology/Principal Findings We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P≤0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. Conclusions/Significance The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the

  4. microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production

    PubMed Central

    Li, Miao; Wang, Jinli; Fang, Yimin; Gong, Sitang; Li, Meiyu; Wu, Minhao; Lai, Xiaomin; Zeng, Gucheng; Wang, Yi; Yang, Kun; Huang, Xi

    2016-01-01

    Macrophages play a crucial role in host innate anti-mycobacterial defense, which is tightly regulated by multiple factors, including microRNAs. Our previous study showed that a panel of microRNAs was markedly up-regulated in macrophages upon mycobacterial infection. Here, we investigated the biological function of miR-146a during mycobacterial infection. miR-146a expression was induced both in vitro and in vivo after Mycobacterium bovis BCG infection. The inducible miR-146a could suppress the inducible nitric oxide (NO) synthase (iNOS) expression and NO generation, thus promoting mycobacterial survival in macrophages. Inhibition of endogenous miR-146a increased NO production and mycobacterial clearance. Moreover, miR-146a attenuated the activation of nuclear factor κB and mitogen-activated protein kinases signaling pathways during BCG infection, which in turn repressed iNOS expression. Mechanistically, miR-146a directly targeted tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) at post-transcriptional level. Silencing TRAF6 decreased iNOS expression and NO production in BCG-infected macrophages, while overexpression of TRAF6 reversed miR-146a-mediated inhibition of NO production and clearance of mycobacteria. Therefore, we demonstrated a novel role of miR-146a in the modulation of host defense against mycobacterial infection by repressing NO production via targeting TRAF6, which may provide a promising therapeutic target for tuberculosis. PMID:27025258

  5. PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements.

    PubMed

    Tajima, Kiyoshi; Enishi, Osamu; Amari, Masahiro; Mitsumori, Makoto; Kajikawa, Hiroshi; Kurihara, Mitsunori; Yanai, Satoshi; Matsui, Hiroki; Yasue, Hiroshi; Mitsuhashi, Tadayoshi; Kawashima, Tomoyuki; Matsumoto, Mitsuto

    2002-10-01

    PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials. PMID:12450143

  6. The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element.

    PubMed Central

    Haynes, S R; Toomey, T P; Leinwand, L; Jelinek, W R

    1981-01-01

    A consensus sequence has been determined for a major interspersed deoxyribonucleic acid repeat in the genome of Chinese hamster ovary cells (CHO cells). This sequence is extensively homologous to (i) the human Alu sequence (P. L. Deininger et al., J. Mol. Biol., in press), (ii) the mouse B1 interspersed repetitious sequence (Krayev et al., Nucleic Acids Res. 8:1201-1215, 1980) (iii) an interspersed repetitious sequence from African green monkey deoxyribonucleic acid (Dhruva et al., Proc. Natl. Acad. Sci. U.S.A. 77:4514-4518, 1980) and (iv) the CHO and mouse 4.5S ribonucleic acid (this report; F. Harada and N. Kato, Nucleic Acids Res. 8:1273-1285, 1980). Because the CHO consensus sequence shows significant homology to the human Alu sequence it is termed the CHO Alu-equivalent sequence. A conserved structure surrounding CHO Alu-equivalent family members can be recognized. It is similar to that surrounding the human Alu and the mouse B1 sequences, and is represented as follows: direct repeat-CHO-Alu-A-rich sequence-direct repeat. A composite interspersed repetitious sequence has been identified. Its structure is represented as follows: direct repeat-residue 47 to 107 of CHO-Alu-non-Alu repetitious sequence-A-rich sequence-direct repeat. Because the Alu flanking sequences resemble those that flank known transposable elements, we think it likely that the Alu sequence dispersed throughout the mammalian genome by transposition. Images PMID:9279371

  7. Computational Finishing of Large Sequence Contigs Reveals Interspersed Nested Repeats and Gene Islands in the rf1-associated Region of Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The architecture of grass genomes varies on multiple levels. Large long terminal repeat (LTR) retrotransposon clusters occupy significant portions of the intergenic regions, and islands of protein-encoding genes are interspersed among the repeat clusters. Hence, advanced assembly techniques are requ...

  8. Comparing Massed-Trial Instruction, Distributed-Trial Instruction, and Task Interspersal to Teach Tacts to Children with Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    Majdalany, Lina M.; Wilder, David A.; Greif, Abigail; Mathisen, David; Saini, Valdeep

    2014-01-01

    Although massed-trial instruction, distributed-trial instruction, and task interspersal have been shown to be effective methods of teaching skills to children with autism spectrum disorders, they have not been directly compared. In the current study, we taught 6 children to tact shapes of countries using these methods to determine which would…

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. Interception of host angiogenic signalling limits mycobacterial growth

    PubMed Central

    Oehlers, Stefan H.; Cronan, Mark R.; Scott, Ninecia R.; Thomas, Monica I.; Okuda, Kazuhide S.; Walton, Eric M.; Beerman, Rebecca W.; Crosier, Philip S.; Tobin, David M.

    2014-01-01

    Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis1,2. Here we examine the development and consequences of vascularisation of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model characterised by organised granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis2. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule VEGFA. Pharmacological inhibition of the VEGF pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergise with the first-line anti-tubercular antibiotic rifampicin as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations3. Our data suggest that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting TB therapy, particularly in extensively drug-resistant disease where current antibiotic regimens are largely ineffective. PMID:25470057

  12. Mechanistic insight into mycobacterial MmpL protein function.

    PubMed

    Székely, R; Cole, S T

    2016-03-01

    Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters. PMID:26710752

  13. Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections.

    PubMed

    Deffert, Christine; Cachat, Julien; Krause, Karl-Heinz

    2014-08-01

    Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)-producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2-derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded - at least in part - contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS. PMID:24916152

  14. Management of nontuberculous mycobacterial infection in the elderly.

    PubMed

    Mirsaeidi, Mehdi; Farshidpour, Maham; Ebrahimi, Golnaz; Aliberti, Stefano; Falkinham, Joseph O

    2014-04-01

    The incidence of nontuberculous mycobacteria (NTM) has increased over the last decades. Elderly people are more susceptible to NTM and experience increased morbidities. NTM incidence is expected to rise due to an increasing elderly population at least up to 2050. Given the importance of NTM infection in the elderly, an increasing interest exists in studying NTM characteristics in the aged population. In this review, we summarize the characteristics of NTM infection among elderly patients. We focus on epidemiology, clinical presentation, and treatment options of NTM in this age group. We highlight the differences in the diagnosis and treatment between rapid and slow growing mycobacterial infections. The current recommendation for treatment of NTM is discussed. We debate if in vitro susceptibility testing has a role in the treatment of NTM. Drug-drug interaction between antibiotics used to treat NTM and other medications, particularly warfarin, is another important issue that we discuss. Finally, we review the prognosis of NTM disease in elderly patients. PMID:24685313

  15. Disseminated nontuberculous mycobacterial infections in sickle cell anemia patients.

    PubMed

    Thorell, Emily A; Sharma, Mukta; Jackson, Mary Anne; Selvarangan, Rangaraj; Woods, Gerald M

    2006-10-01

    Nontuberculous mycobacteria (NTM) are ubiquitous in nature and have been implicated in skin/soft-tissue, pulmonary, middle ear, bone, and surgical/traumatic wound infections. Disseminated disease occurs infrequently and almost exclusively in the immunocompromised. We describe the first 2 reported cases of disseminated Mycobacterium fortuitum infection in teenagers with sickle hemoglobinopathy. Both had central venous catheters (CVCs), frequent admissions for vaso-occlusive painful episode and received hydroxyurea. Diagnosis was confirmed by multiple positive blood cultures and pulmonary dissemination occurred in both. Both had successful treatment after CVC removal and combination drug therapy. Positive cultures persisted in 1 patient due to drug resistance emphasizing the need for accurate susceptibility data. NTM infection should be added to the list of pathogens in sickle cell patients with CVCs and fever. Investigation for disseminated disease should be undertaken based on clinical signs and symptoms. Although some routine blood culture systems can identify NTM, specific mycobacterial blood culture is optimal. Removal of involved CVCs is essential and treatment of NTM must be guided by susceptibilities. As dissemination almost always occurs in those with impaired cellular immunity, human immunodeficiency virus testing should be performed. Hydroxyurea may be a risk factor for dissemination and needs further evaluation. PMID:17023829

  16. [The bacteriology of tuberculosis and non-tuberculosis mycobacterial infections].

    PubMed

    Wyplosz, B; Truffot-Pernot, C; Robert, J; Jarlier, V; Grosset, J

    1997-12-01

    Changing incidence and nature of mycobacterial infections subsequent to the historical regression of tuberculosis and the acquired human immunodeficiency syndrome (AIDS) epidemic, as well as the development of new technical tools for molecular biology, have profoundly modified the methods used for the bacteriological diagnosis of mycobacteria infections. Although microscopic search for acid-fast bacilli, culture and antibiotic resistance tests on Löwenstein-Jensen medium remain the reference methods, more rapid and sophisticated methods are now available. Culture on radiolabeled media using the Bactec system has shortened the delay for positive culture and interpretable antibiotic sensitivity tests. Molecular techniques allow: 1) rapid identification of the most frequently isolated mycobacteria strains, including the most frequent laboratory contaminant M. gordonae, with genome probes; 2) genome typing of M. tuberculosis strains to trace interhuman transmission, detect recurrence or exogenous reinfection or demonstrate laboratory contamination; 3) rapid detection of rifampicin resistance; and 4) direct detection of M. tuberculosis and M. avium in pathological specimens. The role of mycobacteria in the environment causing opportunistic infections, atypical mycobacteria or non-tuberculosis mycobacteria (NTM), particularly the aviaire complex, has grown considerably. Isolation and identification relies on methods used to detect bacilli as well as blood cultures and analysis of fecal matter. NTM are naturally resistant to most of the antituberculosis antibiotics but are sometimes sensitive to aminoglycosides, fluoroquinolones or new macrolides. PMID:9496590

  17. Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis.

    PubMed Central

    Jackett, P S; Bothamley, G H; Batra, H V; Mistry, A; Young, D B; Ivanyi, J

    1988-01-01

    A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease. PMID:2466869

  18. Diversification, evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species.

    PubMed

    Schwichtenberg, Katrin; Wenke, Torsten; Zakrzewski, Falk; Seibt, Kathrin M; Minoche, André; Dohm, Juliane C; Weisshaar, Bernd; Himmelbauer, Heinz; Schmidt, Thomas

    2016-01-01

    Short interspersed nuclear elements (SINEs) are non-autonomous non-long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full-length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs. PMID:26676716

  19. Active and Repressive Chromatin Are Interspersed without Spreading in an Imprinted Gene Cluster in the Mammalian Genome

    PubMed Central

    Regha, Kakkad; Sloane, Mathew A.; Huang, Ru; Pauler, Florian M.; Warczok, Katarzyna E.; Melikant, Balázs; Radolf, Martin; Martens, Joost H.A.; Schotta, Gunnar; Jenuwein, Thomas; Barlow, Denise P.

    2010-01-01

    SUMMARY The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 ± HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome. PMID:17679087

  20. Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation

    SciTech Connect

    Pascale, E.; Valle, E.; Furano, A.V. )

    1990-12-01

    Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation {approximately}80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new one were generated. However, the authors show here that an ancestral rodent L1 family was extensively amplified {approximately}10 million years ago and that the relics of this amplification have persisted in modern murine genomes. This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents The results suggest that repeated amplification of L1 elements is a feature of the evaluation of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages.

  1. Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation.

    PubMed Central

    Pascale, E; Valle, E; Furano, A V

    1990-01-01

    Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation approximately 80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new ones were generated. However, we show here that an ancestral rodent L1 family was extensively amplified approximately 10 million years ago and that the relics (approximately 60,000 copies) of this amplification have persisted in modern murine genomes (Old World rats and mice). This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents. Our results suggest that repeated amplification of L1 elements is a feature of the evolution of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages. Images PMID:2251288

  2. Species distribution in human immunodeficiency virus-related mycobacterial infections: implications for selection of initial treatment.

    PubMed

    Montessori, V; Phillips, P; Montaner, J; Haley, L; Craib, K; Bessuille, E; Black, W

    1996-06-01

    Management of mycobacterial infection is species specific; however, treatment is prompted by positive smears or cultures, often several weeks before species identification. The objective of this study was to determine the species distribution of mycobacterial isolates from various body sites in patients infected with human immunodeficiency virus (HIV). All mycobacterial isolates recovered at St. Paul's Hospital (Vancouver, British Columbia, Canada) from April 1989 to March 1993 were reviewed. Among 357 HIV-positive patients with mycobacterial infections, 64% (96) of the sputum isolates were Mycobacterium avium complex (MAC), 18% were Mycobacterium tuberculosis, and 17% were Mycobacterium kansasii. Lymph node involvement (25 patients) was due to either MAC (72%) or M. tuberculosis (24%). Two hundred ninety-eight episodes of mycobacteremia were due to MAC (98%), M. tuberculosis (1%), and M. kansasii (1%). Similarly, cultures of 84 bone marrow biopsy specimens (99%), 19 intestinal biopsy specimens (100%), and 30 stool specimens (97%) yielded predominantly MAC. These results have implications for initial therapy, particularly in areas where rapid methods for species identification are not readily available. Because of considerable geographic variation, development of guidelines for selection of initial therapy depends on regional determination of species distribution in HIV-related mycobacterial infections. PMID:8783698

  3. Comparative genomics for mycobacterial peptidoglycan remodelling enzymes reveals extensive genetic multiplicity

    PubMed Central

    2014-01-01

    Background Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets. Results We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival. Conclusion PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt. PMID:24661741

  4. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD

    PubMed Central

    Davis, Elizabeth; Chen, James; Leon, Katherine; Darst, Seth A.; Campbell, Elizabeth A.

    2015-01-01

    Escherichia coli has served as the archetypal organism on which the overwhelming majority of biochemical characterizations of bacterial RNA polymerase (RNAP) have been focused; the properties of E. coli RNAP have been accepted as generally representative for all bacterial RNAPs. Here, we directly compare the initiation properties of a mycobacterial transcription system with E. coli RNAP on two different promoters. The detailed characterizations include abortive transcription assays, RNAP/promoter complex stability assays and DNAse I and KMnO4 footprinting. Based on footprinting, we find that promoter complexes formed by E. coli and mycobacterial RNAPs use very similar protein/DNA interactions and generate the same transcription bubbles. However, we find that the open promoter complexes formed by E. coli RNAP on the two promoters tested are highly stable and essentially irreversible (with lifetimes much greater than 1 h), while the open promoter complexes on the same two promoters formed by mycobacterial RNAP are very unstable (lifetimes of about 2 min or less) and readily reversible. We show here that CarD, an essential mycobacterial transcription activator that is not found in E. coli, stabilizes the mycobacterial RNAP/open promoter complexes considerably by preventing transcription bubble collapse. PMID:25510492

  5. A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Santiago, Chile.

    PubMed

    Balcells, María Elvira; García, Patricia; Meza, Paulina; Peña, Carlos; Cifuentes, Marcela; Couvin, David; Rastogi, Nalin

    2015-01-01

    Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or

  6. A First Insight on the Population Structure of Mycobacterium tuberculosis Complex as Studied by Spoligotyping and MIRU-VNTRs in Santiago, Chile

    PubMed Central

    Balcells, María Elvira; García, Patricia; Meza, Paulina; Peña, Carlos; Cifuentes, Marcela; Couvin, David; Rastogi, Nalin

    2015-01-01

    Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or

  7. Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo.

    PubMed

    Levitte, Steven; Adams, Kristin N; Berg, Russell D; Cosma, Christine L; Urdahl, Kevin B; Ramakrishnan, Lalita

    2016-08-10

    The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection. PMID:27512905

  8. Patient Report and Review of Rapidly Growing Mycobacterial Infection after Cardiac Device Implantation

    PubMed Central

    Hirsh, David S.; Goswami, Neela D.

    2016-01-01

    Mycobacterial infections resulting from cardiac implantable electronic devices are rare, but as more devices are implanted, these organisms are increasingly emerging as causes of early-onset infections. We report a patient with an implantable cardioverter-defibrillator pocket and associated bloodstream infection caused by an organism of the Mycobacterium fortuitum group, and we review the literature regarding mycobacterial infections resulting from cardiac device implantations. Thirty-two such infections have been previously described; most (70%) were caused by rapidly growing species, of which M. fortuitum group species were predominant. When managing such infections, clinicians should consider the potential need for extended incubation of routine cultures or dedicated mycobacterial cultures for accurate diagnosis; combination antimicrobial drug therapy, even for isolates that appear to be macrolide susceptible, because of the potential for inducible resistance to this drug class; and the arrhythmogenicity of the antimicrobial drugs traditionally recommended for infections caused by these organisms. PMID:26890060

  9. Comparative Gamma Delta T Cell Immunology: A Focus on Mycobacterial Disease in Cattle

    PubMed Central

    Plattner, Brandon L.; Hostetter, Jesse M.

    2011-01-01

    A theme among many pathogenic mycobacterial species affecting both humans and animals is a prolonged asymptomatic or latent period that can last years to decades. The mechanisms that favor progression to active disease are not well understood. Pathogen containment is often associated with an effective cell-mediated or T-helper 1 immune profile. With certain pathogenic mycobacteria, such as Mycobacterium avium subspecies paratuberculosis, a shift to active clinical disease is associated with loss of T-helper 1 immunity and development of an ineffective humoral or T-helper 2 immune response. Recently γδ T cells have been shown to play a role early in mycobacterial infections and have been hypothesized to influence disease outcome. The purpose of this paper is to compare recent advancements in our understanding of γδ T cells in humans, cattle, and mice and to discuss roles of γδ T cells in host response to mycobacterial infection. PMID:21647391

  10. Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

    PubMed

    Pagán, Antonio J; Yang, Chao-Tsung; Cameron, James; Swaim, Laura E; Ellett, Felix; Lieschke, Graham J; Ramakrishnan, Lalita

    2015-07-01

    The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis. PMID:26159717

  11. Ultrastructural morphologic changes in mycobacterial biofilm in different extreme condition.

    PubMed

    Kumar, Virendra; Sachan, Tarun Kumar; Sharma, Pragya; Rawat, Krishna Dutta

    2015-02-01

    The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 °C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 °C as compared to 30 °C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 °C as compared to 37 and 42 °C. Mycobacterium tuberculosis developed strong biofilm at 37 °C and no biofilm at 30 and 42 °C in Sauton's media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton's medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multi-layered, three-dimensional structure. PMID:25192360

  12. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  13. Who Has Mycobacterial Disease? A Cross Sectional Study in Agropastoral Communities in Tanzania

    PubMed Central

    Kilale, Andrew Martin; Ngadaya, Esther; Muhumuza, Julius; Kagaruki, Gibson Benard; Lema, Yakobo Leonard; Ngowi, Bernard James; Mfinanga, Sayoki Godfrey; Hinderaker, Sven Gudmund

    2016-01-01

    Objective To determine and describe clinical symptoms, demographic characteristics and environmental exposures as determinants of pulmonary mycobacterial diseases among patients examined for tuberculosis in agropastoral communities in Northern Tanzania. Methods This was a cross sectional study. Sputum samples were collected from patients attending three hospitals in Tanzania, and were investigated for pulmonary tuberculosis by microscopy between November 2010 and June 2012. The patients were interviewed about background information, and potential exposure to mycobacteria. Results We examined 1,711 presumptive tuberculosis cases where 936 (54.2%) were males and 775 (45.3%) females. Of all the study participants, 277 (16%) were found to have sputum samples positive for mycobacteria; 228 (13%) were smear positive, 123 (7%) were culture positive and 74 (4%) were positive by both smear microscopy and culture. Of the 123 mycobacterial culture positive, 15 (12.2%) had non-tuberculous mycobacteria. Males were more likely than females to be positive for mycobacteria. Factors associated with mycobacterial disease were loss of appetite, age groups below 41 years, and being a male. Among HIV negative patients, loss of appetite, age below 20 years and being a male were associated with being mycobacterial positive. Among HIV positive patients, males and those patients with a persistently coughing family member were more likely to harbor mycobacteria. Conclusion The findings in this study show that both M. tuberculosis and non-tuberculous mycobacterial strains were prevalent in the study community. Some risk factors were identified. Although the reported predictors may improve screening for mycobacterial diseases, their use requires some precaution. PMID:27213532

  14. Transmission of the FRAXA haplotype from three nonpenetrant brothers to their affected grandsons: an update with AGG interspersion analysis.

    PubMed

    Mogk, R L; Carson, N L; Chudley, A E; Dawson, A J

    1998-01-01

    Recently, we reported on a family showing transmission of the FRAXA gene by three nonpenetrant, normally intelligent, full and half brothers to their affected grandsons [Kirkilionis et al., 1992]. We have reanalyzed this family for CGG repeat size by polymerase chain reaction (PCR) amplification/Southern blot and FMR1 methylation status using EcoRI/BssHII double digests with pE5-1 as the hybridization probe. The half brother was found to have a premutation allele size of 59 CGG repeats. MnlI digestion of PCR products showed the absence of intervening AGG sequences. All of his obligate carrier daughters had CGG alleles ranging from 65 to 90 repeats, with a final expansion of more than 200 repeats in his FRAXA-affected grandson and 131 repeats in his carrier granddaughter. Two full brothers were shown to have inherited a 47-CGG repeat premutation allele. Analysis of one brother showed that he stably transmitted the 47-repeat allele to his daughter. Analysis of the second brother, his daughter, and his granddaughter showed that this allele was meiotically unstable, with the allele size increasing from 47, to 48, to 49 from the father, to the daughter to the granddaughter, respectively. MnlI digestion and DNA sequencing of PCR products showed the absence of intervening AGG sequences. This is the first case in which the lack of AGG interspersions has been associated with instability of a gray zone allele resulting in a one-repeat increase in two successive generations. PMID:9450853

  15. Drug testing in mouse models of tuberculosis and nontuberculous mycobacterial infections.

    PubMed

    Nikonenko, Boris V; Apt, Alexander S

    2013-05-01

    Mice as a species are susceptible to tuberculosis infection while mouse inbred strains present wide spectrum of susceptibility/resistance to this infection. However, non-tuberculosis Mycobacterial infections usually cannot be modeled in mice of common inbred strains. Introduction of specific properties, such as gene mutations, recombinants, targeted gene knockouts significantly extended the use of mice to mimic human Mycobacterial infections, including non-tuberculosis ones. This review describes the available mouse models of tuberculosis and non-tuberculosis infections and drug therapy in these models. Mouse models of non-tuberculosis infections are significantly less developed than tuberculosis models, hampering the development of therapies. PMID:23491715

  16. The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells

    PubMed Central

    Dartois, Véronique

    2015-01-01

    For the successful treatment of pulmonary tuberculosis, drugs need to penetrate complex lung lesions and permeate the mycobacterial cell wall in order to reach their intracellular targets. However, most currently used anti-tuberculosis drugs were introduced into clinical use without considering the pharmacokinetic and pharmacodynamic properties that influence drug distribution, and this has contributed to the long duration and limited success of current therapies. In this Progress article, I describe new methods to quantify and image drug distribution in infected lung tissue and in mycobacterial cells, and I explore how this technology could be used to design optimized multidrug regimens. PMID:24487820

  17. Shifts in Mycobacterial Populations and Emerging Drug-Resistance in West and Central Africa

    PubMed Central

    Fissette, Kristina; de Rijk, Pim; Uwizeye, Cécile; Nduwamahoro, Elie; Goovaerts, Odin; Affolabi, Dissou; Gninafon, Martin; Lingoupou, Fanny M.; Barry, Mamadou Dian; Sow, Oumou; Merle, Corinne; Olliaro, Piero; Ba, Fatoumata; Sarr, Marie; Piubello, Alberto; Noeske, Juergen; Antonio, Martin; Rigouts, Leen; de Jong, Bouke C

    2014-01-01

    In this study, we retrospectively analysed a total of 605 clinical isolates from six West or Central African countries (Benin, Cameroon, Central African Republic, Guinea-Conakry, Niger and Senegal). Besides spoligotyping to assign isolates to ancient and modern mycobacterial lineages, we conducted phenotypic drug-susceptibility-testing for each isolate for the four first-line drugs. We showed that phylogenetically modern Mycobacterium tuberculosis strains are more likely associated with drug resistance than ancient strains and predict that the currently ongoing replacement of the endemic ancient by a modern mycobacterial population in West/Central Africa might result in increased drug resistance in the sub-region. PMID:25493429

  18. A large interspersed repeat found in mouse DNA contains a long open reading frame that evolves as if it encodes a protein.

    PubMed Central

    Martin, S L; Voliva, C F; Burton, F H; Edgell, M H; Hutchison, C A

    1984-01-01

    DNA sequence analysis of a region contained within a large, interspersed repetitive family of mice reveals a long open reading frame. This sequence extends 978 base pairs between two stop codons, creating a reading frame that is open for 326 amino acids. The DNA sequence in this region is conserved between three distantly related Mus species, as well as between mouse and monkey, in a manner that is characteristic of regions undergoing selection for protein function. PMID:6326120

  19. Novel nicotine analogues with potential anti-mycobacterial activity.

    PubMed

    Gandhi, Paresh T; Athmaram, Thimmasandra Narayanappa; Arunkumar, Gundaiah Ramesh

    2016-04-15

    Tuberculosis (TB) is the second leading lethal infectious disease in the world after acquired immuno deficiency (AIDs). We have developed a series of twenty-five novel nicotine analogues with de-addiction property and tested them for their activity against Mycobacterium tuberculosis (MTB). In an effort to increase the specificity of action and directing nicotine analogues to target MTB, four promising compounds were further optimized via molecular docking studies against the Dihydrofolate reductase of MTB. After lead optimization, one nicotine analogue [3-(5-(3fluorophenyl)nicotinoyl)-1-methylpyrrolidin-2-one] exhibited minimum inhibitory concentration of 1μg/mL (2.86nM) against M. tuberculosis (H37Rv strain), a human pathogenic strain of clinically significant importance. Pharmacokinetic analysis of [3-(5-(3fluorophenyl)nicotinoyl)-1methylpyrrolidin-2-one] with lowest MIC value via oral route in Wistar rats revealed that at a dosage of 5mg/kg body weight gave a maximum serum drug concentration (Cmax) of 2.86μg/mL, Tmax of one hour and a half-life (T1/2) of more than 24h and Volume of distribution (Vd) of 27.36L. Whereas the parenteral (intra venous) route showed a Cmax of 3.37μg/mL, Tmax of 0.05h, T1/2 of 24h and Vd equivalent to 23.18L. The acute oral toxicity and repeated oral toxicity studies in female Wistar rats had an LD50>2000mg/kg body weight. Our data suggests that nicotine derivatives developed in the present study has good metabolic stability with tunable pharmacokinetics (PK) with therapeutic potential to combat MTB. However, further in vivo studies for anti-tuberculosis activity and elucidation of mode of action could result in more promising novel drug for treating MTB. To the best of our knowledge this is the first report revealing the anti-mycobacterial potential of nicotine analogue at potential therapeutic concentrations. PMID:26951892

  20. Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens.

    PubMed

    Dechyeva, Daryna; Gindullis, Frank; Schmidt, Thomas

    2003-01-01

    Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences. The tandem repeats are 229-246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin. Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed

  1. Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor

    PubMed Central

    Okudaira, N; Okamura, T; Tamura, M; Iijma, K; Goto, M; Matsunaga, A; Ochiai, M; Nakagama, H; Kano, S; Fujii-Kuriyama, Y; Ishizaka, Y

    2013-01-01

    A single human cell contains more than 5.0 × 105 copies of long interspersed element-1 (L1), 80–100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein β. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis. PMID:23208499

  2. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

    PubMed

    Votintseva, Antonina A; Pankhurst, Louise J; Anson, Luke W; Morgan, Marcus R; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W

    2015-04-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools. PMID:25631807

  3. Acanthamoeba Encephalitis: Isolation of Genotype T1 in Mycobacterial Liquid Culture Medium

    PubMed Central

    Azzam, Rula; Badenoch, Paul R.; Francis, Michelle J.; Fernandez, Charles; Adamson, Penelope J.; Dendle, Claire; Woolley, Ian; Robson, Jenny; Korman, Tony M.

    2014-01-01

    We report a case of Acanthamoeba encephalitis diagnosed from an antemortem brain biopsy specimen, where the organism was first isolated in mycobacterial liquid medium and first identified by using a sequence generated by a commercial panfungal sequencing assay. We correlate susceptibility results with clinical outcome. PMID:25502534

  4. Mycobacterial antigen 85 complex (Ag85) as a target for ficolins and mannose-binding lectin.

    PubMed

    Świerzko, Anna S; Bartłomiejczyk, Marcin A; Brzostek, Anna; Łukasiewicz, Jolanta; Michalski, Mateusz; Dziadek, Jarosław; Cedzyński, Maciej

    2016-06-01

    The pattern recognition molecules (PRMs) able to activate complement via the lectin pathway are suspected to be involved in the interaction between pathogenic Mycobacteria and the host immune response. Recently, we have found strong interactions between 25 and 35kDa mycobacterial cell fractions and mannose-binding lectin (MBL) and ficolins. Here we demonstrate that two biologically important mycobacterial structures, mannosylated lipoarabinomannan (ManLAM) and the antigen 85 (Ag85) complex, induce activation of the lectin pathway of complement. The strong interaction of recombinant MBL with purified ManLAM was confirmed, but no binding of recombinant ficolins (ficolin-1, -2, -3) with this structure was observed. Interestingly, all PRMs tested reacted with the mycobacterial antigen 85 (Ag85) complex. Based on the use of specific inhibitors (mannan for MBL, acetylated bovine serum albumin for ficolin-1 and -2, Hafnia alvei PCM 1200 lipopolysaccharide for ficolin-3), we concluded that carbohydrate-recognition (MBL) and fibrinogen-like domains (ficolins) were involved in these interactions. Our results indicate that the mycobacterial antigen 85 complex is a target for ficolins and MBL. Furthermore, those PRMs also bound to fibronectin and therefore might influence the Ag85 complex-dependent interaction of Mycobacterium with the extracellular matrix. PMID:27141819

  5. Evaluation of Oral Antiseptic Rinsing before Sputum Collection To Reduce Contamination of Mycobacterial Cultures▿

    PubMed Central

    Peres, Renata L.; Palaci, Moisés; Loureiro, Rafaela B.; Dietze, Reynaldo; Johnson, John L.; Golub, Jonathan E.; Ruffino-Netto, A.; Maciel, Ethel L.

    2011-01-01

    To assess whether rinsing with oral antiseptics before sputum collection would reduce contamination of mycobacterial cultures, 120 patients with suspected tuberculosis were randomly assigned to rinse with chlorhexidine or cetylpyridinium mouthwash before collection. The culture contamination rate was significantly lower after rinsing with chlorhexidine before collection, especially for cultures grown in MGIT medium. PMID:21677070

  6. 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. PMID:25995039

  7. Dissecting the membrane cholesterol requirement for mycobacterial entry into host cells.

    PubMed

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

    2015-07-01

    Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-β-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry. PMID:26021693

  8. Primed Mycobacterial Uveitis (PMU): Histologic and Cytokine Characterization of a Model of Uveitis in Rats

    PubMed Central

    Pepple, Kathryn L.; Rotkis, Lauren; Van Grol, Jennifer; Wilson, Leslie; Sandt, Angela; Lam, Deborah L.; Carlson, Eric; Van Gelder, Russell N.

    2015-01-01

    Purpose The purpose of this study was to compare the histologic features and cytokine profiles of experimental autoimmune uveitis (EAU) and a primed mycobacterial uveitis (PMU) model in rats. Methods In Lewis rats, EAU was induced by immunization with interphotoreceptor binding protein peptide, and PMU was induced by immunization with a killed mycobacterial extract followed by intravitreal injection of the same extract. Clinical course, histology, and the cytokine profiles of the aqueous and vitreous were compared using multiplex bead fluorescence immunoassays. Results Primed mycobacterial uveitis generates inflammation 2 days after intravitreal injection and resolves spontaneously 14 days later. CD68+ lymphocytes are the predominant infiltrating cells and are found in the anterior chamber, surrounding the ciliary body and in the vitreous. In contrast to EAU, no choroidal infiltration or retinal destruction is noted. At the day of peak inflammation, C-X-C motif ligand 10 (CXCL10), IL-1β, IL-18, and leptin were induced in the aqueous of both models. Interleukin-6 was induced 2-fold in the aqueous of PMU but not EAU. Cytokines elevated in the aqueous of EAU exclusively include regulated on activation, normal T cell expressed and secreted (RANTES), lipopolysaccharide-induced CXC chemokine (LIX), growth-related oncogene/keratinocyte chemokine (GRO/KC), VEGF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and IL-17A. In the vitreous, CXCL10, GRO/KC, RANTES, and MIP-1α were elevated in both models. Interleukin-17A and IL-18 were elevated exclusively in EAU. Conclusions Primed mycobacterial uveitis generates an acute anterior and intermediate uveitis without retinal involvement. Primed mycobacterial uveitis has a distinct proinflammatory cytokine profile compared with EAU, suggesting PMU is a good complementary model for study of immune-mediated uveitis. CXCL10, a proinflammatory cytokine, was increased in the aqueous and

  9. Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.

    PubMed Central

    Kox, L F; Jansen, H M; Kuijper, S; Kolk, A H

    1997-01-01

    Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary. PMID:9163468

  10. IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements.

    PubMed

    Clément, J M; Wilde, C; Bachellier, S; Lambert, P; Hofnung, M

    1999-11-01

    We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed. PMID:10559158

  11. Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

    PubMed

    Bogunovic, Dusan; Byun, Minji; Durfee, Larissa A; Abhyankar, Avinash; Sanal, Ozden; Mansouri, Davood; Salem, Sandra; Radovanovic, Irena; Grant, Audrey V; Adimi, Parisa; Mansouri, Nahal; Okada, Satoshi; Bryant, Vanessa L; Kong, Xiao-Fei; Kreins, Alexandra; Velez, Marcela Moncada; Boisson, Bertrand; Khalilzadeh, Soheila; Ozcelik, Ugur; Darazam, Ilad Alavi; Schoggins, John W; Rice, Charles M; Al-Muhsen, Saleh; Behr, Marcel; Vogt, Guillaume; Puel, Anne; Bustamante, Jacinta; Gros, Philippe; Huibregtse, Jon M; Abel, Laurent; Boisson-Dupuis, Stéphanie; Casanova, Jean-Laurent

    2012-09-28

    ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes-granulocyte, in particular-reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity. PMID:22859821

  12. Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency

    PubMed Central

    Bogunovic, Dusan; Byun, Minji; Durfee, Larissa A.; Abhyankar, Avinash; Sanal, Ozden; Mansouri, Davood; Salem, Sandra; Radovanovic, Irena; Grant, Audrey V.; Adimi, Parisa; Mansouri, Nahal; Okada, Satoshi; Bryant, Vanessa L.; Kong, Xiao-Fei; Kreins, Alexandra; Velez, Marcela Moncada; Boisson, Bertrand; Khalilzadeh, Soheila; Ozcelik, Ugur; Darazam, Ilad Alavi; Schoggins, John W.; Rice, Charles M.; Al-Muhsen, Saleh; Behr, Marcel; Vogt, Guillaume; Puel, Anne; Bustamante, Jacinta; Gros, Philippe; Huibregtse, Jon M.; Abel, Laurent; Boisson-Dupuis, Stéphanie; Casanova, Jean-Laurent

    2012-01-01

    ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. We describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes — granulocytes in particular — reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of Nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity. PMID:22859821

  13. The lta4h Locus Modulates Susceptibility to Mycobacterial Infection in Zebrafish and Humans

    PubMed Central

    Tobin, David M.; Vary, Jay C.; Ray, John P.; Walsh, Gregory S.; Dunstan, Sarah J.; Bang, Nguyen D.; Hagge, Deanna A.; Khadge, Saraswoti; King, Mary-Claire; Hawn, Thomas R.; Moens, Cecilia B.; Ramakrishnan, Lalita

    2010-01-01

    SUMMARY Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A4 hydrolase, which catalyzes the final step in the synthesis of leukotriene B4 (LTB4), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB4 reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB4 production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection. PMID:20211140

  14. A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages.

    PubMed

    Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

    2016-09-01

    Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in Raw264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of Raw264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the Raw264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

  15. Mycobacterial MazG Safeguards Genetic Stability via Housecleaning of 5-OH-dCTP

    PubMed Central

    Fan, Xiao-Yong; Ma, Hui; Zhao, Guo-Ping

    2013-01-01

    Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo. PMID:24339782

  16. A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages

    PubMed Central

    Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

    2016-01-01

    Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in RAW264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of RAW264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the RAW264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

  17. Nontuberculous Mycobacterial Disease in Children – Epidemiology, Diagnosis & Management at a Tertiary Center

    PubMed Central

    MacGregor, Duncan; Gonis, Gena; Leslie, David; Sedda, Luigi; Ritz, Nicole; Connell, Tom; Curtis, Nigel

    2016-01-01

    Background There are limited data on the epidemiology, diagnosis and optimal management of nontuberculous mycobacterial (NTM) disease in children. Methods Retrospective cohort study of NTM cases over a 10-year-period at a tertiary referral hospital in Australia. Results A total of 140 children with NTM disease, including 107 with lymphadenitis and 25 with skin and soft tissue infections (SSTIs), were identified. The estimated incidence of NTM disease was 0.6–1.6 cases / 100,000 children / year; no increasing trend was observed over the study period. Temporal analyses revealed a seasonal incidence cycle around 12 months, with peaks in late winter/spring and troughs in autumn. Mycobacterium-avium-complex accounted for most cases (77.8%), followed by Mycobacterium ulcerans (14.4%) and Mycobacterium marinum (3.3%). Polymerase chain reaction testing had higher sensitivity than culture and microscopy for acid-fast bacilli (92.0%, 67.2% and 35.7%, respectively). The majority of lymphadenitis cases underwent surgical excision (97.2%); multiple recurrences in this group were less common in cases treated with clarithromycin and rifampicin compared with clarithromycin alone or no anti-mycobacterial drugs (0% versus 7.1%; OR:0.73). SSTI recurrences were also less common in cases treated with two anti-mycobacterial drugs compared with one or none (10.5% versus 33.3%; OR:0.23). Conclusions There was seasonal variation in the incidence of NTM disease, analogous to recently published observations in tuberculosis, which have been linked to seasonal variation in vitamin D. Our finding that anti-mycobacterial combination therapy was associated with a reduced risk of recurrences in patients with NTM lymphadenitis or SSTI requires further confirmation in prospective trials. PMID:26812154

  18. Genotyping and clinical characteristics of multidrug and extensively drug-resistant tuberculosis in a tertiary care tuberculosis hospital in China

    PubMed Central

    2013-01-01

    Background There is a lack of information on the clinical characteristics of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB in the Jiangxi Province of China; furthermore, data have not been reported on the utility of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analyses in genotyping Mycobacterium tuberculosis strains isolated from this region. The aim of this study was to analyse the clinical features of patients with MDR and XDR TB from Jiangxi Province and to evaluate the discriminatory power of the 15-loci MIRU-VNTR method. Methods A retrospective study was conducted on patients diagnosed with MDR and XDR TB at the Jiangxi Chest Hospital from July 2010 to June 2011. The RD105 deletion-targeted multiplex PCR (DTM-PCR) and the 15-loci MIRU-VNTR method were used to determine the genetic background of the identified MDR and XDR M. tuberculosis clinical isolates. Results Of 804 M. tuberculosis clinical isolates, 159 (159/804, 19.8%) of the isolates were identified as MDR with first-line drug susceptibility testing. Of the 123 available MDR isolates, 13 (13/123, 10.6%) were XDR. The RD105 deletion-targeted multiplex PCR method identified 85 (85/110, 77.3%) MDR and 12 (12/13, 92.3%) XDR isolates as the Beijing genotype. MIRU-VNTR cluster analysis demonstrated that 101 MDR and 13 XDR strains had unique genotype patterns; the remaining 9 MDR strains were in 4 clusters, namely 1 cluster with 3 strains and 3 clusters with 2 strains, resulting in a low clustering rate (4.06%). The Hunter-Gaston discriminatory index (HGDI) of the 15-loci MIRU-VNTR method was as high as 0.992. In addition, clinical surveys showed that 87 (87/110, 79.1%) MDR TB patients and 10 (10/13, 76.9%) XDR TB patients had been previously treated. Diabetes mellitus was the most common comorbidity in both MDR TB (16/110, 14.5%) and XDR TB (2/13, 15.4%) patients. Conclusions Based on our preliminary data, the MDR and XDR M

  19. A close-up on the epidemiology and transmission of multidrug-resistant tuberculosis in Poland.

    PubMed

    Jagielski, T; Brzostek, A; van Belkum, A; Dziadek, J; Augustynowicz-Kopeć, E; Zwolska, Z

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) poses a serious challenge to the global control of the disease. The purpose of this study was to characterize MDR-TB patients from Poland and to determine the extent of MDR-TB disease attributable to recent transmission. The study included all 46 patients diagnosed with MDR-TB in Poland in 2004 and followed up for 6 years (until 2011). For each patient, sociodemographic and clinical characteristics, treatment outcomes, and bacteriological data were collected by the review of medical and laboratory records. Mycobacterium tuberculosis isolates from all patients were characterized using spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, IS6110 restriction fragment length polymorphism (RFLP) analysis, and sequencing analysis of drug resistance-associated loci (katG, mabA-inhA, rpoβ, rpsL, and embB). The majority of patients were male (86.9%), 40-64 years of age (60.8%), with a history of TB treatment (84.8%), and producing smear-positive sputa (86.9%). Twenty-two (47.8%) patients suffered from concomitant diseases and 28 (60.8%) were alcohol abusers. Treatment outcome assessment revealed that 8 (17.4%) patients were cured or completed therapy, while 15 (32.6%) died of TB, 11 (23.9%) defaulted, 8 (17.4%) failed, and 1 (2.2%) was transferred and lost to follow-up. Upon genotyping, 10 (21.7%) isolates were allocated in four clusters. These were further subdivided by mutational profiling. Overall, in 6 (13%) patients, MDR-TB was a result of recent transmission. For 4 (8.7%) of these patients, a direct epidemiological link was established. The study shows that the transmission of MDR-TB occurs at a low rate in Poland. Of urgent need is the implementation of a policy of enforced treatment of MDR-TB patients in Poland. PMID:25037868

  20. Genetic Diversity of Mycobacterium tuberculosis Isolates from Assam, India: Dominance of Beijing Family and Discovery of Two New Clades Related to CAS1_Delhi and EAI Family Based on Spoligotyping and MIRU-VNTR Typing

    PubMed Central

    Devi, Kangjam Rekha; Bhutia, Rinchenla; Bhowmick, Shovonlal; Mukherjee, Kaustab; Mahanta, Jagadish; Narain, Kanwar

    2015-01-01

    Tuberculosis (TB) is one of the major public health concerns in Assam, a remote state located in the northeastern (NE) region of India. The present study was undertaken to explore the circulating genotypes of Mycobacterium tuberculosis complex (MTBC) in this region. A total of 189 MTBC strains were collected from smear positive pulmonary tuberculosis cases from different designated microscopy centres (DMC) from various localities of Assam. All MTBC isolates were cultured on Lowenstein-Jensen (LJ) media and subsequently genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Spoligotyping of MTBC isolates revealed 89 distinct spoligo patterns. The most dominant MTBC strain belonged to Beijing lineage and was represented by 35.45% (n = 67) of total isolates, followed by MTBC strains belonging to Central Asian-Delhi (CAS/Delhi) lineage and East African Indian (EAI5) lineage. In addition, in the present study 43 unknown spoligo patterns were detected. The discriminatory power of spoligotyping was found to be 0.8637 based on Hunter Gaston Discriminatory Index (HGDI). On the other hand, 24-loci MIRU-VNTR typing revealed that out of total 189 MTBC isolates from Assam 185 (97.9%) isolates had unique MIRU-VNTR profiles and 4 isolates grouped into 2 clusters. Phylogenetic analysis of 67 Beijing isolates based on 24-loci MIRU-VNTR typing revealed that Beijing isolates from Assam represent two major groups, each comprising of several subgroups. Neighbour-Joining (NJ) phylogenetic tree analysis based on combined spoligotyping and 24-loci MIRU-VNTR data of 78 Non-Beijing isolates was carried out for strain lineage identification as implemented by MIRU-VNTRplus database. The important lineages of MTBC identified were CAS/CAS1_Delhi (41.02%, n = 78) and East-African-Indian (EAI, 33.33%). Interestingly, phylogenetic analysis of orphan (23.28%) MTBC spoligotypes revealed that majority of these orphan

  1. SITVITWEB--a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology.

    PubMed

    Demay, Christophe; Liens, Benjamin; Burguière, Thomas; Hill, Véronique; Couvin, David; Millet, Julie; Mokrousov, Igor; Sola, Christophe; Zozio, Thierry; Rastogi, Nalin

    2012-06-01

    Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units-variable number of DNA tandem repeats (MIRU-VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU-VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a global vision of MTC genetic diversity worldwide based on 62,582 clinical isolates corresponding to 153 countries of patient origin (105 countries of isolation). We report a total of 7105 spoligotype patterns (corresponding to 58,180 clinical isolates) - grouped into 2740 shared-types or spoligotype international types (SIT) containing 53,816 clinical isolates and 4364 orphan patterns. Interestingly, only 7% of the MTC isolates worldwide were orphans whereas more than half of SITed isolates (n=27,059) were restricted to only 24 most prevalent SITs. The database also contains a total of 2379 MIRU patterns (from 8161 clinical isolates) from 87 countries of patient origin (35 countries of isolation); these were grouped in 847 shared-types or MIRU international types (MIT) containing 6626 isolates and 1533 orphan patterns. Lastly, data on 5-locus exact tandem repeats (ETRs) were available on 4626 isolates from 59 countries of patient origin (22 countries of isolation); a total of 458 different VNTR patterns were observed - split into 245 shared-types or VNTR International Types (VIT) containing 4413 isolates) and 213 orphan patterns. Datamining of SITVITWEB further allowed to update

  2. Genetic Diversity of Mycobacterium tuberculosis Isolates from Assam, India: Dominance of Beijing Family and Discovery of Two New Clades Related to CAS1_Delhi and EAI Family Based on Spoligotyping and MIRU-VNTR Typing.

    PubMed

    Devi, Kangjam Rekha; Bhutia, Rinchenla; Bhowmick, Shovonlal; Mukherjee, Kaustab; Mahanta, Jagadish; Narain, Kanwar

    2015-01-01

    Tuberculosis (TB) is one of the major public health concerns in Assam, a remote state located in the northeastern (NE) region of India. The present study was undertaken to explore the circulating genotypes of Mycobacterium tuberculosis complex (MTBC) in this region. A total of 189 MTBC strains were collected from smear positive pulmonary tuberculosis cases from different designated microscopy centres (DMC) from various localities of Assam. All MTBC isolates were cultured on Lowenstein-Jensen (LJ) media and subsequently genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Spoligotyping of MTBC isolates revealed 89 distinct spoligo patterns. The most dominant MTBC strain belonged to Beijing lineage and was represented by 35.45% (n = 67) of total isolates, followed by MTBC strains belonging to Central Asian-Delhi (CAS/Delhi) lineage and East African Indian (EAI5) lineage. In addition, in the present study 43 unknown spoligo patterns were detected. The discriminatory power of spoligotyping was found to be 0.8637 based on Hunter Gaston Discriminatory Index (HGDI). On the other hand, 24-loci MIRU-VNTR typing revealed that out of total 189 MTBC isolates from Assam 185 (97.9%) isolates had unique MIRU-VNTR profiles and 4 isolates grouped into 2 clusters. Phylogenetic analysis of 67 Beijing isolates based on 24-loci MIRU-VNTR typing revealed that Beijing isolates from Assam represent two major groups, each comprising of several subgroups. Neighbour-Joining (NJ) phylogenetic tree analysis based on combined spoligotyping and 24-loci MIRU-VNTR data of 78 Non-Beijing isolates was carried out for strain lineage identification as implemented by MIRU-VNTRplus database. The important lineages of MTBC identified were CAS/CAS1_Delhi (41.02%, n = 78) and East-African-Indian (EAI, 33.33%). Interestingly, phylogenetic analysis of orphan (23.28%) MTBC spoligotypes revealed that majority of these orphan

  3. Emerging Tuberculosis Pathogen Hijacks Social Communication Behavior in the Group-Living Banded Mongoose (Mungos mungo)

    PubMed Central

    Sanderson, Claire E.; Larsen, Michelle H.; Robbe-Austerman, Suelee; Williams, Mark C.; Palmer, Mitchell V.

    2016-01-01

    ABSTRACT An emerging Mycobacterium tuberculosis complex (MTC) pathogen, M. mungi, infects wild banded mongooses (Mungos mungo) in Northern Botswana, causing significant mortality. This MTC pathogen did not appear to be transmitted through a primary aerosol or oral route. We utilized histopathology, spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), quantitative PCR (qPCR), and molecular markers (regions of difference [RDs] from various MTC members, including region of difference 1 [RD1] from M. bovis BCG [RD1BCG], M. microti [RD1mic], and M. pinnipedii [RD1seal], genes Rv1510 [RD4], Rv1970 [RD7], Rv3877/8 [RD1], and Rv3120 [RD12], insertion element IS1561, the 16S RNA gene, and gene Rv0577 [cfp32]), including the newly characterized mongoose-specific deletion in RD1 (RD1mon), in order to demonstrate the presence of M. mungi DNA in infected mongooses and investigate pathogen invasion and exposure mechanisms. M. mungi DNA was identified in 29% of nasal planum samples (n = 52), 56% of nasal rinses and swabs (n = 9), 53% of oral swabs (n = 19), 22% of urine samples (n = 23), 33% of anal gland tissue (n = 18), and 39% of anal gland secretions (n = 44). The occurrence of extremely low cycle threshold values obtained with qPCR in anal gland and nasal planum samples indicates that high levels of M. mungi can be found in these tissue types. Histological data were consistent with these results, suggesting that pathogen invasion occurs through breaks in the nasal planum and/or skin of the mongoose host, which are in frequent contact with anal gland secretions and urine during olfactory communication behavior. Lesions in the lung, when present, occurred only with disseminated disease. No environmental sources of M. mungi DNA could be found. We report primary environmental transmission of an MTC pathogen that occurs in association with social communication behavior. PMID:27165798

  4. Control of Mycobacterial Infections in Mice Expressing Human Tumor Necrosis Factor (TNF) but Not Mouse TNF.

    PubMed

    Olleros, Maria L; Chavez-Galan, Leslie; Segueni, Noria; Bourigault, Marie L; Vesin, Dominique; Kruglov, Andrey A; Drutskaya, Marina S; Bisig, Ruth; Ehlers, Stefan; Aly, Sahar; Walter, Kerstin; Kuprash, Dmitry V; Chouchkova, Miliana; Kozlov, Sergei V; Erard, François; Ryffel, Bernard; Quesniaux, Valérie F J; Nedospasov, Sergei A; Garcia, Irene

    2015-09-01

    Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo, providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF. PMID:26123801

  5. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells

    PubMed Central

    Fischer, Karsten; Scotet, Emmanuel; Niemeyer, Marcus; Koebernick, Heidrun; Zerrahn, Jens; Maillet, Sophie; Hurwitz, Robert; Kursar, Mischo; Bonneville, Marc; Kaufmann, Stefan H. E.; Schaible, Ulrich E.

    2004-01-01

    A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-γ production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d. PMID:15243159

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

    SciTech Connect

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

    2010-09-17

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

  7. Comparative evaluation of in vitro human macrophage models for mycobacterial infection study.

    PubMed

    Mendoza-Coronel, E; Castañón-Arreola, M

    2016-08-01

    Macrophages are phagocytic cells that play a key role maintaining the homeostasis of many tissues. Their function is essential for controlling and eradicating infecting mycobacteria. Human monocytic cell lines such as THP-1 and U937 have provided interesting insights into how mycobacteria subvert the host cell response. However, immortalized cell lines could bring some disadvantages. Here we compare the response of THP-1 and U937 cell lines with human monocyte-derived macrophages (hMDMs) to determine functional differences during infection with different mycobacterial phenotypes (virulent Mycobacterium tuberculosis H37Rv and Mycobacterium bovis, and attenuated M. bovis BCG). The findings of this study indicate that the U937 cell line displays a significantly lower phagocytic capacity than hMDMs and THP-1 macrophages, regardless of the mycobacterial strain. In all cell models, interferon-γ activation leads to up-regulation of interleukin-12 and nitrite production. However, the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of U937 and THP-1 cell lines induces a significant tumor necrosis factor-α production in resting macrophages. However, this state of activation has no effect on the control of intracellular growth of mycobacteria. Moreover, U937 cells show more discrepancies with hMDM than THP-1. This study demonstrates that THP-1 macrophages exhibit closer functional similarities to hMDMs in response to mycobacterial infection, regardless of the strain. PMID:27307103

  8. Control of Mycobacterial Infections in Mice Expressing Human Tumor Necrosis Factor (TNF) but Not Mouse TNF

    PubMed Central

    Olleros, Maria L.; Chavez-Galan, Leslie; Segueni, Noria; Bourigault, Marie L.; Vesin, Dominique; Kruglov, Andrey A.; Drutskaya, Marina S.; Bisig, Ruth; Ehlers, Stefan; Aly, Sahar; Walter, Kerstin; Kuprash, Dmitry V.; Chouchkova, Miliana; Kozlov, Sergei V.; Erard, François; Ryffel, Bernard; Quesniaux, Valérie F. J.; Nedospasov, Sergei A.

    2015-01-01

    Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo, providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF. PMID:26123801

  9. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production.

    PubMed

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection. PMID:27251437

  10. Perspectives on mycobacterial vacuole-to-cytosol translocation: the importance of cytosolic access.

    PubMed

    Simeone, Roxane; Majlessi, Laleh; Enninga, Jost; Brosch, Roland

    2016-08-01

    Mycobacterium tuberculosis, the infectious agent of human tuberculosis is a master player in circumventing the defense mechanisms of the host immune system. The host-pathogen interaction in the case of an infection with M. tuberculosis is highly complex, involving dedicated mycobacterial virulence factors as well as the action of the innate and adapted immune systems, which determine the outcome of infection. Macrophages play a key role in this process through internalizing the bacterium in a phagosomal vacuole. While this action has normally the function of eliminating invading bacteria, M. tuberculosis employs efficient strategies to prevent its extermination. The question on how-and-where the bacterium succeeds in doing so has interested generations of scientists and still remains a fascinating and important research subject focused on mycobacterial lipids, secretion systems and other contributing factors. This topic is also central to the longstanding and partially controversial discussion on mycobacterial phagosomal rupture and vacuole-to-cytosol translocation, to be reviewed here in more detail. PMID:27247079

  11. Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals.

    PubMed

    TAKEYA, K; HISATSUNE, K

    1963-01-01

    Takeya, Kenji (Kyushu University, Fukuoka, Japan) and Kazuhito Hisatsune. Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals. J. Bacteriol. 85:16-23. 1963.-Several methods of preparation of mycobacterial cell walls were examined, and the grinding method with glass powder, using Dry Ice, was found to give fairly good cell-wall preparations. "Paired fibrous structures" were clearly seen on the purified cell wall. The appearance of the cell wall as revealed by the electron microscope was not altered by digestion with trypsin, pronase, or pronase in 5% alcoholic solution, nor by treatment with 95% alcohol, acetone-alcohol mixture, or ether-alcohol mixture. By treatment with alcoholic KOH solution, the fibrous structure was removed. The remaining thin layer of the cell wall was tentatively designated the "basal layer" of the mycobacterial cell wall. The fibers appeared also to be removed by chloroform treatment. Nagarse digestion seemed to solubilize some constituents of the cell wall. The cell wall lost its shape and rigidity after lysozyme digestion. PMID:13984703

  12. Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes.

    PubMed

    Smith, Victoria L; Jackson, Liam; Schorey, Jeffrey S

    2015-09-15

    Exosomes are extracellular vesicles of endocytic origin that function in intercellular communication. Our previous studies indicate that exosomes released from Mycobacterium tuberculosis-infected macrophages contain soluble mycobacterial proteins. However, it was unclear how these secreted proteins were targeted to exosomes. In this study, we determined that exosome production by the murine macrophage cell line RAW264.7 requires the endosomal sorting complexes required for transport and that trafficking of mycobacterial proteins from phagocytosed bacilli to exosomes was dependent on protein ubiquitination. Moreover, soluble mycobacterial proteins, when added exogenously to RAW264.7 or human HEK293 cells, were endocytosed, ubiquitinated, and released via exosomes. This suggested that endocytosed proteins could be recycled from cells through exosomes. This hypothesis was supported using the tumor-associated protein He4, which, when endocytosed by RAW264.7 or HEK293 cells, was transported to exosomes in a ubiquitin-dependent manner. Our data suggest that ubiquitination is a modification sufficient for trafficking soluble proteins within the phagocytic/endocytic network to exosomes. PMID:26246139

  13. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production

    PubMed Central

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection. PMID:27251437

  14. The vesicle-associated function of NOD2 as a link between Crohn's disease and mycobacterial infection.

    PubMed

    Nabatov, Alexey A

    2015-01-01

    Although Crohn's disease (CD) etiology remains unclear, a growing body of evidence suggests that CD may include an infectious component, with Mycobacterium avium subsp. paratuberculosis (MAP) being the most likely candidate for this role. However, the molecular mechanism of the MAP involvement in CD pathogenesis remains unclear. The polymorphism of the NOD2 gene, coding for an intracellular pattern recognition receptor, is a factor of predisposition to mycobacterial infections and CD. Recent findings on NOD2 interactions and functions provide the missing pieces in the puzzle of a NOD2-mediated mechanism common for mycobacterial infections and CD. Implications of these new findings for the development of a better understanding and treatments of CD and mycobacterial infections are discussed. PMID:25653718

  15. Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

    PubMed Central

    Kreins, Alexandra Y.; Ciancanelli, Michael J.; Okada, Satoshi; Kong, Xiao-Fei; Ramírez-Alejo, Noé; Kilic, Sara Sebnem; El Baghdadi, Jamila; Nonoyama, Shigeaki; Mahdaviani, Seyed Alireza; Ailal, Fatima; Bousfiha, Aziz; Mansouri, Davood; Nievas, Elma; Ma, Cindy S.; Rao, Geetha; Bernasconi, Andrea; Sun Kuehn, Hye; Niemela, Julie; Stoddard, Jennifer; Deveau, Paul; Cobat, Aurelie; El Azbaoui, Safa; Sabri, Ayoub; Lim, Che Kang; Sundin, Mikael; Avery, Danielle T.; Halwani, Rabih; Grant, Audrey V.; Boisson, Bertrand; Bogunovic, Dusan; Itan, Yuval; Moncada-Velez, Marcela; Martinez-Barricarte, Ruben; Migaud, Melanie; Deswarte, Caroline; Alsina, Laia; Kotlarz, Daniel; Klein, Christoph; Muller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Cormier-Daire, Valerie; Rose-John, Stefan; Picard, Capucine; Hammarstrom, Lennart; Puel, Anne; Al-Muhsen, Saleh; Abel, Laurent; Chaussabel, Damien; Rosenzweig, Sergio D.; Minegishi, Yoshiyuki; Tangye, Stuart G.; Bustamante, Jacinta; Casanova, Jean-Laurent

    2015-01-01

    Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/β, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17+ T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/β. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans. PMID:26304966

  16. Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome.

    PubMed

    Kreins, Alexandra Y; Ciancanelli, Michael J; Okada, Satoshi; Kong, Xiao-Fei; Ramírez-Alejo, Noé; Kilic, Sara Sebnem; El Baghdadi, Jamila; Nonoyama, Shigeaki; Mahdaviani, Seyed Alireza; Ailal, Fatima; Bousfiha, Aziz; Mansouri, Davood; Nievas, Elma; Ma, Cindy S; Rao, Geetha; Bernasconi, Andrea; Sun Kuehn, Hye; Niemela, Julie; Stoddard, Jennifer; Deveau, Paul; Cobat, Aurelie; El Azbaoui, Safa; Sabri, Ayoub; Lim, Che Kang; Sundin, Mikael; Avery, Danielle T; Halwani, Rabih; Grant, Audrey V; Boisson, Bertrand; Bogunovic, Dusan; Itan, Yuval; Moncada-Velez, Marcela; Martinez-Barricarte, Ruben; Migaud, Melanie; Deswarte, Caroline; Alsina, Laia; Kotlarz, Daniel; Klein, Christoph; Muller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Cormier-Daire, Valerie; Rose-John, Stefan; Picard, Capucine; Hammarstrom, Lennart; Puel, Anne; Al-Muhsen, Saleh; Abel, Laurent; Chaussabel, Damien; Rosenzweig, Sergio D; Minegishi, Yoshiyuki; Tangye, Stuart G; Bustamante, Jacinta; Casanova, Jean-Laurent; Boisson-Dupuis, Stéphanie

    2015-09-21

    Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/β, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17(+) T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/β. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans. PMID:26304966

  17. Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase.

    PubMed

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

    2008-09-01

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

  18. Characterization and comparison of mycobacterial antigens by two-dimensional immunoelectrophoresis.

    PubMed

    Roberts, D B; Wright, G L; Affronti, L F; Reich, M

    1972-10-01

    Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of antigens is subjected to electrophoresis first through an agarose matrix in one direction and secondly through an antiserum-agarose matrix at right angles to the first direction, was evaluated as a tool for analysis of mycobacterial antigens. Cell extracts from four species of mycobacteria, Mycobacterium tuberculosis (four strains), M. bovis strain BCG, M. scrofulaceum, and M. phlei, were assayed by 2D-IEP with four anti-mycobacterial antisera. Besides displaying the precipitin curves in a more easily interpreted format than did conventional immunoelectrophoresis (IEP), 2D-IEP offered greater sensitivity in terms of numbers of precipitin curves when like reactions were compared with IEP patterns. As many as 60 immunoprecipitates were observed on 2D-IEP slides compared to 18 on comparable IEP plates. Technical reproducibility of patterns from run to run was excellent. Other parameters, such as the influence of using different batches of antigen on the pattern, are discussed. Each of the cell extract antigens gave a unique pattern of precipitin peaks which could be easily differentiated from the patterns given by the other mycobacterial cell extracts when reacted with any of the antisera in 2D-IEP. Since both the species and strains of mycobacteria could be easily and reproducibly differentiated solely on the basis of two-dimensional immunoelectrophoretic patterns obtained with any of the antisera employed in this study, it may be possible, by using IEP, to differentiate and identify all species and strains of mycobacteria with one standard, highly sensitive antiserum, rather than a battery of antisera. PMID:4628899

  19. Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG.

    PubMed

    Zahrt, T C; Song, J; Siple, J; Deretic, V

    2001-03-01

    In several bacteria, the catalase-peroxidase gene katG is under positive control by oxyR, a transcriptional regulator of the peroxide stress response. The Mycobacterium tuberculosis genome also contains sequences corresponding to oxyR, but this gene has been inactivated in the tubercle bacillus because of the presence of multiple mutations and deletions. Thus, M. tuberculosis katG and possibly other parts of the oxidative stress response in this organism are either not regulated or are controlled by a factor different from OxyR. The mycobacterial FurA is a homologue of the ferric uptake regulator Fur and is encoded by a gene located immediately upstream of katG. Here, we examine the possibility that FurA regulates katG expression. Inactivation of furA on the Mycobacterium smegmatis chromosome, a mycobacterial species that also lacks an oxyR homologue, resulted in derepression of katG, concomitant with increased resistance of the furA mutant to H2O2. In addition, M. smegmatis furA::Km(r) was more sensitive to the front-line antituberculosis agent isonicotinic acid hydrazide (INH) compared with the parental furA+ strain. The phenotypic manifestations were specific, as the mutant strain did not show altered sensitivity to organic peroxides, and both H2O2 and INH susceptibility profiles were complemented by the wild-type furA+ gene. We conclude that FurA is a second regulator of oxidative stress response in mycobacteria and that it negatively controls katG. In species lacking a functional oxyR, such as M. tuberculosis and M. smegmatis, FurA appears to be a dominant regulator affecting mycobacterial physiology and intracellular survival. PMID:11251835

  20. Mycobacterial Pan-Genome Analysis Suggests Important Role of Plasmids in the Radiation of Type VII Secretion Systems

    PubMed Central

    Dumas, Emilie; Christina Boritsch, Eva; Vandenbogaert, Mathias; Rodríguez de la Vega, Ricardo C.; Thiberge, Jean-Michel; Caro, Valerie; Gaillard, Jean-Louis; Heym, Beate; Girard-Misguich, Fabienne; Brosch, Roland; Sapriel, Guillaume

    2016-01-01

    In mycobacteria, various type VII secretion systems corresponding to different ESX (ESAT-6 secretory) types, are contributing to pathogenicity, iron acquisition, and/or conjugation. In addition to the known chromosomal ESX loci, the existence of plasmid-encoded ESX systems was recently reported. To investigate the potential role of ESX-encoding plasmids on mycobacterial evolution, we analyzed a large representative collection of mycobacterial genomes, including both chromosomal and plasmid-borne sequences. Data obtained for chromosomal ESX loci confirmed the previous five classical ESX types and identified a novel mycobacterial ESX-4-like type, termed ESX-4-bis. Moreover, analysis of the plasmid-encoded ESX loci showed extensive diversification, with at least seven new ESX profiles, identified. Three of them (ESX-P clusters 1–3) were found in multiple plasmids, while four corresponded to singletons. Our phylogenetic and gene-order-analyses revealed two main groups of ESX types: 1) ancestral types, including ESX-4 and ESX-4-like systems from mycobacterial and non-mycobacterial actinobacteria and 2) mycobacteria-specific ESX systems, including ESX-1-2-3-5 systems and the plasmid-encoded ESX types. Synteny analysis revealed that ESX-P systems are part of phylogenetic groups that derived from a common ancestor, which diversified and resulted in the different ESX types through extensive gene rearrangements. A converging body of evidence, derived from composition bias-, phylogenetic-, and synteny analyses points to a scenario in which ESX-encoding plasmids have been a major driving force for acquisition and diversification of type VII systems in mycobacteria, which likely played (and possibly still play) important roles in the adaptation to new environments and hosts during evolution of mycobacterial pathogenesis. PMID:26748339

  1. Mycobacterial Pan-Genome Analysis Suggests Important Role of Plasmids in the Radiation of Type VII Secretion Systems.

    PubMed

    Dumas, Emilie; Christina Boritsch, Eva; Vandenbogaert, Mathias; Rodríguez de la Vega, Ricardo C; Thiberge, Jean-Michel; Caro, Valerie; Gaillard, Jean-Louis; Heym, Beate; Girard-Misguich, Fabienne; Brosch, Roland; Sapriel, Guillaume

    2016-02-01

    In mycobacteria, various type VII secretion systems corresponding to different ESX (ESAT-6 secretory) types, are contributing to pathogenicity, iron acquisition, and/or conjugation. In addition to the known chromosomal ESX loci, the existence of plasmid-encoded ESX systems was recently reported. To investigate the potential role of ESX-encoding plasmids on mycobacterial evolution, we analyzed a large representative collection of mycobacterial genomes, including both chromosomal and plasmid-borne sequences. Data obtained for chromosomal ESX loci confirmed the previous five classical ESX types and identified a novel mycobacterial ESX-4-like type, termed ESX-4-bis. Moreover, analysis of the plasmid-encoded ESX loci showed extensive diversification, with at least seven new ESX profiles, identified. Three of them (ESX-P clusters 1-3) were found in multiple plasmids, while four corresponded to singletons. Our phylogenetic and gene-order-analyses revealed two main groups of ESX types: 1) ancestral types, including ESX-4 and ESX-4-like systems from mycobacterial and non-mycobacterial actinobacteria and 2) mycobacteria-specific ESX systems, including ESX-1-2-3-5 systems and the plasmid-encoded ESX types. Synteny analysis revealed that ESX-P systems are part of phylogenetic groups that derived from a common ancestor, which diversified and resulted in the different ESX types through extensive gene rearrangements. A converging body of evidence, derived from composition bias-, phylogenetic-, and synteny analyses points to a scenario in which ESX-encoding plasmids have been a major driving force for acquisition and diversification of type VII systems in mycobacteria, which likely played (and possibly still play) important roles in the adaptation to new environments and hosts during evolution of mycobacterial pathogenesis. PMID:26748339

  2. Rapid synthesis of linear homologous oligoarabinofuranosides related to mycobacterial lipoarabinomannan and a neoglycoconjugate thereof.

    PubMed

    Podvalnyy, Nikita M; Chizhov, Alexander O; Zinin, Alexander I; Kononov, Leonid O

    2016-08-01

    Rapid and simple synthesis of oligosaccharides related to one of the terminal motifs of mycobacterial lipoarabinomannan is described. An array of homologous linear α(1 → 5)-linked oligoarabinofuranosides with 4-(2-chloroethoxy)phenyl aglycon and selectively unprotected 5-OH group at the non-reducing end was obtained by oligomerization of 3-O-benzoyl β-D-arabinofuranose 1,2,5-orthobenzoate. Subsequent introduction of β(1 → 2)-linked arabinofuranose disaccharide moiety by step-wise glycosylation furnished the target oligosaccharides which were conjugated with bovine serum albumin. PMID:27267065

  3. Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction

    PubMed Central

    Chen, Tingting; Blanc, Caroline; Eder, Anke Z.; Prados-Rosales, Rafael; Souza, Ana Camila Oliveira; Kim, Ryung S.; Glatman-Freedman, Aharona; Joe, Maju; Bai, Yu; Lowary, Todd L.; Tanner, Rachel; Brennan, Michael J.; Fletcher, Helen A.; McShane, Helen; Casadevall, Arturo; Achkar, Jacqueline M.

    2016-01-01

    Background. The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. Methods. Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. Results. Immunoglobulin G (IgG) responses to AM increased significantly 4–8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. Conclusions. Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans. PMID:27056953

  4. Crystal structures of Mycobacterial MeaB and MMAA-like GTPases.

    PubMed

    Edwards, Thomas E; Baugh, Loren; Bullen, Jameson; Baydo, Ruth O; Witte, Pam; Thompkins, Kaitlin; Phan, Isabelle Q H; Abendroth, Jan; Clifton, Matthew C; Sankaran, Banumathi; Van Voorhis, Wesley C; Myler, Peter J; Staker, Bart L; Grundner, Christoph; Lorimer, Donald D

    2015-06-01

    The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB. PMID:25832174

  5. Crystal structures of Mycobacterial MeaB and MMAA-like GTPases

    PubMed Central

    Baugh, Loren; Bullen, Jameson; Baydo, Ruth O.; Witte, Pam; Thompkins, Kaitlin; Phan, Isabelle Q.H.; Abendroth, Jan; Clifton, Matthew C.; Sankaran, Banumathi; Van Voorhis, Wesley C.; Myler, Peter J.; Staker, Bart L.; Grundner, Christoph; Lorimer, Donald D.

    2015-01-01

    The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB. PMID:25832174

  6. Active site of mycobacterial dUTPase: Structural characteristics and a built-in sensor

    SciTech Connect

    Varga, Balazs; Barabas, Orsolya; Takacs, Eniko; Nagy, Nikolett; Nagy, Peter; Vertessy, Beata G.

    2008-08-15

    dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, {alpha},{beta}-imido-dUTP and Mg{sup 2+} at 1.5 A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K{sub d} for {alpha},{beta}-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.

  7. Developments on drug delivery systems for the treatment of mycobacterial infections.

    PubMed

    Gaspar, M M; Cruz, A; Fraga, A G; Castro, A G; Cruz, M E M; Pedrosa, J

    2008-01-01

    The clinical management of tuberculosis and other mycobacterial diseases with antimycobacterial chemotherapy remains a difficult task. The classical treatment protocols are long-lasting; the drugs reach mycobacteria-infected macrophages in low amounts and/or do not persist long enough to develop the desired antimycobacterial effect; and the available agents induce severe toxic effects. Nanotechnology has provided a huge improvement to pharmacology through the designing of drug delivery systems able to target phagocytic cells infected by intracellular pathogens, such as mycobacteria. Liposomes and nanoparticles of polymeric nature represent two of the most efficient drug carrier systems that after in vivo administration are endocytosed by phagocytic cells and then release the carried agents into these cells. This article reviews the relevant publications describing the effectiveness of the association of antimycobacterial agents with liposomes or nanoparticles for the treatment of mycobacterioses, particularly for Mycobacterium tuberculosis and M. avium infections. The increased therapeutic index of antimycobacterial drugs; the reduction of dosing frequency; and the improvement of solubility of hydrophobic agents, allowing the administration of higher doses, have been demonstrated in experimental infections. These advantages may lead to new therapeutic protocols that will improve patient compliance and, consequently, lead to a more successful control of mycobacterial infections. The potential therapeutic advantages resulting from the use of non-invasive administration routes for nanoparticulate systems are also discussed. PMID:18473884

  8. Development of a murine mycobacterial growth inhibition assay for evaluating vaccines against Mycobacterium tuberculosis.

    PubMed

    Parra, Marcela; Yang, Amy L; Lim, JaeHyun; Kolibab, Kristopher; Derrick, Steven; Cadieux, Nathalie; Perera, Liyanage P; Jacobs, William R; Brennan, Michael; Morris, Sheldon L

    2009-07-01

    The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability. PMID:19458207

  9. The internal organization of mycobacterial partition assembly: does the DNA wrap a protein core?

    PubMed

    Qian, Shuo; Dean, Rebecca; Urban, Volker S; Chaudhuri, Barnali N

    2012-01-01

    Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A "DNA outside, protein inside" mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA. PMID:23285150

  10. A Redox Regulatory System Critical for Mycobacterial Survival in Macrophages and Biofilm Development

    PubMed Central

    Wolff, Kerstin A.; de la Peña, Andres H.; Nguyen, Hoa T.; Pham, Thanh H.; Amzel, L. Mario; Gabelli, Sandra B.; Nguyen, Liem

    2015-01-01

    Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments. PMID:25884716

  11. Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.

    PubMed

    Egen, Jackson G; Rothfuchs, Antonio Gigliotti; Feng, Carl G; Winter, Nathalie; Sher, Alan; Germain, Ronald N

    2008-02-01

    Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations formed a relatively immobile cellular matrix that interacted with a highly dynamic effector T cell population. The efficient recruitment of these T cells was highly dependent on TNF-alpha-derived signals, which also maintained the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape. PMID:18261937

  12. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle

    PubMed Central

    Ishikawa, Eri; Ishikawa, Tetsuaki; Morita, Yasu S.; Toyonaga, Kenji; Yamada, Hisakata; Takeuchi, Osamu; Kinoshita, Taroh; Akira, Shizuo; Yoshikai, Yasunobu

    2009-01-01

    Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6′-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM. PMID:20008526

  13. Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

    USGS Publications Warehouse

    Ramsay, J.M.; Watral, V.; Schreck, C.B.; Kent, M.L.

    2009-01-01

    Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

  14. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    SciTech Connect

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-08-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.

  15. Dynamics of Mycobacteriophage-Mycobacterial Host Interaction: Evidence for Secondary Mechanisms for Host Lethality

    PubMed Central

    Samaddar, Sourabh; Grewal, Rajdeep Kaur; Sinha, Saptarshi; Ghosh, Shrestha

    2015-01-01

    Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms. PMID:26475112

  16. Mycobacterial growth and sensitivity to H2O2 killing in human monocytes in vitro.

    PubMed Central

    Laochumroonvorapong, P; Paul, S; Manca, C; Freedman, V H; Kaplan, G

    1997-01-01

    The intracellular growth and susceptibilities to killing by H2O2 in cultured human monocytes of a number of mycobacterial species including laboratory strains and clinical isolates of Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin (BCG) and a clinical isolate of Mycobacterium avium-M. intracellulare were examined. The clinical isolate of M. avium-M. intracellulare did not replicate in freshly explanted monocytes (generation time of >400 h); BCG replicated with a generation time of 95 h, and M. tuberculosis strains CDC551, H37Rv, and H37Ra replicated with generation times of 24, 35, and 37 h, respectively, during the 4-day growth assay. When cultured in monocytes for 4 days, the mycobacteria were variably sensitive to H2O2-induced killing. A positive correlation between the generation time and percent killing of intracellular bacilli was observed. By comparison, mycobacterial strains were similarly sensitive to H2O2 treatment in cell-free culture media and in sonicated cell suspensions. Using a number of inhibitors of reactive oxygen intermediates we determined that other than catalase the inhibitors tested did not affect H2O2-induced killing of intracellular mycobacteria. Our studies suggest that the killing of mycobacteria growing in human monocytes in vitro by the addition of exogenous H2O2 is dependent on the susceptibility to a peroxide-induced killing pathway as well as on the intracellular growth rate of the mycobacteria. PMID:9353075

  17. Mycobacterial P1-Type ATPases Mediate Resistance to Zinc Poisoning in Human Macrophages

    PubMed Central

    Botella, Hélène; Peyron, Pascale; Levillain, Florence; Poincloux, Renaud; Poquet, Yannick; Brandli, Irène; Wang, Chuan; Tailleux, Ludovic; Tilleul, Sylvain; Charrière, Guillaume M.; Waddell, Simon J.; Foti, Maria; Lugo-Villarino, Geanncarlo; Gao, Qian; Maridonneau-Parini, Isabelle; Butcher, Philip D.; Castagnoli, Paola Ricciardi; Gicquel, Brigitte; de Chastellier, Chantal; Neyrolles, Olivier

    2011-01-01

    Summary Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P1-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race. PMID:21925112

  18. Incidence, characteristics, and treatment outcomes of mycobacterial diseases in transplant recipients.

    PubMed

    Yoo, Jung-Wan; Jo, Kyung-Wook; Kim, Sung-Han; Lee, Sang-Oh; Kim, Jae Joong; Park, Su-Kil; Lee, Je-Hwan; Han, Duck Jong; Hwang, Shin; Lee, SeungGyu; Shim, Tae Sun

    2016-05-01

    The incidence, clinical characteristics, and treatment outcomes of tuberculosis (TB) and nontuberculous mycobacterial (NTM) disease developed after transplantation (TPL) in transplant recipients were investigated retrospectively. Between 1996 and 2013, 7342 solid-organ transplantation and 1266 hematopoietic stem cell transplantation were performed at a tertiary referral center in South Korea. Among them, TB and NTM disease developed in 130 and 22 patients, respectively. The overall incidence of TB was 257.4 cases/100 000 patient-years (95% confidence interval [CI], 215.1-305.7) and that of NTM disease was 42.7 cases/100 000 patient-years (95% CI, 26.8-64.7). The median interval from organ TPL to the development of mycobacterial disease was 8.5 months (95% CI, 6.3-11.4) in recipients with TB patients and 24.2 months (95% CI, 13.5-55.7) in those with NTM, respectively. Among NTM patients, Mycobacterium avium-intracellulare complex was the most common causative organism, and nodular bronchiectatic type (77.8%) was the most frequent radiologic feature. Favorable treatment outcome was achieved in 83.7% (95% CI, 76.4-89.1) and 68.8% (95% CI, 44.4-85.8) of TB and NTM patients, respectively (P = 0.166). In conclusion, the overall incidence of TB was higher than that of NTM disease in transplant recipients and treatment outcomes were favorable in both drug-susceptible TB and NTM patients. PMID:26840221

  19. Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids*

    PubMed Central

    Vats, Archana; Singh, Anil Kumar; Mukherjee, Raju; Chopra, Tarun; Ravindran, Madhu Sudhan; Mohanty, Debasisa; Chatterji, Dipankar; Reyrat, Jean-Marc; Gokhale, Rajesh S.

    2012-01-01

    Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. PMID:22798073

  20. A chemically synthesized peptide which elicits humoral and cellular immune responses to mycobacterial antigens.

    PubMed Central

    Minden, P; Houghten, R A; Spear, J R; Shinnick, T M

    1986-01-01

    Monoclonal antibodies directed to Mycobacterium bovis BCG (BCG) and to M. tuberculosis H37Rv (H37Rv) were used in conjunction with affinity chromatography to prepare a mycobacterial component which was designated BCG-a. A synthetic peptide antigen was prepared based on the amino acid sequence of BCG-a and was designated BCG-a-P. Significant immunological similarities were found between BCG-a-P and antigens in extracts of BCG and H37Rv but not between BCG-a-P and antigens of nontuberculous mycobacteria. An enzyme-linked immunosorbent assay detected antibodies to BCG-a-P in sera from rabbits that had been immunized with BCG and H37Rv sonicates. In Western blot analysis, antibodies to BCG-a-P reacted to 10,000-molecular-weight components of extracts of BCG and H37Rv. Delayed cutaneous hypersensitivity reactions to BCG-a-P were elicited in guinea pigs immunized with sonicates of BCG and H37Rv but were weak or nonexistent in unimmunized animals or in animals immunized with sonicates of nontuberculous mycobacteria. This study points out the feasibility of using monoclonal antibodies to prepare and characterize synthetic mycobacterial peptides with a potential for immunodiagnostic purposes. Images PMID:3744551

  1. The characterization of DINE-1, a short, interspersed repetitive element present on chromosome and in the centric heterochromatin of Drosophila melanogaster.

    PubMed

    Locke, J; Howard, L T; Aippersbach, N; Podemski, L; Hodgetts, R B

    1999-11-01

    The banded portion of chromosome 4 (the "dot" chromosome) in Drosophila melanogaster displays some properties of beta-heterochromatin, which is normally found within the centric domain of the chromosomes. The nature and distribution of repetitive elements on chromosome 4 could play a role in the establishment of this unusual chromatin configuration. We describe here one such element: a short, interspersed repetitive sequence named DINE-1. Determination of a consensus sequence for the element reveals that there are two conserved regions (A and B) separated by a highly variable spacer. The conserved sequences are approximately 400 bp long but degenerate at both ends, opening the possibility that a yet-to-be-discovered mother element may be present in the genome. DINE-1 bears few of the properties of the mammalian short interspersed elements (SINEs) to which it bears a superficial resemblance in size. It does not appear to be the product of reverse transcription and lacks any polymerase III promoter consensus. The elements are not flanked by target site duplications and their termini lack direct or inverted repeats, suggesting that they themselves are not transposable. Our analysis of cosmid clones from chromosome 4, and elsewhere in the genome, revealed that the euchromatic locations of DINE-1 are almost exclusively confined to chromosome 4. In situ hybridization of a DINE-1 probe to polytene chromosomes confirmed the preferential distribution along 4, in addition to its presence in the centric heterochromatin. This unusual genomic distribution of bias toward chromosome 4 is also seen in the sibling species, D. simulans, whose dot chromosomes exhibit poorly resolved polytene bands and lack crossing over during meiosis like those of D. melanogaster. However, the dot chromosome of D. virilis, which exhibits a well-defined banded structure on polytene chromosomes and can cross over, has only a single, discrete site of DINE-1 element hybridization. The presence of DINE-1

  2. Evaluation of Performance of the Real-Q NTM-ID Kit for Rapid Identification of Eight Nontuberculous Mycobacterial Species

    PubMed Central

    Huh, Hee Jae; Park, Kyung Sun; Jang, Mi-Ae; Kim, Ji-Youn; Kwon, Hyeon Jeong

    2014-01-01

    We evaluated a multiplex real-time PCR and melting curve analysis assay (Real-Q NTM-ID kit; Biosewoom, Seoul, South Korea) for the identification of eight common nontuberculous mycobacterial species, using 30 type strains and 230 consecutive clinical isolates. The concordance rate of this assay with multigene sequence-based typing was 97.0% (223/230 isolates). PMID:25165078

  3. Synthesis of Pt-Loaded Self-Interspersed Anatase TiO2 with a Large Fraction of (001) Facets for Efficient Photocatalytic Nitrobenzene Degradation.

    PubMed

    Wang, Wei-Kang; Chen, Jie-Jie; Li, Wen-Wei; Pei, Dan-Ni; Zhang, Xing; Yu, Han-Qing

    2015-09-16

    TiO2 is capable of directly utilizing solar energy for sustainable energy harvest and water purification. Facet-dependent performance of TiO2 has attracted enormous interests due to its tunable photocatalytic activity toward photoredox transformations, but information about the noble-metal-loaded TiO2 for its facet-dependent photocatalytic performance, especially in pollutant degradation systems, is limited. In this work, inspired by our previous theoretical calculations about the roles of the crystal surface in Pt-loaded TiO2 in its enhanced photocatalytic capacity, TiO2 nanocrystals with interspersed polyhedron nanostructures and coexposed (001) and (101) surfaces as a support of Pt nanoparticles are prepared in a simple and relatively green route. Also, their performance for photocatalytic degradation of nitrobenzene (NB), a model organic pollutant, is explored. The experimental results demonstrate that the NB photodegradation and photoconversion efficiencies are significantly enhanced by uniformly loading Pt nanoparticles on the crystal surfaces, but the Pt nanoparticles deposited on only the (101) surface have no contribution to the improved NB photodegradation. Furthermore, the liquid chromatography mass spectrometry results also show that NB photodegradation tends to proceed on the (001) surface of Pt/TiO2 for the generation of nitrophenol intermediates through the photooxidation pathway. This work provides a new route to design and construct advanced photocatalysts toward pollutant photoredox conversions and deepens our fundamental understanding about crystal surface engineering. PMID:26308282

  4. Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence.

    PubMed

    Bachellier, S; Clément, J M; Hofnung, M; Gilson, E

    1997-03-01

    A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed. PMID:9055066

  5. Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

    PubMed Central

    McCarthy, L; Hunter, K; Schalkwyk, L; Riba, L; Anson, S; Mott, R; Newell, W; Bruley, C; Bar, I; Ramu, E

    1995-01-01

    The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7777502

  6. Short interspersed nuclear elements (SINEs) are abundant in Solanaceae and have a family-specific impact on gene structure and genome organization.

    PubMed

    Seibt, Kathrin M; Wenke, Torsten; Muders, Katja; Truberg, Bernd; Schmidt, Thomas

    2016-05-01

    Short interspersed nuclear elements (SINEs) are highly abundant non-autonomous retrotransposons that are widespread in plants. They are short in size, non-coding, show high sequence diversity, and are therefore mostly not or not correctly annotated in plant genome sequences. Hence, comparative studies on genomic SINE populations are rare. To explore the structural organization and impact of SINEs, we comparatively investigated the genome sequences of the Solanaceae species potato (Solanum tuberosum), tomato (Solanum lycopersicum), wild tomato (Solanum pennellii), and two pepper cultivars (Capsicum annuum). Based on 8.5 Gbp sequence data, we annotated 82 983 SINE copies belonging to 10 families and subfamilies on a base pair level. Solanaceae SINEs are dispersed over all chromosomes with enrichments in distal regions. Depending on the genome assemblies and gene predictions, 30% of all SINE copies are associated with genes, particularly frequent in introns and untranslated regions (UTRs). The close association with genes is family specific. More than 10% of all genes annotated in the Solanaceae species investigated contain at least one SINE insertion, and we found genes harbouring up to 16 SINE copies. We demonstrate the involvement of SINEs in gene and genome evolution including the donation of splice sites, start and stop codons and exons to genes, enlargement of introns and UTRs, generation of tandem-like duplications and transduction of adjacent sequence regions. PMID:26996788

  7. Duplication of the gamma-globin gene mediated by L1 long interspersed repetitive elements in an early ancestor of simian primates.

    PubMed Central

    Fitch, D H; Bailey, W J; Tagle, D A; Goodman, M; Sieu, L; Slightom, J L

    1991-01-01

    Regions surrounding the single gamma-globin gene of galago and the duplicated gamma 1- and gamma 2-globin genes of gibbon, rhesus monkey, and spider monkey were sequenced and aligned with those from humans. Contrary to previous studies, spider monkey was found to have not one but two gamma-globin genes, only one of which (gamma 2) is functional. The reconstructed evolutionary history of the gamma-globin genes and their flanking sequences traces their origin to a tandem duplication of a DNA segment approximately 5.5 kilobases long that occurred before catarrhine primates (humans, apes, and Old World monkeys) diverged from platyrrhines (New World monkeys), much earlier than previously thought. This reconstructed molecular history also reveals that the duplication resulted from an unequal homologous crossover between two related L1 long interspersed repetitive elements, one upstream and one downstream of the single ancestral gamma-globin gene. Perhaps facilitated by the redundancy resulting from the duplication, the gamma-globin genes escaped the selective constraints of embryonically functioning genes and evolved into fetally functioning genes. This view is supported by the finding that a burst of nonsynonymous substitutions occurred in the gamma-globin genes while they became restructured for fetal expression in the common ancestor of platyrrhines and catarrhines. PMID:1908094

  8. Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2

    PubMed Central

    2011-01-01

    Background Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. Results We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. Conclusion Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual

  9. Lung surfactant dysfunction in tuberculosis: effect of mycobacterial tubercular lipids on dipalmitoylphosphatidylcholine surface activity.

    PubMed

    Chimote, G; Banerjee, R

    2005-11-10

    In pulmonary tuberculosis, Mycobacterium tuberculosis bacteria reside in the alveoli and are in close proximity with the alveolar surfactant. Mycolic acid in its free form and as cord factor, constitute the major lipids of the mycobacterial cell wall. They can detach from the bacteria easily and are known to be moderately surface active. We hypothesize that these surface-active mycobacterial cell wall lipids could interact with the pulmonary surfactant and result in lung surfactant dysfunction. In this study, the major phospholipid of the lung surfactant, dipalmitoylphosphatidylcholine (DPPC) and binary mixtures of DPPC:phosphatidylglycerol (PG) in 9:1 and 7:3 ratios were modelled as lung surfactant monolayers and the inhibitory potential of mycolic acid and cord factor on the surface activity of DPPC and DPPC:PG mixtures was evaluated using Langmuir monolayers. The mycobacterial lipids caused common profile changes in all the isotherms: increase in minimum surface tension, compressibility and percentage area change required for change in surface tension from 30 to 10 mN/m. Higher minimum surface tension values were achieved in the presence of mycolic acid (18.2+/-0.7 mN/m) and cord factor (13.28+/-1.2 mN/m) as compared to 0 mN/m, achieved by pure DPPC film. Similarly higher values of compressibility (0.375+/-0.005 m/mN for mycolic acid:DPPC and 0.197+/-0.003 m/mN for cord factor:DPPC monolayers) were obtained in presence of mycolic acid and cord factor. Thus, mycolic acid and cord factor were said to be inhibitory towards lung surfactant phospholipids. Higher surface tension and compressibility values in presence of tubercular lipids are suggestive of an unstable and fluid surfactant film, which will fail to achieve low surface tensions and can contribute to alveolar collapse in patients suffering from pulmonary tuberculosis. In conclusion a biophysical inhibition of lung surfactant may play a role in the pathogenesis of tuberculosis and may serve as a target for

  10. Mycobacterial Infections

    MedlinePlus

    ... similar to tuberculosis: Cough Weight loss Coughing up blood or mucus Weakness or fatigue Fever and chills Night sweats Lack of appetite and weight loss Medicines can treat these infections, but often more than one is needed to cure the infection.