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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. 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

  8. 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

  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. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  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 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

  12. 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

  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. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. '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

  10. ‘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

  11. 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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  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. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  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. 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

  18. 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

  19. 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

  20. 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

  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. 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

  3. 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

  4. 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

  5. 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

  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. 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.

  19. 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

  20. 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

  1. 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

  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. 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

  8. 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

  9. 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

  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. 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

  13. 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

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  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. 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

  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 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  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

    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. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  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. 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

  13. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  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. 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

  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. 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

  6. 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

  7. 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

  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 culture

    MedlinePlus

    ... test to look for the bacteria that cause tuberculosis and similar infections. How the Test is Performed ... order this test if you have signs of tuberculosis or a related infection. Normal Results If there ...

  11. Mycobacterial Infections

    MedlinePlus

    ... many different kinds. The most common one causes tuberculosis. Another one causes leprosy. Still others cause infections ... aren't "typical" because they don't cause tuberculosis. But they can still harm people, especially people ...

  12. 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.

  13. Mycobacterial polysaccharides. II. Comparison of polysaccharides from strains of four species of mycobacteria.

    PubMed

    Birnbaum, S E; Affronti, L F

    1969-10-01

    Evidence from chemical and serological studies indicates that a cellular heteropolysaccharide, also found in lipid extracts and culture filtrate, is present as a group antigen in Mycobacterium tuberculosis H37Ra and in other strains of mycobacteria representing M. kansasii, scotochromogenic and Battey strains. Polysaccharides from the four strains contain the same main sugars, arabinose, and galactose, as revealed by thin-layer chromatography and spectrophotometric studies. In Ouchterlony gel diffusions, bands of identity are produced between the polysaccharides by using rabbit antiserum prepared against any of the four mycobacteria. Immune adsorption studies also confirm the presence of identical antigenic determinant groups. In skin tests with tuberculopolysaccharide I, a skin reaction of about equal size was elicited in guinea pigs sensitized with either M. tuberculosis H37Ra or heterologous mycobacterial antigens in Freund's incomplete adjuvant. In animals sensitized with M. tuberculosis H37Ra, skin tests with both homologous and heterologous polysaccharides elicited similar responses. PMID:4981066

  14. Leveraging Advances in Tuberculosis Diagnosis and Treatment to Address Nontuberculous Mycobacterial Disease

    PubMed Central

    Zhao, Yanlin; Rubin, Eric J.

    2016-01-01

    The nontuberculous mycobacteria (NTM), defined as any mycobacterial pathogen other than Mycobacterium tuberculosis or Mycobacterium leprae, are a diverse group of pathogens that collectively cause a substantive but often unappreciated worldwide burden of illness. Although NTMs may cause illness similar to M. tuberculosis, these pathogens generally do not respond to classic tuberculosis (TB) drug regimens, resulting in misdiagnosis and poor treatment, particularly in resource-poor settings. Although a few high-quality epidemiologic surveys have been made on the topic, existing evidence suggests that NTM-associated disease is much more common than previously thought: more common than TB in the industrialized world and likely increasing in prevalence globally. Despite this evidence, these organisms remain markedly understudied, and few international grants support basic science and clinical research. Here we suggest that the considerable efforts in developing new treatments and diagnostics for TB can be harnessed in the fight against NTM-associated illnesses. PMID:26886068

  15. Two Episodes of Cutaneous Non-Tuberculous Mycobacterial Infection in a Patient with Psoriasis

    PubMed Central

    Chan, Wai Sze Agnes; Tee, Shang-Ian; Chandran, Nisha Su Yien; Pan, Jiun Yit

    2015-01-01

    Non-tuberculous mycobacteria (NTM) are a group of environmental pathogens, which cause a broad spectrum of disease. The incidence of NTM infection is increasing, especially in immunocompromized patients. The past three decades also saw a rapid increase in the incidence of NTM infection involving otherwise healthy subjects. We report a case of cutaneous NTM infection in a 79-year-old Chinese woman, who was receiving methotrexate for psoriasis. Mycobacterial culture grew Mycobacterium abscessus, and the lesions cleared with a combination of oral clarithromycin, ciprofloxacin and doxycycline. Interestingly, she then developed a second episode of cutaneous NTM infection with Mycobacterium haemophilum over the same body region, five years after stoppage of methotrexate. Both episodes were separated in time and involved different species, indicating that they were independent from each other. We further discuss the risk factors for cutaneous NTM infection, treatment, and highlight the need for diagnostic vigilance. PMID:26236445

  16. The acylation state of mycobacterial lipomannans modulates innate immunity response through toll-like receptor 2.

    PubMed

    Gilleron, Martine; Nigou, Jérôme; Nicolle, Delphine; Quesniaux, Valérie; Puzo, Germain

    2006-01-01

    Detection of Mycobacterium tuberculosis antigens by professional phagocytes via toll-like receptors (TLR) contributes to controlling chronic M. tuberculosis infection. Lipomannans (LM), which are major lipoglycans of the mycobacterial envelope, were recently described as agonists of TLR2 with potent activity on proinflammatory cytokine regulation. LM correspond to a heterogeneous population of acyl- and glyco-forms. We report here the purification and the complete structural characterization of four LM acyl-forms from Mycobacterium bovis BCG using MALDI MS and 2D (1)H-(31)P NMR analyses. All this biochemical work provided the tools to investigate the implication of LM acylation degree on its proinflammatory activity. The latter was ascribed to the triacylated LM form, essentially an agonist of TLR2, using TLR2/TLR1 heterodimers for signaling. Altogether, these findings shed more light on the molecular basis of LM recognition by TLR. PMID:16426970

  17. Two Episodes of Cutaneous Non-Tuberculous Mycobacterial Infection in a Patient with Psoriasis.

    PubMed

    Chan, Wai Sze Agnes; Tee, Shang-Ian; Chandran, Nisha Su Yien; Pan, Jiun Yit

    2015-05-21

    Non-tuberculous mycobacteria (NTM) are a group of environmental pathogens, which cause a broad spectrum of disease. The incidence of NTM infection is increasing, especially in immunocompromized patients. The past three decades also saw a rapid increase in the incidence of NTM infection involving otherwise healthy subjects. We report a case of cutaneous NTM infection in a 79-year-old Chinese woman, who was receiving methotrexate for psoriasis. Mycobacterial culture grew Mycobacterium abscessus, and the lesions cleared with a combination of oral clarithromycin, ciprofloxacin and doxycycline. Interestingly, she then developed a second episode of cutaneous NTM infection with Mycobacterium haemophilum over the same body region, five years after stoppage of methotrexate. Both episodes were separated in time and involved different species, indicating that they were independent from each other. We further discuss the risk factors for cutaneous NTM infection, treatment, and highlight the need for diagnostic vigilance. PMID:26236445

  18. Leveraging Advances in Tuberculosis Diagnosis and Treatment to Address Nontuberculous Mycobacterial Disease.

    PubMed

    Raju, Ravikiran M; Raju, Sagar M; Zhao, Yanlin; Rubin, Eric J

    2016-03-01

    The nontuberculous mycobacteria (NTM), defined as any mycobacterial pathogen other than Mycobacterium tuberculosis or Mycobacterium leprae, are a diverse group of pathogens that collectively cause a substantive but often unappreciated worldwide burden of illness. Although NTMs may cause illness similar to M. tuberculosis, these pathogens generally do not respond to classic tuberculosis (TB) drug regimens, resulting in misdiagnosis and poor treatment, particularly in resource-poor settings. Although a few high-quality epidemiologic surveys have been made on the topic, existing evidence suggests that NTM-associated disease is much more common than previously thought: more common than TB in the industrialized world and likely increasing in prevalence globally. Despite this evidence, these organisms remain markedly understudied, and few international grants support basic science and clinical research. Here we suggest that the considerable efforts in developing new treatments and diagnostics for TB can be harnessed in the fight against NTM-associated illnesses. PMID:26886068

  19. Highly Deviated Asymmetric Division in Very Low Proportion of Mycobacterial Mid-log Phase Cells

    PubMed Central

    Vijay, Srinivasan; Mukkayyan, Nagaraja; Ajitkumar, Parthasarathi

    2014-01-01

    In this study, we show that about 20% of the septating Mycobacterium smegmatis and Mycobacterium xenopi cells in the exponential phase populationdivideasymmetrically, with an unusually high deviation (17 ± 4%) in the division site from the median, to generate short cells and long cells, thereby generating population heterogeneity. This mode of division is very different from the symmetric division of themajority (about 80%) of the septating cells in the Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG exponential phase population, with 5-10% deviation in the division site from the mid-cell site, as reported by recent studies. The short cells and the long cells further grew and divided to generate a population. We speculate that the generation of the short cells and the long cells through the highly deviated asymmetric divisionin the low proportions of mycobacterial population may have a role in stress tolerance. PMID:24949109

  20. Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential.

    PubMed

    Wehenkel, Annemarie; Bellinzoni, Marco; Graña, Martin; Duran, Rosario; Villarino, Andrea; Fernandez, Pablo; Andre-Leroux, Gwénaëlle; England, Patrick; Takiff, Howard; Cerveñansky, Carlos; Cole, Stewart T; Alzari, Pedro M

    2008-01-01

    Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds. PMID:17869195

  1. Functional plasticity and allosteric regulation of α-ketoglutarate decarboxylase in central mycobacterial metabolism.

    PubMed

    Wagner, Tristan; Bellinzoni, Marco; Wehenkel, Annemarie; O'Hare, Helen M; Alzari, Pedro M

    2011-08-26

    The α-ketoglutarate dehydrogenase (KDH) complex is a major regulatory point of aerobic energy metabolism. Mycobacterium tuberculosis was reported to lack KDH activity, and the putative KDH E1o component, α-ketoglutarate decarboxylase (KGD), was instead assigned as a decarboxylase or carboligase. Here, we show that this protein does in fact sustain KDH activity, as well as the additional two reactions, and these multifunctional properties are shared by the Escherichia coli homolog, SucA. We also show that the mycobacterial enzyme is finely regulated by an additional acyltransferase-like domain and by the action of acetyl-CoA, a powerful allosteric activator able to enhance the concerted protein motions observed during catalysis. Our results uncover the functional plasticity of a crucial node in bacterial metabolism, which may be important for M. tuberculosis during host infection. PMID:21867916

  2. Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides.

    PubMed

    Hanashima, Shinya; Götze, Sebastian; Liu, Yan; Ikeda, Akemi; Kojima-Aikawa, Kyoko; Taniguchi, Naoyuki; Varón Silva, Daniel; Feizi, Ten; Seeberger, Peter H; Yamaguchi, Yoshiki

    2015-07-01

    ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical-shift perturbation experiments with uniformly (15)N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p. PMID:25919894

  3. Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

    PubMed

    Kumar, Santosh; Mittal, Ekansh; Deore, Sapna; Kumar, Anil; Rahman, Aejazur; Krishnasastry, Musti V

    2015-01-01

    The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms. PMID:26347855

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

    PubMed

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

    2015-12-10

    Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis. PMID:26211627

  5. The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection.

    PubMed

    Torraca, Vincenzo; Cui, Chao; Boland, Ralf; Bebelman, Jan-Paul; van der Sar, Astrid M; Smit, Martine J; Siderius, Marco; Spaink, Herman P; Meijer, Annemarie H

    2015-03-01

    The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system. PMID:25573892

  6. Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination.

    PubMed

    Markova, Nadya; Slavchev, Georgi; Michailova, Lilia

    2015-01-01

    Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features ("fried eggs" colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine. PMID:25874947

  7. Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination

    PubMed Central

    Markova, Nadya; Slavchev, Georgi; Michailova, Lilia

    2015-01-01

    Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features (“fried eggs” colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine. PMID:25874947

  8. Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species

    SciTech Connect

    Siddiqi, S.H.; Hwangbo, C.C.; Silcox, V.; Good, R.C.; Snider, D.E. Jr.; Middlebrook, G.

    1984-10-01

    Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on /sup 14/CO/sub 2/ evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of /sup 14/CO/sub 2/ evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

  9. The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection

    PubMed Central

    Torraca, Vincenzo; Cui, Chao; Boland, Ralf; Bebelman, Jan-Paul; van der Sar, Astrid M.; Smit, Martine J.; Siderius, Marco; Spaink, Herman P.; Meijer, Annemarie H.

    2015-01-01

    The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system. PMID:25573892

  10. Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression.

    PubMed

    Blokpoel, Marian C J; O'Toole, Ronan; Smeulders, Marjan J; Williams, Huw D

    2003-08-01

    Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria. PMID:12782376

  11. Nontuberculous mycobacterial pulmonary disease mimicking lung cancer: Clinicoradiologic features and diagnostic implications.

    PubMed

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

    2016-06-01

    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

  12. Gene encoded antimicrobial peptides, a template for the design of novel anti-mycobacterial drugs.

    PubMed

    Carroll, James; Field, Des; O'Connor, Paula M; Cotter, Paul D; Coffey, Aidan; Hill, Colin; Ross, R Paul; O'Mahony, Jim

    2010-01-01

    Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility.The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis (CIT05/03) and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp. hominissuis by 28% and Mycobacterium avium subsp. paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%.Nisin S, nisin T and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria. PMID:21468208

  13. Drug resistance pattern of mycobacterial isolates in HIV and non-HIV population in South India

    PubMed Central

    Shivaswamy, Umamaheshwari; Neelambike, Sumana M

    2016-01-01

    Background: Emergence of drug resistance has complicated the treatment of tuberculosis (TB). WHO reports India to be one among 27 “high burden” multidrug-resistant (MDR) TB countries. Objective: To diagnose TB and detect drug resistance of mycobacterial isolates in acid-fast bacilli (AFB) smear negative HIV reactive patients (Group A) and compare them with HIV seropositive AFB smear positive (Group B) and HIV-seronegative AFB positive cases (Group C). Materials and Methods: Clinical specimens collected in all groups were processed as per the standard protocol except blood, which was processed by lysis centrifugation technique. They were then inoculated with Lowenstein-Jensen media and the isolates obtained were subjected to drug susceptibility test (DST) by proportion method and genotype MTBDR plus assay. Results: In Group A, 162 patients were included. Of the 443 clinical samples collected, 76 mycobacterial strains were obtained from 67 (41%) patients. Of these, 50 (65.8%) were sensitive to all drugs and 26 (34.2%) resistant to one or more anti-tubercular drugs. Antibiogram of Group A when compared with Group B and C showed that the MDR rate 6.6%, 6.7% and 8% respectively) did not differ much; but resistance to at least single drug was (26 [34.2%], 3 [10%], and 8 [16%]), respectively. Conclusion: Our study suggests that HIV has no influence on the anti-tubercular resistance pattern, but increased MDR rate along with HIV in high TB burden setting stresses the need for early diagnosis and DST in providing proper regimens and improve prognosis. PMID:26933303

  14. Mendelian Susceptibility to Mycobacterial Disease due to IL-12Rβ1 Deficiency in Three Iranian Children

    PubMed Central

    SARRAFZADEH, Shokouh Azam; MAHLOOJIRAD, Maryam; NOURIZADEH, Maryam; CASANOVA, Jean-Laurent; POURPAK, Zahra; BUSTAMANTE, Jacinta; MOIN, Mostafa

    2016-01-01

    Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare inheritance syndrome, characterized by a disseminated infection with mycobacterium in children following BCG vaccination at birth. Regarding the vaccination program in Iran, it may consider as a public health problem. The pathogenesis of MSMD is dependent on either insufficient production of IFN-gamma (γ) or inadequate response to it. Here, we want to introduce three cases including two siblings and one girl from two unrelated families with severe mycobacterial infections referred to Immunology, Asthma and Allergy Research Institute (IAARI), from 2013 to 2015; their MSMD was confirmed by both cytokine assessment and genetic analysis. Regarding the clinical features of the patients, cell proliferation against a mitogen and BCG antigen was ordered in a lymphocyte transformation test (LTT) setting. ELISA was performed for the measurement of IL-12p70 and IFN-γ in whole blood samples activated by BCG + recombinant human IFN-γ and BCG + recombinant human IL-12, respectively. In contrast to mitogen, the antigen-dependent proliferation activity of the patients’ leukocytes was significantly lower than that in normal range. We identified a homozygous mutation in IL12RB1 gene for two kindred who had a homozygous mutation affecting an essential splice site. For the third patient, a novel frameshift deletion in IL12RB1 gene was found. The genetic study results confirmed the impaired function of stimulated lymphocytes to release IFN-γ following stimulation with BCG+IL-12 while the response to rhIFN-γ for IL-12p70 production was relatively intact. Our findings show that cellular and molecular assessments are needed for precise identification of immunodeficiency disorders especially those without clear-cut diagnostic criteria. PMID:27141500

  15. Mendelian Susceptibility to Mycobacterial Disease due to IL-12Rβ1 Deficiency in Three Iranian Children

    PubMed Central

    SARRAFZADEH, Shokouh azam; MAHLOOJIRAD, Maryam; NOURIZADEH, Maryam; CASANOVA, Jean-Laurent; POURPAK, Zahra; BUSTAMANTE, Jacinta; MOIN, Mostafa

    2016-01-01

    Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare inheritance syndrome, characterized by a disseminated infection with mycobacterium in children following BCG vaccination at birth. Regarding the vaccination program in Iran, it may consider as a public health problem. The pathogenesis of MSMD is dependent on either insufficient production of IFN-gamma (γ) or inadequate response to it. Here, we want to introduce three cases including two siblings and one girl from two unrelated families with severe mycobacterial infections referred to Immunology, Asthma and Allergy Research Institute (IAARI), from 2013 to 2015; their MSMD was confirmed by both cytokine assessment and genetic analysis. Regarding the clinical features of the patients, cell proliferation against a mitogen and BCG antigen was ordered in a lymphocyte transformation test (LTT) setting. ELISA was performed for the measurement of IL-12p70 and IFN- γ in whole blood samples activated by BCG + recombinant human IFN-γ and BCG + recombinant human IL-12, respectively. In contrast to mitogen, the antigen-dependent proliferation activity of the patients’ leukocytes was significantly lower than that in normal range. We identified a homozygous mutation in IL12RB1 gene for two kindred who had a homozygous mutation affecting an essential splice site. For the third patient, a novel frameshift deletion in IL12RB1 gene was found. The genetic study results confirmed the impaired function of stimulated lymphocytes to release IFN-γ following stimulation with BCG+IL-12 while the response to rhIFN-γ for IL-12p70 production was relatively intact. Our findings show that cellular and molecular assessments are needed for precise identification of immunodeficiency disorders especially those without clear-cut diagnostic criteria. PMID:27114990

  16. A comparative analysis of the DNA recombination repair pathway in mycobacterial genomes.

    PubMed

    Singh, Amandeep; Bhagavat, Raghu; Vijayan, M; Chandra, Nagasuma

    2016-07-01

    In prokaryotes, repair by homologous recombination provides a major means to reinstate the genetic information lost in DNA damage. Recombination repair pathway in mycobacteria has multiple differences as compared to that in Escherichia coli. Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the same protein from different strains suggests the importance of RecO-RecA and RecFOR-RecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New annotations obtained from the analysis, include identification of a protein with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. New insights into the binding of small molecules to the relevant proteins are provided by binding pocket analysis using three dimensional structural models. Analysis of the various features of the recombination repair pathway, presented here, is likely to provide a framework for further exploring stress response and emergence of drug resistance in mycobacteria. PMID:27450012

  17. Mendelian Susceptibility to Mycobacterial Disease due to IL-12Rβ1 Deficiency in Three Iranian Children.

    PubMed

    Sarrafzadeh, Shokouh Azam; Mahloojirad, Maryam; Nourizadeh, Maryam; Casanova, Jean-Laurent; Pourpak, Zahra; Bustamante, Jacinta; Moin, Mostafa

    2016-03-01

    Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare inheritance syndrome, characterized by a disseminated infection with mycobacterium in children following BCG vaccination at birth. Regarding the vaccination program in Iran, it may consider as a public health problem. The pathogenesis of MSMD is dependent on either insufficient production of IFN-gamma (γ) or inadequate response to it. Here, we want to introduce three cases including two siblings and one girl from two unrelated families with severe mycobacterial infections referred to Immunology, Asthma and Allergy Research Institute (IAARI), from 2013 to 2015; their MSMD was confirmed by both cytokine assessment and genetic analysis. Regarding the clinical features of the patients, cell proliferation against a mitogen and BCG antigen was ordered in a lymphocyte transformation test (LTT) setting. ELISA was performed for the measurement of IL-12p70 and IFN-γ in whole blood samples activated by BCG + recombinant human IFN-γ and BCG + recombinant human IL-12, respectively. In contrast to mitogen, the antigen-dependent proliferation activity of the patients' leukocytes was significantly lower than that in normal range. We identified a homozygous mutation in IL12RB1 gene for two kindred who had a homozygous mutation affecting an essential splice site. For the third patient, a novel frameshift deletion in IL12RB1 gene was found. The genetic study results confirmed the impaired function of stimulated lymphocytes to release IFN-γ following stimulation with BCG+IL-12 while the response to rhIFN-γ for IL-12p70 production was relatively intact. Our findings show that cellular and molecular assessments are needed for precise identification of immunodeficiency disorders especially those without clear-cut diagnostic criteria. PMID:27141500

  18. Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach

    SciTech Connect

    Liu, J.; Rezonzew, R. |; Stanton, V.P. Jr.

    1995-03-20

    We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome. 81 refs., 8 figs., 3 tabs.

  19. Pretreatment long interspersed element (LINE)-1 methylation levels, not early hypomethylation under treatment, predict hematological response to azacitidine in elderly patients with acute myeloid leukemia

    PubMed Central

    Cross, Michael; Bach, Enrica; Tran, Thao; Krahl, Rainer; Jaekel, Nadja; Niederwieser, Dietger; Junghanss, Christian; Maschmeyer, Georg; Al-Ali, Haifa Kathrin

    2013-01-01

    Background Epigenetic modulations, including changes in DNA cytosine methylation, are implicated in the pathogenesis and progression of acute myeloid leukemia (AML). Azacitidine is a hypomethylating agent that is incorporated into RNA as well as DNA. Thus, there is a rationale to its use in patients with AML. We determined whether baseline and/or early changes in the methylation of long interspersed element (LINE)-1 or CDH13 correlate with bone marrow blast clearance, hematological response, or survival in patients with AML treated with azacitidine. Methods An open label, phase I/II trial was performed in 40 AML patients (median bone marrow blast count was 42%) unfit for intensive chemotherapy treated with azacitidine 75 mg/m2/day subcutaneously for 5 days every 4 weeks. Bone marrow mononuclear cell samples were taken on day 0 (pretreatment) and day 15 during the first treatment cycle; LINE-1 and CDH13 methylation levels were quantified by methylation-specific, semiquantitative, real-time polymerase chain reaction. Results Treatment with azacitidine significantly reduced LINE-1 but not CDH13 methylation levels over the first cycle (P < 0.0001). Absolute LINE-1 methylation levels tended to be lower on day 0 (P = 0.06) and day 15 of cycle 1 (P = 0.03) in patients who went on to achieve subsequent complete remission, partial remission or hematological improvement versus patients with stable disease. However, the decrease in LINE-1 methylation over the first treatment cycle did not correlate with subsequent response (P = 0.31). Baseline methylation levels of LINE-1 or CDH13 did not correlate with disease-related prognostic factors, including cytogenetic risk, relapsed/refractory AML, or presence of NPM1 or FLT3 mutations. No correlation was observed between LINE-1 or CDH13 methylation levels and overall survival. Conclusion Analysis of baseline LINE-1 methylation levels may help identify elderly AML patients who are most likely to respond to azacitidine therapy. PMID

  20. The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat

    PubMed Central

    Kralovicova, Jana; Patel, Alpa; Searle, Mark; Vorechovsky, Igor

    2015-01-01

    Splice-site selection is controlled by secondary structure through sequestration or approximation of splicing signals in primary transcripts but the exact role of even the simplest and most prevalent structural motifs in exon recognition remains poorly understood. Here we took advantage of a single-hairpin exon that was activated in a mammalian-wide interspersed repeat (MIR) by a mutation stabilizing a terminal triloop, with splice sites positioned close to each other in a lower stem of the hairpin. We first show that the MIR exon inclusion in mRNA correlated inversely with hairpin stabilities. Employing a systematic manipulation of unpaired regions without altering splice-site configuration, we demonstrate a high correlation between exon inclusion of terminal tri- and tetraloop mutants and matching tri-/tetramers in splicing silencers/enhancers. Loop-specific exon inclusion levels and enhancer/silencer associations were preserved across primate cell lines, in 4 hybrid transcripts and also in the context of a distinct stem, but only if its loop-closing base pairs were shared with the MIR hairpin. Unlike terminal loops, splicing activities of internal loop mutants were predicted by their intramolecular Watson-Crick interactions with the antiparallel strand of the MIR hairpin rather than by frequencies of corresponding trinucleotides in splicing silencers/enhancers. We also show that splicing outcome of oligonucleotides targeting the MIR exon depend on the identity of the triloop adjacent to their antisense target. Finally, we identify proteins regulating MIR exon recognition and reveal a distinct requirement of adjacent exons for C-terminal extensions of Tra2α and Tra2β RNA recognition motifs. PMID:25826413

  1. Epigenetic modification of long interspersed elements-1 in cumulus cells of mature and immature oocytes from patients with polycystic ovary syndrome

    PubMed Central

    Wasinarom, Artisa; Sereepapong, Wisan; Sirayapiwat, Porntip; Rattanatanyong, Prakasit; Mutirangura, Apiwat

    2016-01-01

    Objective The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. Methods The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. Results The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. Conclusion These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS. PMID:27358825

  2. Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis.

    PubMed

    Taus, Francesco; Santucci, Marilina B; Greco, Emanuela; Morandi, Matteo; Palucci, Ivana; Mariotti, Sabrina; Poerio, Noemi; Nisini, Roberto; Delogu, Giovanni; Fraziano, Maurizio

    2015-01-01

    A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination. PMID:26023779

  3. Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

    PubMed Central

    Taus, Francesco; Santucci, Marilina B.; Greco, Emanuela; Morandi, Matteo; Palucci, Ivana; Mariotti, Sabrina; Poerio, Noemi; Nisini, Roberto; Delogu, Giovanni; Fraziano, Maurizio

    2015-01-01

    A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination. PMID:26023779

  4. Plasma Membrane Profiling Reveals Upregulation of ABCA1 by Infected Macrophages Leading to Restriction of Mycobacterial Growth

    PubMed Central

    Long, Jing; Basu Roy, Robindra; Zhang, Yanjia J.; Antrobus, Robin; Du, Yuxian; Smith, Duncan L.; Weekes, Michael P.; Javid, Babak

    2016-01-01

    The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection. We applied plasma membrane profiling, a technique that combines quantitative mass spectrometry with selective cell surface aminooxy-biotinylation, to Bacille Calmette–Guérin (BCG)-infected THP-1 macrophages. We quantified 559 PM proteins in BCG-infected THP-1 cells. One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1. We showed that ABCA1 was upregulated on the macrophage cell-surface following infection with pathogenic mycobacteria and knockdown of ABCA1 resulted in increased mycobacterial survival within macrophages, suggesting that it may be a novel mycobacterial host-restriction factor. PMID:27462310

  5. Growth inhibition of Mycobacterium smegmatis by prodrugs of deoxyxylulose phosphate reducto-isomerase inhibitors, promising anti-mycobacterial agents.

    PubMed

    Ponaire, Sarah; Zinglé, Catherine; Tritsch, Denis; Grosdemange-Billiard, Catherine; Rohmer, Michel

    2012-05-01

    Since Mycobacterium tuberculosis sets up several multiple anti-tuberculosis drug resistance mechanisms, development of new drugs with innovative target is urgent. The methylerythritol phosphate pathway (MEP) involved in the biosynthesis of essential metabolites for the survival of mycobacteria, represents such a target. Fosmidomycin 1a and FR900098 1b, two inhibitors of DXR, do not affect the viability of M. tuberculosis cells, due to a lack of uptake. To overcome the absence of the mycobacterial cell wall crossing of these compounds, we synthesized and tested the inhibition potency of acyloxymethyl phosphonate esters as prodrugs of fosmidomycin 1a, FR900098 1b and their analogs 2a and 2b on Mycobacterium smegmatis. Only the prodrugs 4b-6b inhibit the bacterial growth and could be effective anti-mycobacterial agents. PMID:22405649

  6. A Mycobacterial Perspective on Tuberculosis in West Africa: Significant Geographical Variation of M. africanum and Other M. tuberculosis Complex Lineages

    PubMed Central

    Gehre, Florian; Kumar, Samrat; Kendall, Lindsay; Ejo, Mebrat; Secka, Oumie; Ofori-Anyinam, Boatema; Abatih, Emmanuel; Antonio, Martin; Berkvens, Dirk; de Jong, Bouke C.

    2016-01-01

    Background Phylogenetically distinct Mycobacterium tuberculosis lineages differ in their phenotypes and pathogenicity. Consequently, understanding mycobacterial population structures phylogeographically is essential for design, interpretation and generalizability of clinical trials. Comprehensive efforts are lacking to date to establish the West African mycobacterial population structure on a sub-continental scale, which has diagnostic implications and can inform the design of clinical TB trials. Methodology/Principal Findings We collated novel and published genotyping (spoligotyping) data and classified spoligotypes into mycobacterial lineages/families using TBLineage and Spotclust, followed by phylogeographic analyses using statistics (logistic regression) and lineage axis plot analysis in GenGIS, in which a phylogenetic tree constructed in MIRU-VNTRplus was analysed. Combining spoligotyping data from 16 previously published studies with novel data from The Gambia, we obtained a total of 3580 isolates from 12 countries and identified 6 lineages comprising 32 families. By using stringent analytical tools we demonstrate for the first time a significant phylogeographic separation between western and eastern West Africa not only of the two M. africanum (West Africa 1 and 2) but also of several major M. tuberculosis sensu stricto families, such as LAM10 and Haarlem 3. Moreover, in a longitudinal logistic regression analysis for grouped data we showed that M. africanum West Africa 2 remains a persistent health concern. Conclusions/Significance Because of the geographical divide of the mycobacterial populations in West Africa, individual research findings from one country cannot be generalized across the whole region. The unequal geographical family distribution should be considered in placement and design of future clinical trials in West Africa. PMID:26964059

  7. Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells

    PubMed Central

    Adlakha, Nidhi; Vir, Pooja; Verma, Indu

    2012-01-01

    Background: Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tb). In lungs, alveolar macrophages and type II alveolar epithelial cells serve as a replicative niche for this pathogen. Secretory proteins released by actively replicating tubercle bacilli are known to interact with host cells at the initial stages of infection. To understand the role of these cells in TB pathogenesis, it is important to identify the mycobacterial components involved in interaction with alveolar epithelial cells. Materials and Methods: We fractionated the whole secretory proteome of M. tb H37Rv into 10 narrow molecular mass fractions (A1-A10; <20 kDa to >90 kDa) that were studied for their binding potential with A549; type II alveolar epithelial cell line. We also studied the consequences of this interaction in terms of change in epithelial cell viability by MTT assay and cytokine release by ELISA. Results: Our results show that several mycobacterial proteins bind and confer cytolysis in epithelial cells. Amongst all the fractions, proteins ranging from 35-45 kDa (A5) exhibited highest binding to A549 cells with a consequence of cytolysis of these cells. This fraction (A5) also led to release of various cytokines important in anti-mycobacterial immunity. Conclusion: Fraction A5 (35-45 kDa) of mycobacterial secretory proteome play an important role in mediating M. tb interaction with type II alveolar epithelial cells with the consequences detrimental for the TB pathogenesis. Further studies are being carried out to identify the candidate proteins from this region. PMID:23243342

  8. Design and synthesis of triazolopyrimidine acylsulfonamides as novel anti-mycobacterial leads acting through inhibition of acetohydroxyacid synthase.

    PubMed

    Patil, Vikas; Kale, Manoj; Raichurkar, Anandkumar; Bhaskar, Brahatheeswaran; Prahlad, Dwarakanath; Balganesh, Meenakshi; Nandan, Santosh; Shahul Hameed, P

    2014-05-01

    Novel triazolopyrimidine acylsulfonamides class of antimycobacterial agents, which are mycobacterial acetohydroxyacid synthase (AHAS) inhibitors were designed by hybridization of known AHAS inhibitors such as sulfonyl urea and triazolopyrimidine sulfonamides. This Letter describes the synthesis and SAR studies of this class of molecules by variation of two parts of the molecule, the phenyl and triazolopyrimidine rings. SAR study describes optimisation of enzyme potency, whole cell potency and evidence of mechanism of action. PMID:24703230

  9. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells.

    PubMed

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D; Piccirilli, Joseph A; Moody, D Branch; Adams, Erin J

    2014-10-28

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  10. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

    PubMed Central

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

    2014-01-01

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  11. Complications after video-assisted thoracic surgery in patients with pulmonary nontuberculous mycobacterial lung disease who underwent preoperative pulmonary rehabilitation

    PubMed Central

    Morino, Akira; Murase, Kazuma; Yamada, Katsuo

    2015-01-01

    [Purpose] Video-assisted thoracic surgery and preoperative pulmonary rehabilitation are effective in preventing postoperative complications in patients with cardiopulmonary disease. The present study aims to elucidate the presence of postoperative pneumonia and atelectasis in patients with nontuberculous mycobacterial lung disease who underwent lung resection with video-assisted thoracic surgery and preoperative pulmonary rehabilitation. [Subjects and Methods] Nineteen patients with nontuberculous mycobacterial lung disease who had undergone lung resection with video-assisted thoracic surgery and preoperative pulmonary rehabilitation were enrolled in this study. The presence of postoperative pneumonia and atelectasis was evaluated, and preoperative and postoperative pulmonary functions were compared. [Results] Postoperative pneumonia and postoperative atelectasis were not observed. Decreases of pulmonary function were 5.9% (standard deviation, 8.5) in forced vital capacity (percent predicted) and 9.6% (standard deviation, 11.1) in forced expiratory volume in 1 s (percent predicted). [Conclusion] The present study indicates that the combination of lung resection with video-assisted thoracic surgery and preoperative pulmonary rehabilitation in patients with nontuberculous mycobacterial lung disease may be effective in preventing postoperative complications. PMID:26357436

  12. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline.

    PubMed

    Preiss, Laura; Langer, Julian D; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E G; Koul, Anil; Meier, Thomas

    2015-05-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring's ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens. PMID:26601184

  13. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline

    PubMed Central

    Preiss, Laura; Langer, Julian D.; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E. G.; Koul, Anil; Meier, Thomas

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring’s ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens. PMID:26601184

  14. Isolation and characterization of recombinant lambda gt11 bacteriophages expressing eight different mycobacterial antigens of potential immunological relevance.

    PubMed Central

    Andersen, A B; Worsaae, A; Chaparas, S D

    1988-01-01

    A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies. The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species. Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens. Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques. PaeA and PaeB are distinct proteins but were shown to share an epitope. A similar condition was observed between PafA and PafB. Among the phages isolated, two groups expressed epitopes specific for M. tuberculosis and Mycobacterium bovis BCG. One group of phages produced an antigenic determinant which is found in M. tuberculosis and Mycobacterium marinum but not in M. bovis BCG. Images PMID:2451643

  15. The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

    PubMed Central

    Wilson, Gillian J.; Marakalala, Mohlopheni J.; Hoving, Jennifer C.; van Laarhoven, Arjan; Drummond, Rebecca A.; Kerscher, Bernhard; Keeton, Roanne; van de Vosse, Esther; Ottenhoff, Tom H.M.; Plantinga, Theo S.; Alisjahbana, Bachti; Govender, Dhirendra; Besra, Gurdyal S.; Netea, Mihai G.; Reid, Delyth M.; Willment, Janet A.; Jacobs, Muazzam; Yamasaki, Sho; van Crevel, Reinout; Brown, Gordon D.

    2015-01-01

    Summary The interaction of microbes with pattern recognition receptors (PRRs) is essential for protective immunity. While many PRRs that recognize mycobacteria have been identified, none is essentially required for host defense in vivo. Here, we have identified the C-type lectin receptor CLECSF8 (CLEC4D, MCL) as a key molecule in anti-mycobacterial host defense. Clecsf8−/− mice exhibit higher bacterial burdens and increased mortality upon M. tuberculosis infection. Additionally, Clecsf8 deficiency is associated with exacerbated pulmonary inflammation, characterized by enhanced neutrophil recruitment. Clecsf8−/− mice show reduced mycobacterial uptake by pulmonary leukocytes, but infection with opsonized bacteria can restore this phagocytic defect as well as decrease bacterial burdens. Notably, a CLECSF8 polymorphism identified in humans is associated with an increased susceptibility to pulmonary tuberculosis. We conclude that CLECSF8 plays a non-redundant role in anti-mycobacterial immunity in mouse and in man. PMID:25674984

  16. Induction of mycobacterial proteins during phagocytosis and heat shock: a time interval analysis.

    PubMed

    Alavi, M R; Affronti, L F

    1994-05-01

    Mycobacterium tuberculosis survives macrophage bactericidal activities by mechanisms that may include induction of stress proteins. We sought to determine whether the synthesis of any mycobacterial proteins is increased during phagocytosis and whether any of these proteins are also up-regulated during heat shock. Protein synthesis by M. tuberculosis H37Ra during phagocytosis by the mouse macrophage cell line IC-21, and during heat shock at 45 and 48 degrees C, was monitored at various time intervals using 35S-labeled methionine/cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest the existence of certain common elements in the stress response of mycobacteria to the three stress stimuli. This apparent similarity was best characterized by the up-regulation of a 25-kDa protein after exposure to each of the stress conditions. Furthermore, this 25-kDa protein and a 37-kDa protein that was also synthesized during phagocytosis appeared to be extracellular because they were preferentially solubilized when infected macrophages were lysed with 0.5% NP-40. PMID:8182341

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

    PubMed Central

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

    2013-01-01

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

  18. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3.

    PubMed

    Haapaniemi, Emma M; Kaustio, Meri; Rajala, Hanna L M; van Adrichem, Arjan J; Kainulainen, Leena; Glumoff, Virpi; Doffinger, Rainer; Kuusanmäki, Heikki; Heiskanen-Kosma, Tarja; Trotta, Luca; Chiang, Samuel; Kulmala, Petri; Eldfors, Samuli; Katainen, Riku; Siitonen, Sanna; Karjalainen-Lindsberg, Marja-Liisa; Kovanen, Panu E; Otonkoski, Timo; Porkka, Kimmo; Heiskanen, Kaarina; Hänninen, Arno; Bryceson, Yenan T; Uusitalo-Seppälä, Raija; Saarela, Janna; Seppänen, Mikko; Mustjoki, Satu; Kere, Juha

    2015-01-22

    The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity. PMID:25349174

  19. Identification of a Non-Pentapeptide Region Associated with Rapid Mycobacterial Evolution

    PubMed Central

    Warholm, Per; Light, Sara

    2016-01-01

    A large portion of the coding capacity of Mycobacterium tuberculosis is devoted to the production of proteins containing several copies of the pentapeptide-2 repeat, namely the PE/PPE_MPTR proteins. Protein domain repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. They are not as common in prokaryotes, compared to eukaryotes, but the enrichment of pentapeptide-2 repeats in Mycobacteria constitutes an exception to that rule. The genes encoding the PE/PPE_MPTR proteins have undergone many rearrangements and here we have identified the expansion patterns across the Mycobacteria. We have performed a reclassification of the PE/PPE_MPTR proteins using cohesive regions rather than sparse domain architectures. It is clear that these proteins have undergone large insertions of several pentapeptide-2 domains appearing adjacent to one another in a repetitive pattern. Further, we have identified a non-pentapeptide motif associated with rapid mycobacterial evolution. The sequence composition of this region suggests a different structure compared to pentapeptide-2 repeats. By studying the evolution of the PE/PPE_MPTR proteins, we have distinguished features pertaining to tuberculosis-inducing species. Further studies of the non-pentapeptide region associated with repeat expansions promises to shed light on the pathogenicity of Mycobacterium tuberculosis. PMID:27149271

  20. The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus

    PubMed Central

    Striebel, Frank; Hunkeler, Moritz; Summer, Heike; Weber-Ban, Eilika

    2010-01-01

    Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin-like protein (Pup) that is recognized by the N-terminal coiled-coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup-tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N-terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant. PMID:20203624

  1. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3

    PubMed Central

    Haapaniemi, Emma M.; Kaustio, Meri; Rajala, Hanna L. M.; van Adrichem, Arjan J.; Kainulainen, Leena; Glumoff, Virpi; Doffinger, Rainer; Kuusanmäki, Heikki; Heiskanen-Kosma, Tarja; Trotta, Luca; Chiang, Samuel; Kulmala, Petri; Eldfors, Samuli; Katainen, Riku; Siitonen, Sanna; Karjalainen-Lindsberg, Marja-Liisa; Kovanen, Panu E.; Otonkoski, Timo; Porkka, Kimmo; Heiskanen, Kaarina; Hänninen, Arno; Bryceson, Yenan T.; Uusitalo-Seppälä, Raija; Saarela, Janna; Seppänen, Mikko; Kere, Juha

    2015-01-01

    The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked–like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4−CD8−) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity. PMID:25349174

  2. Mycobacterium tuberculosis Rv1265 promotes mycobacterial intracellular survival and alters cytokine profile of the infected macrophage.

    PubMed

    Luo, Hongping; Zeng, Jie; Huang, Qinqin; Liu, Minqiang; Abdalla, Abualgasim Elgaili; Xie, Longxiang; Wang, Huan; Xie, Jianping

    2016-03-01

    Mycobacterium tuberculosis cAMP and underlying regulatory network are crucial for its survival and thrive in the presence of numerous stresses mounted by the host. Our studies mainly focus on the cAMP-induced M. tuberculosis gene Rv1265, which was shown to be up-regulated under hypoxia and during macrophage infection by addition of exogenous cAMP. To explore the role of Rv1265 in host-pathogen interactions, Rv1265 was expressed in a non-pathogenic Mycobacterium smegmatis. We found that Rv1265 was associated with cell envelope and can up-regulate some cell wall fatty acid components, especially the C26:0. The survival of the recombinant Ms_Rv1265 was enhanced within macrophages and under stress conditions such as low pH and SDS. Macrophages infected with Ms_Rv1265 increased transcription of pro-inflammatory cytokines IL-1β, IL-6, and IL-12 P40 and anti-inflammatory cytokine IL-10 possibly through activation of NF-κB and ERK1/2 pathway. Our findings indicate that Rv1265 can enhance mycobacterial survival within macrophages, and perturb the cytokine profile of macrophage. PMID:26156642

  3. Heme Oxygenase-1 Regulates Inflammation and Mycobacterial Survival in Human Macrophages during Mycobacterium tuberculosis Infection.

    PubMed

    Scharn, Caitlyn R; Collins, Angela C; Nair, Vidhya R; Stamm, Chelsea E; Marciano, Denise K; Graviss, Edward A; Shiloh, Michael U

    2016-06-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth. PMID:27183573

  4. Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

    PubMed

    Bos, Kirsten I; Harkins, Kelly M; Herbig, Alexander; Coscolla, Mireia; Weber, Nico; Comas, Iñaki; Forrest, Stephen A; Bryant, Josephine M; Harris, Simon R; Schuenemann, Verena J; Campbell, Tessa J; Majander, Kerttu; Wilbur, Alicia K; Guichon, Ricardo A; Wolfe Steadman, Dawnie L; Cook, Della Collins; Niemann, Stefan; Behr, Marcel A; Zumarraga, Martin; Bastida, Ricardo; Huson, Daniel; Nieselt, Kay; Young, Douglas; Parkhill, Julian; Buikstra, Jane E; Gagneux, Sebastien; Stone, Anne C; Krause, Johannes

    2014-10-23

    Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean. PMID:25141181

  5. Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis

    PubMed Central

    Bos, Kirsten I.; Harkins, Kelly M.; Herbig, Alexander; Coscolla, Mireia; Weber, Nico; Comas, Iñaki; Forrest, Stephen A.; Bryant, Josephine M.; Harris, Simon R.; Schuenemann, Verena J.; Campbell, Tessa J.; Majander, Kerrtu; Wilbur, Alicia K.; Guichon, Ricardo A.; Wolfe Steadman, Dawnie L.; Cook, Della Collins; Niemann, Stefan; Behr, Marcel A.; Zumarraga, Martin; Bastida, Ricardo; Huson, Daniel; Nieselt, Kay; Young, Douglas; Parkhill, Julian; Buikstra, Jane E.; Gagneux, Sebastien; Stone, Anne C.; Krause, Johannes

    2015-01-01

    Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact1. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World2. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch3, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean. PMID:25141181

  6. [Uncommon mycobacterial infections in domestic and zoo animals: four cases with special emphasis on pathology].

    PubMed

    Steiger, K; Ellenberger, C; Schüppel, K F; Richter, E; Schmerbach, K; Krautwald-Junghanns, M E; Wünnemann, K; Eulenberger, K; Schoon, H A

    2003-09-01

    Infections caused by classical tubercle bacilli are rare during the last years. Nevertheless, diseases caused by other mycobacteria have to be considered clinically and in diagnostic pathology especially in cases of immunosuppression and due to their potential zoonosis risk. An infection by mycobacteria was diagnosed in four animals (Mayotte Maki, Blue-headed Parrot, Patagonian sealion, Beagle) necropsied between 1995 and 2002 in the Institute of Veterinary-Pathology of the University of Leipzig. The Maki, the blue-headed parrot and the dog showed a disseminated character of the disease caused by Mycobacterium genavense (monkey and bird) resp. Mycobacterium avium (dog), while an open chronical tuberculosis of the lungs due to a pathogenic member of Mycobacterium tuberculosis complex was observed in the seal. All these bacteria are potential causes of zoonoses. So, if granulomatous or disseminated histiocytic alterations are detected in diagnostic pathology, mycobacterial infections should always be included in differential diagnoses and require careful aetiological investigations by histopathological and bacteriological methods. PMID:14560447

  7. Revisiting tuberculous pleurisy: pleural fluid characteristics and diagnostic yield of mycobacterial culture in an endemic area

    PubMed Central

    Ruan, Sheng-Yuan; Chuang, Yu-Chung; Lin, Jou-Wei; Chien, Jung-Yien; Huang, Chun-Ta; Kuo, Yao-Wen; Lee, Li-Na; Yu, Chong-Jen J

    2012-01-01

    Background Tuberculous pleurisy is traditionally indicated by extreme lymphocytosis in pleural fluid and low yield of effusion culture. However, there is considerable inconsistency among previous study results. In addition, these data should be updated due to early effusion studies and advances in culture methods. Methods From January 2004 to June 2009, patients with tuberculous pleurisy were retrospectively identified from the mycobacteriology laboratories and the pathology and tuberculosis registration databases of two hospitals in Taiwan where tuberculosis is endemic. Pleural fluid characteristics and yields of mycobacterial cultures using liquid media were evaluated. Results A total of 382 patients with tuberculous pleurisy were identified. The median lymphocyte percentage of total cells in pleural fluids was 84% (IQR 64–95%) and 17% of cases had a lymphocyte percentage of <50%. The lymphocyte percentage was negatively associated with the probability of a positive effusion culture (OR 0.97; 95% CI 0.96 to 0.99). The diagnostic yields were 63% for effusion culture, 48% for sputum culture, 79% for the combination of effusion and sputum cultures, and 74% for histological examination of pleural biopsy specimens. Conclusion The degree of lymphocyte predominance in tuberculous pleurisy was lower than was previously thought. The lymphocyte percentage in pleural fluid was negatively associated with the probability of a positive effusion culture. With the implementation of a liquid culture method, the sensitivity of effusion culture was much higher than has been previously reported, and the combination of effusion and sputum cultures provided a good diagnostic yield. PMID:22436167

  8. Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

    PubMed

    Pule, Caroline M; Sampson, Samantha L; Warren, Robin M; Black, Philippa A; van Helden, Paul D; Victor, Tommie C; Louw, Gail E

    2016-01-01

    The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy. PMID:26472768

  9. The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

    PubMed

    Su, Hong-Wei; Zhu, Jun-Hao; Li, Hao; Cai, Rong-Jun; Ealand, Christopher; Wang, Xun; Chen, Yu-Xiang; Kayani, Masood Ur Rehman; Zhu, Ting F; Moradigaravand, Danesh; Huang, Hairong; Kana, Bavesh D; Javid, Babak

    2016-01-01

    Although regulation of translation fidelity is an essential process(1-7), diverse organisms and organelles have differing requirements of translational accuracy(8-15), and errors in gene translation serve an adaptive function under certain conditions(16-20). Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB(21-25), but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation. PMID:27564922

  10. The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS).

    PubMed

    Horsburgh, C R; Selik, R M

    1989-01-01

    We analyzed cases of disseminated nontuberculous mycobacterial infection (DNTM) in patients with AIDS reported to the Centers for Disease Control. Between 1981 and 1987, 2,269 cases were reported. In 96% of cases, infection was caused by M. avium complex (MAC). The number of cases has risen steadily since 1981, but the rate as a percentage of AIDS cases has remained stable at 5.5%. DNTM was seen less frequently in AIDS cases with Kaposi's sarcoma than in other AIDS cases (p less than 0.01). Rates of DNTM were lower in Hispanics and declined with age but were not significantly different by patient sex or means of acquiring HIV infection. Rates of disseminated MAC varied by geographic region from 3.9% to 7.8% (p less than 0.0001). As assessed by helper/suppressor T-cell ratios, AIDS patients with DNTM were not more immunologically impaired than those with other opportunistic infections. Life table analysis revealed that AIDS patients with DNTM survived a shorter time (median, 7.4 months) than did other AIDS patients (median, 13.3 months; p less than 0.0001). We conclude that DNTM is acquired by unpreventable environmental exposures. Because DNTM adversely affects survival of AIDS patients, effective therapeutic agents must be vigorously sought. PMID:2912355

  11. DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

    PubMed Central

    Santos, Rubens R; Sartori, Alexandrina; Lima, Deison S; Souza, Patrícia RM; Coelho-Castelo, Arlete AM; Bonato, Vânia LD; Silva, Célio L

    2009-01-01

    Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. PMID:19754943

  12. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function. PMID:22094466

  13. Autophagy-Related Proteins Target Ubiquitin-Free Mycobacterial Compartment to Promote Killing in Macrophages

    PubMed Central

    Bah, Aïcha; Lacarrière, Camille; Vergne, Isabelle

    2016-01-01

    Autophagy is a lysosomal degradative process that plays essential functions in innate immunity, particularly, in the clearance of intracellular bacteria such as Mycobacterium tuberculosis. The molecular mechanisms involved in autophagy activation and targeting of mycobacteria, in innate immune responses of macrophages, are only partially characterized. Autophagy targets pathogenic M. tuberculosis via a cytosolic DNA recognition- and an ubiquitin-dependent pathway. In this report, we show that non-pathogenic M. smegmatis induces a robust autophagic response in THP-1 macrophages with an up regulation of several autophagy-related genes. Autophagy activation relies in part on recognition of mycobacteria by Toll-like receptor 2 (TLR2). Notably, LC3 targeting of M. smegmatis does not rely on membrane damage, ubiquitination, or autophagy receptor recruitment. Lastly, M. smegmatis promotes recruitment of several autophagy proteins, which are required for mycobacterial killing. In conclusion, our study uncovered an alternative autophagic pathway triggered by mycobacteria which involves cell surface recognition but not bacterial ubiquitination. PMID:27242971

  14. Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis.

    PubMed

    Mougous, Joseph D; Petzold, Christopher J; Senaratne, Ryan H; Lee, Dong H; Akey, David L; Lin, Fiona L; Munchel, Sarah E; Pratt, Matthew R; Riley, Lee W; Leary, Julie A; Berger, James M; Bertozzi, Carolyn R

    2004-08-01

    Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection. PMID:15258569

  15. The Ser/Thr Protein Kinase PknB Is Essential for Sustaining Mycobacterial Growth▿

    PubMed Central

    Fernandez, Pablo; Saint-Joanis, Brigitte; Barilone, Nathalie; Jackson, Mary; Gicquel, Brigitte; Cole, Stewart T.; Alzari, Pedro M.

    2006-01-01

    The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc2155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs. PMID:16980473

  16. Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish.

    PubMed

    Vibe, Carina Beatrice; Fenaroli, Federico; Pires, David; Wilson, Steven Ray; Bogoeva, Vanya; Kalluru, Raja; Speth, Martin; Anes, Elsa; Griffiths, Gareth; Hildahl, Jon

    2016-08-01

    Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo. PMID:26573343

  17. Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection

    PubMed Central

    Renna, Maurizio; Schaffner, Catherine; Brown, Karen; Shang, Shaobin; Tamayo, Marcela Henao; Hegyi, Krisztina; Grimsey, Neil J.; Cusens, David; Coulter, Sarah; Cooper, Jason; Bowden, Anne R.; Newton, Sandra M.; Kampmann, Beate; Helm, Jennifer; Jones, Andrew; Haworth, Charles S.; Basaraba, Randall J.; DeGroote, Mary Ann; Ordway, Diane J.; Rubinsztein, David C.; Floto, R. Andres

    2011-01-01

    Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens. PMID:21804191

  18. Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

    PubMed Central

    Floss, Doreen Manuela; Mockey, Michael; Zanello, Galliano; Brosson, Damien; Diogon, Marie; Frutos, Roger; Bruel, Timothée; Rodrigues, Valérie; Garzon, Edwin; Chevaleyre, Claire; Berri, Mustapha; Salmon, Henri; Conrad, Udo; Dedieu, Laurence

    2010-01-01

    This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis. PMID:20414351

  19. Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation.

    PubMed

    Deffert, Christine; Schäppi, Michela G; Pache, Jean-Claude; Cachat, Julien; Vesin, Dominique; Bisig, Ruth; Ma Mulone, Xiaojuan; Kelkka, Tiina; Holmdahl, Rikard; Garcia, Irene; Olleros, Maria L; Krause, Karl-Heinz

    2014-09-01

    Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (∼ 50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-α, IFN-γ, IL-17 and IL-12) early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and

  20. Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

    PubMed Central

    Vordermeier, H. Martin; Hewinson, R. Glyn; Wilkinson, Robert J.; Wilkinson, Katalin A.; Gideon, Hannah P.; Young, Douglas B.; Sampson, Samantha L.

    2012-01-01

    The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology. PMID:22870206

  1. A spatial epidemiological analysis of nontuberculous mycobacterial infections in Queensland, Australia

    PubMed Central

    2014-01-01

    Background The epidemiology of infections with nontuberculous mycobacteria (NTM) has been changing and the incidence has been increasing in some settings. The main route of transmission to humans is considered to be from the environment. We aimed to describe spatial clusters of cases of NTM infections and to identify associated climatic, environmental and socio-economic variables. Methods NTM data were obtained from the Queensland Mycobacterial Reference Laboratory for the period 2001–2011. A Bayesian spatial conditional autoregressive model was constructed at the postcode level, with covariates including soil variables, maximum, mean and minimum rainfall and temperature, income (proportion of population earning < $32,000 and < $52,000) and land use category. Results Significant clusters of NTM infection were identified in the central Queensland region overlying the Surat sub-division of the Great Artesian Basin, as well as in the lower North Queensland Local Government Area known as the Whitsunday region. Our models estimated an expected increase of 21% per percentage increase of population earning < $52,000 (95% CI 9–34%) and an expected decrease of 13% for every metre increase of average topsoil depth for risk of Mycobacterium intracellulare infection (95% CI -3 – -22%). There was an estimated increase of 79% per mg/m3 increase of soil bulk density (95% CI 26–156%) and 19% decrease for every percentage increase in population earning < $32,000 for risk of M. kansasii infection (95% CI -3 – -49%). Conclusions There were distinct spatial clusters of M. kansasii, M. intracellulare and M. abscessus infections in Queensland, and a number of socio-ecological, economic and environmental factors were found to be associated with NTM infection risk. PMID:24885916

  2. Increased Incidence of Cutaneous Nontuberculous Mycobacterial Infection, 1980 to 2009: A Population-Based Study

    PubMed Central

    Wentworth, Ashley B.; Drage, Lisa A.; Wengenack, Nancy L.; Wilson, John W.; Lohse, Christine M.

    2013-01-01

    Objectives To determine the incidence and clinical characteristics of cutaneous nontuberculous mycobacterial (NTM) infection during the past 30 years and whether the predominant species have changed. Patients and Methods Using Rochester Epidemiology Project data, we identified Olmsted County, Minnesota, residents with cutaneous NTM infections between January 1, 1980, and December 31, 2009, examining the incidence of infection, patient demographic and clinical features, the mycobacterium species, and therapy. Results Forty patients (median age, 47 years; 58% female [23 of 40]) had positive NTM cultures plus 1 or more clinical signs. The overall age- and sex-adjusted incidence of cutaneous NTM infection was 1.3 per 100,000 person-years (95% CI, 0.9–1.7 per 100,000 person-years). The incidence increased with age at diagnosis (P = .003) and was higher in 2000 to 2009 (2.0 per 100,000 person-years; 95% CI, 1.3–2.8 per 100,000 person-years) than in 1980 to 1999 (0.7 per 100,000 person-years; 95% CI, 0.3–1.1 per 100,000 person-years) (P = .002). The distal extremities were the most common sites of infection (27 of 39 patients [69%]). No patient had human immunodeficiency virus infection, but 23% (9 of 39) were immunosuppressed. Of the identifiable causes, traumatic injuries were the most frequent (22 of 29 patients [76%]). The most common species were Mycobacterium marinum (17 of 38 patients [45%]) and Mycobacterium chelonae/Mycobacterium abscessus (12 of 38 patients [32%]). In the past decade (2000–2009), 15 of 24 species (63%) were rapidly growing mycobacteria compared with only 4 of 14 species (29%) earlier (1980–1999) (P = .04). Conclusion The incidence of cutaneous NTM infection increased nearly 3-fold during the study period. Rapidly growing mycobacteria were predominant during the past decade. PMID:23218797

  3. Preliminary Results of Bedaquiline as Salvage Therapy for Patients With Nontuberculous Mycobacterial Lung Disease

    PubMed Central

    Wallace, Richard J.; Benwill, Jeana L.; Taskar, Varsha; Brown-Elliott, Barbara A.; Thakkar, Foram; Aksamit, Timothy R.; Griffith, David E.

    2015-01-01

    BACKGROUND: Bedaquiline is an oral antimycobacterial agent belonging to a new class of drugs called diarylquinolines. It has low equivalent minimal inhibitory concentrations for Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) lung disease, especially Mycobacterium avium complex (MAC) and Mycobacterium abscessus (Mab). Bedaquiline appears to be effective for the treatment of multidrug-resistant TB but has not been tested clinically for NTM disease. METHODS: We describe a case series of off-label use of bedaquiline for treatment failure lung disease caused by MAC or Mab. Only patients whose insurance would pay for the drug were included. Fifteen adult patients were selected, but only 10 (six MAC, four Mab) could obtain bedaquiline. The 10 patients had been treated for 1 to 8 years, and all were on treatment at the start of bedaquiline therapy. Eighty percent had macrolide-resistant isolates (eight of 10). The patients were treated with the same bedaquiline dosage as that used in TB trials and received the best available companion drugs (mean, 5.0 drugs). All patients completed 6 months of therapy and remain on bedaquiline. RESULTS: Common side effects included nausea (60%), arthralgias (40%), and anorexia and subjective fever (30%). No abnormal ECG findings were observed with a mean corrected QT interval lengthening of 2.4 milliseconds at 6 months. After 6 months of therapy, 60% of patients (six of 10) had a microbiologic response, with 50% (five of 10) having one or more negative cultures. CONCLUSIONS: This small preliminary report demonstrates potential clinical and microbiologic activity of bedaquiline in patients with advanced MAC or Mab lung disease but the findings require confirmation with larger studies. PMID:25675393

  4. Lack of Adherence to Evidence-based Treatment Guidelines for Nontuberculous Mycobacterial Lung Disease

    PubMed Central

    Prevots, D. Rebecca; Gallagher, Jack; Heap, Kylee; Gupta, Renu; Griffith, David

    2014-01-01

    Rationale: The 2007 American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) recommend that patients with pulmonary nontuberculous mycobacterial (PNTM) disease caused by Mycobacterium avium complex (MAC) or M. abscessus be treated with a macrolide-based multidrug antibiotic regimen until sputum culture negative for 1 year. After 6 years, the degree of adherence to recommended guidelines among physicians remains unknown. Objective: To describe antibiotic treatment practices among physicians treating patients with PNTM in the United States. Methods: A nationally representative sample of 1,286 U.S. physicians was contacted in December 2011 through January 2012; 582 of the responding physicians were treating patients with PNTM and were eligible to participate. Physicians were asked to extract medical record data on the last four patients they treated in the past year with PNTM disease from either MAC or M. abscessus. Treatment patterns were assessed for all patients by NTM species and physician specialty, and compared with the 2007 recommended ATS/IDSA guidelines. Main Results: Questionnaires were completed by 349 physicians on 915 patients with PNTM, including 744 (81%) with MAC and 174 (19%) with M. abscessus; 3 patients were positive for both. Physicians treated 76 (44%) patients with M. abscessus and 411 (55%) patients with MAC. Only 13% of antibiotic regimens prescribed to patients with MAC met ATS/IDSA guidelines, 56% did not include a macrolide, and 16% were for macrolide monotherapy. Among patients with M. abscessus, 64% of regimens prescribed did not include a macrolide. Conclusions: Adherence to the 2007 ATS/IDSA guidelines for treating PNTM disease is poor. Across all physician specialties evaluated, suboptimal or potentially harmful antibiotic regimens were commonly prescribed. PMID:24236749

  5. Chronic Gastrointestinal Nematode Infection Mutes Immune Responses to Mycobacterial Infection Distal to the Gut.

    PubMed

    Obieglo, Katja; Feng, Xiaogang; Bollampalli, Vishnu Priya; Dellacasa-Lindberg, Isabel; Classon, Cajsa; Österblad, Markus; Helmby, Helena; Hewitson, James P; Maizels, Rick M; Gigliotti Rothfuchs, Antonio; Nylén, Susanne

    2016-03-01

    Helminth infections have been suggested to impair the development and outcome of Th1 responses to vaccines and intracellular microorganisms. However, there are limited data regarding the ability of intestinal nematodes to modulate Th1 responses at sites distal to the gut. In this study, we have investigated the effect of the intestinal nematode Heligmosomoides polygyrus bakeri on Th1 responses to Mycobacterium bovis bacillus Calmette-Guérin (BCG). We found that H. polygyrus infection localized to the gut can mute BCG-specific CD4(+) T cell priming in both the spleen and skin-draining lymph nodes. Furthermore, H. polygyrus infection reduced the magnitude of delayed-type hypersensitivity (DTH) to PPD in the skin. Consequently, H. polygyrus-infected mice challenged with BCG had a higher mycobacterial load in the liver compared with worm-free mice. The excretory-secretory product from H. polygyrus (HES) was found to dampen IFN-γ production by mycobacteria-specific CD4(+) T cells. This inhibition was dependent on the TGF-βR signaling activity of HES, suggesting that TGF-β signaling plays a role in the impaired Th1 responses observed coinfection with worms. Similar to results with mycobacteria, H. polygyrus-infected mice displayed an increase in skin parasite load upon secondary infection with Leishmania major as well as a reduction in DTH responses to Leishmania Ag. We show that a nematode confined to the gut can mute T cell responses to mycobacteria and impair control of secondary infections distal to the gut. The ability of intestinal helminths to reduce DTH responses may have clinical implications for the use of skin test-based diagnosis of microbial infections. PMID:26819205

  6. Chronic Gastrointestinal Nematode Infection Mutes Immune Responses to Mycobacterial Infection Distal to the Gut

    PubMed Central

    Obieglo, Katja; Feng, Xiaogang; Bollampalli, Vishnu Priya; Dellacasa-Lindberg, Isabel; Classon, Cajsa; Österblad, Markus; Helmby, Helena; Hewitson, James P.; Maizels, Rick M.

    2016-01-01

    Helminth infections have been suggested to impair the development and outcome of Th1 responses to vaccines and intracellular microorganisms. However, there are limited data regarding the ability of intestinal nematodes to modulate Th1 responses at sites distal to the gut. In this study, we have investigated the effect of the intestinal nematode Heligmosomoides polygyrus bakeri on Th1 responses to Mycobacterium bovis bacillus Calmette–Guérin (BCG). We found that H. polygyrus infection localized to the gut can mute BCG-specific CD4+ T cell priming in both the spleen and skin-draining lymph nodes. Furthermore, H. polygyrus infection reduced the magnitude of delayed-type hypersensitivity (DTH) to PPD in the skin. Consequently, H. polygyrus–infected mice challenged with BCG had a higher mycobacterial load in the liver compared with worm-free mice. The excretory–secretory product from H. polygyrus (HES) was found to dampen IFN-γ production by mycobacteria-specific CD4+ T cells. This inhibition was dependent on the TGF-βR signaling activity of HES, suggesting that TGF-β signaling plays a role in the impaired Th1 responses observed coinfection with worms. Similar to results with mycobacteria, H. polygyrus–infected mice displayed an increase in skin parasite load upon secondary infection with Leishmania major as well as a reduction in DTH responses to Leishmania Ag. We show that a nematode confined to the gut can mute T cell responses to mycobacteria and impair control of secondary infections distal to the gut. The ability of intestinal helminths to reduce DTH responses may have clinical implications for the use of skin test–based diagnosis of microbial infections. PMID:26819205

  7. Engineering new mycobacterial vaccine design for HIV–TB pediatric vaccine vectored by lysine auxotroph of BCG

    PubMed Central

    Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

    2014-01-01

    In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961

  8. Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis.

    PubMed

    Bhatt, Apoorva; Kremer, Laurent; Dai, Annie Z; Sacchettini, James C; Jacobs, William R

    2005-11-01

    Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development. PMID:16267284

  9. Mycobacterial disease in cats in Great Britain: I. Culture results, geographical distribution and clinical presentation of 339 cases.

    PubMed

    Gunn-Moore, Danièlle A; McFarland, Sarah E; Brewer, Jacqueline I; Crawshaw, Timothy R; Clifton-Hadley, Richard S; Kovalik, Marcel; Shaw, Darren J

    2011-12-01

    This study investigated 339 cases of feline mycobacterial disease from cats with cutaneous lesions or masses found at exploratory laparotomy. Tissue samples were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study assessed which species of culturable mycobacteria were involved, where the cats lived, and their clinical presentation (physical findings, serum biochemistry, radiography, feline leukaemia virus and feline immunodeficiency virus status). Mycobacterium microti was cultured from 19%, Mycobacterium bovis 15%, Mycobacterium avium 7%, non-M avium non-tuberculous mycobacteria 6%, with no growth in 53% of samples. M microti, M bovis and M avium were found in almost mutually exclusive clusters within Great Britain (GB) (ie, M bovis in South-West England/Wales/Welsh Border, M avium in eastern England and M microti south of London and in South-West Scotland). While differences were seen in the clinical presentation and distribution of lesions caused by the different infections, these were not sufficiently different to be diagnostic. Cats commonly presented with single or multiple cutaneous lesions (74%), which were sometimes ulcerated or discharging, located most frequently on the head (54%). Lymph nodes were usually involved (47%); typically the submandibular nodes. Systemic or pulmonary signs were rarely seen (10-16%). When a cat is suspected of having mycobacteriosis, accurate identification of the species involved helps to determine appropriate action. Our findings show that knowing the cat's geographic location can be helpful, while the nature of the clinical presentation is less useful. Most cases of feline mycobacterial disease in GB are cutaneous. PMID:22079343

  10. Comparative genomic analysis of Mycobacterium iranicum UM_TJL against representative mycobacterial species suggests its environmental origin

    PubMed Central

    Tan, Joon Liang; Ngeow, Yun Fong; Wee, Wei Yee; Wong, Guat Jah; Ng, Hien Fuh; Choo, Siew Woh

    2014-01-01

    Mycobacterium iranicum is a newly reported mycobacterial species. We present the first comparative study of M. iranicum UM_TJL and other mycobacteria. We found M. iranicum to have a close genetic association with environmental mycobacteria infrequently associated with human infections. Nonetheless, UM_TJL is also equipped with many virulence genes (some of which appear to be the consequence of transduction-related gene transfer) that have been identified in established human pathogens. Taken all together, our data suggest that M. iranicum is an environmental bacterium adapted for pathogenicity in the human host. This comparative study provides important clues and forms the basis for future functional studies on this mycobacterium. PMID:25417557

  11. An unusual outbreak of nontuberculous mycobacteria in hospital respiratory wards: Association with nontuberculous mycobacterial colonization of hospital water supply network.

    PubMed

    D'Antonio, Salvatore; Rogliani, Paola; Paone, Gregorino; Altieri, Alfonso; Alma, Mario Giuseppe; Cazzola, Mario; Puxeddu, Ermanno

    2016-06-01

    The incidence and prevalence of pulmonary nontuberculous mycobacterial (NTM) infection is increasing worldwide arousing concerns that NTM infection may become a serious health challenge. We recently observed a significant increase of NTM-positive sputa samples from patients referred to respiratory disease wards of a large tertiary hospital in Rome. A survey to identify possible NTM contamination revealed a massive presence of NTM in the hospital water supply network. After decontamination procedures, NTM presence dropped both in water pipelines and sputa samples. We believe that this observation should encourage water network surveys for NTM contamination and prompt decontamination procedures should be considered to reduce this potential source of infection. PMID:27242241

  12. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    PubMed Central

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-01-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis. PMID:27457620

  13. Tuberculous lymphadenitis: Comparison of cytomorphology, Ziehl–Neelsen staining, and rapid mycobacterial culture at a pediatric superspecialty hospital

    PubMed Central

    Mahana, Sonam; Tomar, Reena; Agrawal, Rawi; Saksena, Rushika; Manchanda, Vikas; Gupta, Ruchika

    2016-01-01

    Background: To evaluate and compare the role of Ziehl–Neelsen (ZN) staining and mycobacterial culture in diagnosis of tuberculous lymphadenitis. Materials and Methods: A total of 56 fine needle aspirations (FNAs) from patients who were clinically suspected to have tuberculous lymphadenitis were included. Acid-fast Bacilli detection was attempted by ZN staining on smears as well as culture on Middlebrook 7H9 broth. Percentage positivity of both smears and culture was calculated. Results: Of the 56 cases, 46 showed cytomorphological features consistent with tuberculosis (TB). The most common pattern was only necrosis in 37 cases followed by necrotizing granulomas in 13 cases. ZN-stained smears were positive in 40 cases while culture was positive in only 27 cases. The highest smear and culture positivity was noted in cases with only necrosis. In six cases, diagnosis of TB was made on culture alone since smear was negative in these cases. Conclusion: FNA is a reliable technique for early and accurate diagnosis of tuberculous lymphadenitis in many cases. Mycobacterial culture by newer rapid techniques can assist in bacillary detection in smear-negative cases and also allows for drug sensitivity testing. Hence, culture should be resorted to in such cases.

  14. Effect of intestinal resection on serum antibodies to the mycobacterial 45/48 kilodalton doublet antigen in Crohn's disease.

    PubMed Central

    Kreuzpaintner, G; Das, P K; Stronkhorst, A; Slob, A W; Strohmeyer, G

    1995-01-01

    Interest in the role of mycobacterial infection in Crohn's disease has been revived by the cultural detection of Mycobacterium paratuberculosis in patients with Crohn's disease. This hypothesis was examined serologically using assays with high specificity for Crohn's disease. The effect of intestinal resection on serum antibodies specific for Crohn's disease was investigated with an immunoblot assay and an enzyme linked immunosorbent assay using the 45/48 kilodalton doublet antigen of Mycobacterium tuberculosis. Antibodies were detected in 64.7% of patients with Crohn's disease (n = 17), 10% of patients with ulcerative colitis (n = 10), 5% of patients with carcinoma of the colon (n = 20), and none of 10 healthy subjects with the immunoblot assay. Statistical comparison of the Crohn's disease patients with each control group resulted in p = 0.0000236. Immunoglobulin G was essentially unchanged 75 days (mean) after surgery. After more than 180 days, however, the antibody response was reduced in all of five patients studied, and was no longer demonstrable in two of them (40%). Simultaneously, the Crohn's disease activity index (CDAI) decreased. Both the high specificity of this assay for Crohn's disease and the diminished antibody response after intestinal resection in parallel with decreased CDAI support a mycobacterial aetiology of Crohn's disease. Images Figure 1 Figure 2 Figure 3 PMID:7590431

  15. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

    PubMed

    Abdallah, Abdallah M; Bestebroer, Jovanka; Savage, Nigel D L; de Punder, Karin; van Zon, Maaike; Wilson, Louis; Korbee, Cees J; van der Sar, Astrid M; Ottenhoff, Tom H M; van der Wel, Nicole N; Bitter, Wilbert; Peters, Peter J

    2011-11-01

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. PMID:21957139

  16. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    NASA Astrophysics Data System (ADS)

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-07-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

  17. Fasciola hepatica infection reduces Mycobacterium bovis burden and mycobacterial uptake and suppresses the pro-inflammatory response.

    PubMed

    Garza-Cuartero, L; O'Sullivan, J; Blanco, A; McNair, J; Welsh, M; Flynn, R J; Williams, D; Diggle, P; Cassidy, J; Mulcahy, G

    2016-07-01

    Bovine tuberculosis (BTB), caused by Mycobacterium bovis, has an annual incidence in cattle of 0.5% in the Republic of Ireland and 4.7% in the UK, despite long-standing eradication programmes being in place. Failure to achieve complete eradication is multifactorial, but the limitations of diagnostic tests are significant complicating factors. Previously, we have demonstrated that Fasciola hepatica infection, highly prevalent in these areas, induced reduced sensitivity of the standard diagnostic tests for BTB in animals co-infected with F. hepatica and M. bovis. This was accompanied by a reduced M. bovis-specific Th1 immune response. We hypothesized that these changes in co-infected animals would be accompanied by enhanced growth of M. bovis. However, we show here that mycobacterial burden in cattle is reduced in animals co-infected with F. hepatica. Furthermore, we demonstrate a lower mycobacterial recovery and uptake in blood monocyte-derived macrophages (MDM) from F. hepatica-infected cattle which is associated with suppression of pro-inflammatory cytokines and a switch to alternative activation of macrophages. However, the cell surface expression of TLR2 and CD14 in MDM from F. hepatica-infected cattle is increased. These findings reflecting the bystander effect of helminth-induced downregulation of pro-inflammatory responses provide insights to understand host-pathogen interactions in co-infection. PMID:27108767

  18. Mycobacterial infection in Northern snakehead (Channa argus) from the Potomac River catchment

    USGS Publications Warehouse

    Densmore, Christine L.; Iwanowicz, L.R.; Henderson, A.P.; Iwanowicz, D.D.; Odenkirk, J.S.

    2016-01-01

    The Northern snakehead, Channa argus (Cantor), is a non-native predatory fish that has become established regionally in some temperate freshwater habitats within the United States. Over the past decade, Northern snakehead populations have developed within aquatic ecosystems throughout the eastern USA, including the Potomac River system within Virginia, Maryland and Washington, D.C. Since this species was initially observed in this region in 2002, the population has expanded considerably (Odenkirk & Owens 2007). In the Chesapeake Bay watershed, populations of Northern snakehead exist in the lower Potomac River and Rappahannock Rivers on the Western shore of the Bay, and these fish have also been found in middle or upper reaches of river systems on the Eastern shore of the Bay, including the Nanticoke and Wicomico Rivers among others. Over the past several years, many aspects of Northern snakehead life history in the Potomac River have been described, including range and dispersal patterns, microhabitat selection and diet (Lapointe, Thorson & Angermeier 2010; Saylor, Lapointe & Angermeier 2012; Lapointe, Odenkirk & Angermeier 2013). However, comparatively little is known about their health status including susceptibility to parasitism and disease and their capacity to serve as reservoirs of disease for native wildlife. Although considered hardy by fisheries biologists, snakehead fish have demonstrated susceptibility to a number of described piscine diseases within their native range and habitat in Asia. Reported pathogens of significance in snakehead species in Asia include snakehead rhabdovirus (Lio-Po et al. 2000), aeromonad bacteria (Zheng, Cao & Yang 2012), Nocardia (Wang et al. 2007) andMycobacterium spp. (Chinabut, Limsuwan & Chantatchakool 1990; ). Mycobacterial isolates recovered from another snakehead species (Channa striata) in the previous studies have included M. marinum and M. fortuitum, as identified through molecular

  19. Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages

    PubMed Central

    George, Remo; Cavalcante, Renata; Jr, Celso Carvalho; Marques, Elyana; Waugh, Jonathan B; Unlap, M Tino

    2015-01-01

    Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis (MTB). Once the pathogen enters the body, it subverts the host immune defenses and thrives for extended periods of time within the host macrophages in the lung granulomas, a condition called latent tuberculosis (LTB). Persons with LTB are prone to reactivation of the disease when the body’s immunity is compromised. Currently there are no reliable and effective diagnosis and treatment options for LTB, which necessitates new research in this area. The mycobacterial proteins and genes mediating the adaptive responses inside the macrophage is largely yet to be determined. Recently, it has been shown that the mce operon genes are critical for host cell invasion by the mycobacterium and for establishing a persistent infection in both in vitro and in mouse models of tuberculosis. The YrbE and Mce proteins which are encoded by the MTB mce operons display high degrees of homology to the permeases and the surface binding protein of the ABC transports, respectively. Similarities in structure and cell surface location impute a role in cell invasion at cholesterol rich regions and immunomodulation. The mce4 operon is also thought to encode a cholesterol transport system that enables the mycobacterium to derive both energy and carbon from the host membrane lipids and possibly generating virulence mediating metabolites, thus enabling the bacteria in its long term survival within the granuloma. Various deletion mutation studies involving individual or whole mce operon genes have shown to be conferring varying degrees of attenuation of infectivity or at times hypervirulence to the host MTB, with the deletion of mce4A operon gene conferring the greatest degree of attenuation of virulence. Antisense technology using synthetic siRNAs has been used in knocking down genes in bacteria and over the years this has evolved into a powerful tool for

  20. Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study

    PubMed Central

    Pankhurst, Louise J; del Ojo Elias, Carlos; Votintseva, Antonina A; Walker, Timothy M; Cole, Kevin; Davies, Jim; Fermont, Jilles M; Gascoyne-Binzi, Deborah M; Kohl, Thomas A; Kong, Clare; Lemaitre, Nadine; Niemann, Stefan; Paul, John; Rogers, Thomas R; Roycroft, Emma; Smith, E Grace; Supply, Philip; Tang, Patrick; Wilcox, Mark H; Wordsworth, Sarah; Wyllie, David; Xu, Li; Crook, Derrick W

    2016-01-01

    of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. Interpretation We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. Funding National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin. PMID:26669893

  1. Predicting the subcellular localization of mycobacterial proteins by incorporating the optimal tripeptides into the general form of pseudo amino acid composition.

    PubMed

    Zhu, Pan-Pan; Li, Wen-Chao; Zhong, Zhe-Jin; Deng, En-Ze; Ding, Hui; Chen, Wei; Lin, Hao

    2015-02-01

    Mycobacterium tuberculosis is a bacterium that causes tuberculosis, one of the most prevalent infectious diseases. Predicting the subcellular localization of mycobacterial proteins in this bacterium may provide vital clues for the prediction of protein function as well as for drug discovery and design. Therefore, a computational method that can predict the subcellular localization of mycobacterial proteins with high precision is highly desirable. We propose a computational method to predict the subcellular localization of mycobacterial proteins. An objective and strict benchmark dataset was constructed after collecting 272 non-redundant proteins from the universal protein resource (the UniProt database). Subsequently, a novel feature selection strategy based on binomial distribution was used to optimize the feature vector. Finally, a subset containing 219 chosen tripeptide features was imported into a support vector machine-based method to estimate the performance of the dataset in accurately and sensitively identifying these proteins. We found that the proposed method gave a maximum overall accuracy of 89.71% with an average accuracy of 81.12% in the jackknife cross-validation. The results indicate that our prediction method gave an efficient and powerful performance when compared with other published methods. We made the proposed method available on a purpose built Web server called MycoSub that is freely accessible at . We anticipate that MycoSub will become a useful tool for studying the functions of mycobacterial proteins and for designing and developing anti-mycobacterium drugs. PMID:25437899

  2. MUSASHI-Mediated Expression of JMJD3, a H3K27me3 Demethylase, Is Involved in Foamy Macrophage Generation during Mycobacterial Infection

    PubMed Central

    Singh, Vikas; Karnam, Anupama; Mukherjee, Tanushree; Mahadik, Kasturi; Parikh, Pankti; Singh, Amit; Rajmani, R. S.; Ramachandra, Subbaraya G.; Balaji, Kithiganahalli Narayanaswamy

    2016-01-01

    Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity. PMID:27532872

  3. Specific recognition of mycobacterial protein and peptide antigens by gamma-delta T cell subsets following infection with virulent Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Promoting effective immunity to Mycobacterium bovis infection is a challenge that is of interest to the fields of human and animal medicine alike. We report that 'd T cells from virulent M. bovis infected cattle respond specifically and directly to complex, protein and non-protein mycobacterial anti...

  4. Rituximab as Successful Adjunct Treatment in a Patient With Disseminated Nontuberculous Mycobacterial Infection Due to Acquired Anti–Interferon-γ Autoantibody

    PubMed Central

    Czaja, Christopher A.; Merkel, Patricia A.; Chan, Edward D.; Lenz, Laurel L.; Wolf, Molly L.; Alam, Rafeul; Frankel, Stephen K.; Fischer, Aryeh; Gogate, Shaila; Perez-Velez, Carlos M.; Knight, Vijaya

    2014-01-01

    An acquired immune deficiency due to interferon gamma (IFN-γ) autoantibodies was diagnosed in a 78-year-old Japanese man with treatment-refractory disseminated nontuberculous mycobacterial infection. In addition to standard antimycobacterial therapy, he was successfully treated with rituximab to eliminate B cells and thereby the autoantibody. Subsequently, he obtained a sustained remission from infection. PMID:24336756

  5. Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR.

    PubMed

    Mehta, Promod K; Singh, Netrapal; Dharra, Renu; Dahiya, Bhawna; Sharma, Suman; Sheoran, Abhishek; Gupta, Krishna B; Chaudhary, Dhruva; Mehta, Neeru; Varma-Basil, Mandira

    2016-08-01

    Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients. PMID:27164021

  6. MUSASHI-Mediated Expression of JMJD3, a H3K27me3 Demethylase, Is Involved in Foamy Macrophage Generation during Mycobacterial Infection.

    PubMed

    Holla, Sahana; Prakhar, Praveen; Singh, Vikas; Karnam, Anupama; Mukherjee, Tanushree; Mahadik, Kasturi; Parikh, Pankti; Singh, Amit; Rajmani, R S; Ramachandra, Subbaraya G; Balaji, Kithiganahalli Narayanaswamy

    2016-08-01

    Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity. PMID:27532872

  7. Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG

    PubMed Central

    Nakedi, Kehilwe C.; Nel, Andrew J. M.; Garnett, Shaun; Blackburn, Jonathan M.; Soares, Nelson C.

    2015-01-01

    Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. PMID:25904896

  8. Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

    PubMed

    Nakedi, Kehilwe C; Nel, Andrew J M; Garnett, Shaun; Blackburn, Jonathan M; Soares, Nelson C

    2015-01-01

    Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. PMID:25904896

  9. Mycobacterial disease in a population of 339 cats in Great Britain: II. Histopathology of 225 cases, and treatment and outcome of 184 cases.

    PubMed

    Gunn-Moore, Danièlle A; McFarland, Sarah E; Schock, Alex; Brewer, Jacqueline I; Crawshaw, Tim R; Clifton-Hadley, Richard S; Shaw, Darren J

    2011-12-01

    This study investigated 339 cases of feline mycobacterial infection, with histopathology findings from 225 cases, and treatment and outcome information from 184 cases. Tissue samples from cats with cutaneous lesions or suspicious masses at exploratory laparotomy were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study reviewed the files for information about histopathology, treatment and outcome, and blindly reviewed histopathological changes (including staining for acid-fast bacteria [AFB]) in a sub-set of 45 cases. When a cat is suspected of having a mycobacterial infection, accurate identification of the species involved helps to determine possible treatment options and prognosis. The study confirmed that histopathology and the presence of AFB are useful tools in the recognition of mycobacterial infection. Unfortunately, they did little to help determine the species of mycobacteria involved. The study identified a group of cats that were negative for AFB at the primary laboratory, but from which mycobacteria could be cultured; commonly Mycobacterium bovis or Mycobacterium microti. The study also identified a group of cats which where culture negative, despite typical signs of mycobacterial infection and positive AFB staining. Many cases responded favourably to treatment (56% of the cases where information was available), and many cats gained complete remission (42%). However, relapses were common (64%) and often followed by pulmonary and/or systemic spread that may have resulted from treatment with short courses of single drugs. This study shows that the diagnosis and treatment of feline mycobacteriosis is complex and challenging. PMID:22061264

  10. A hybrid soft solar cell based on the mycobacterial porin MspA linked to a sensitizer-viologen Diad.

    PubMed

    Perera, Ayomi S; Subbaiyan, Navaneetha K; Kalita, Mausam; Wendel, Sebastian O; Samarakoon, Thilani N; D'Souza, Francis; Bossmann, Stefan H

    2013-05-01

    A prototype of a nano solar cell containing the mycobacterial channel protein MspA has been successfully designed. MspA, an octameric transmembrane channel protein from Mycobacterium smegmatis, is one of the most stable proteins known to date. Eight Ruthenium(II) aminophenanthroline-viologen maleimide Diads (Ru-Diads) have been successfully bound to the MspA mutant MspAA96C via cysteine-maleimide bonds. MspA is known to form double layers in which it acts as nanoscopic surfactant. The nanostructured layer that is formed by (Ru-Diad)8MspA at the TiO2 electrode is photochemically active. The resulting "protein nano solar cell" features an incident photon conversion efficiency of 1% at 400 nm. This can be regarded as a proof-of-principle that stable proteins can be successfully integrated into the design of solar cells. PMID:23611424

  11. The FHA-containing protein GarA acts as a phosphorylation-dependent molecular switch in mycobacterial signaling.

    PubMed

    England, Patrick; Wehenkel, Annemarie; Martins, Sonia; Hoos, Sylviane; André-Leroux, Gwénaëlle; Villarino, Andrea; Alzari, Pedro M

    2009-01-22

    Fork-head associated (FHA) domains are widely found in bacteria, but their cellular functions remain unclear. Here, we focus on Mycobacterium tuberculosis GarA, an FHA-containing protein conserved in actinomycetes that is phosphorylated by different Ser/Thr protein kinases. Using various physicochemical approaches, we show that phosphorylation significantly stabilizes GarA, and that its FHA domain interacts strongly with the phosphorylated N-terminal extension. Altogether, our results indicate that phosphorylation triggers an intra-molecular protein closure, blocking the phosphothreonine-binding site and switching off the regulatory properties of GarA. The model can explain the reported functions of this mycobacterial protein as regulator of glycogen degradation and glutamate metabolism. PMID:19114043

  12. The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

    PubMed Central

    Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H.; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R.; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V.

    2015-01-01

    PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture. PMID:26136255

  13. Identification of a Novel Mycobacterial Arabinosyltransferase Activity Which Adds an Arabinosyl Residue to α-d-Mannosyl Residues.

    PubMed

    Angala, Shiva Kumar; McNeil, Michael R; Zou, Lu; Liav, Avraham; Zhang, Junfeng; Lowary, Todd L; Jackson, Mary

    2016-06-17

    The arabinosyltransferases responsible for the biosynthesis of the arabinan domains of two abundant heteropolysaccharides of the cell envelope of all mycobacterial species, lipoarabinomannan and arabinogalactan, are validated drug targets. Using a cell envelope preparation from Mycobacterium smegmatis as the enzyme source and di- and trimannoside synthetic acceptors, we uncovered a previously undetected arabinosyltransferase activity. Thin layer chromatography, GC/MS, and LC/MS/MS analyses of the major enzymatic product are consistent with the transfer of an arabinose residue to the 6 position of the terminal mannosyl residue at the nonreducing end of the acceptors. The newly identified enzymatic activity is resistant to ethambutol and could correspond to the priming arabinosyl transfer reaction that occurs during lipoarabinomannan biosynthesis. PMID:27045860

  14. A Novel Inhibitor of Gyrase B Is a Potent Drug Candidate for Treatment of Tuberculosis and Nontuberculosis Mycobacterial Infections

    PubMed Central

    Jones, Steven M.; Hanzelka, Brian L.; Perola, Emanuele; Shoen, Carolyn M.; Cynamon, Michael H.; Ngwane, Andile H.; Wiid, Ian J.; van Helden, Paul D.; Betoudji, Fabrice; Nuermberger, Eric L.; Thomson, John A.

    2014-01-01

    New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 μg/ml and 0.08 to 5.48 μg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 μg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 μg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections. PMID:25534737

  15. Mapping of multiple HLA class II-restricted T-cell epitopes of the mycobacterial 70-kilodalton heat shock protein.

    PubMed Central

    Oftung, F; Geluk, A; Lundin, K E; Meloen, R H; Thole, J E; Mustafa, A S; Ottenhoff, T H

    1994-01-01

    By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules. PMID:7525484

  16. Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery.

    PubMed

    Poen, Sinothai; Nakatani, Yoshio; Opel-Reading, Helen K; Lassé, Moritz; Dobson, Renwick C J; Krause, Kurt L

    2016-05-01

    Glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria and has been a favoured target in pharmaceutical drug design efforts. It has recently been proven to be essential in Mycobacterium tuberculosis, the causative organism of tuberculosis, a disease for which new medications are urgently needed. In the present study, we have determined the protein crystal structures of MurI from both M. tuberculosis and Mycobacterium smegmatis in complex with D-glutamate to 2.3 Å and 1.8 Å resolution respectively. These structures are conserved, but reveal differences in their active site architecture compared with that of other MurI structures. Furthermore, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a new drug design effort will be needed to develop inhibitors. A new type of MurI dimer arrangement has been observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found to date. The mycobacterial MurI dimer is tightly associated, with a KD in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. We created triple mutants of this interface in the M. smegmatis glutamate racemase (D26R/R105A/G194R or E) that have appreciable activity (kcat=0.056-0.160 min(-1) and KM=0.26-0.51 mM) and can be utilized to screen proposed antimicrobial candidates for inhibition. PMID:26964898

  17. A novel inhibitor of gyrase B is a potent drug candidate for treatment of tuberculosis and nontuberculosis mycobacterial infections.

    PubMed

    Locher, Christopher P; Jones, Steven M; Hanzelka, Brian L; Perola, Emanuele; Shoen, Carolyn M; Cynamon, Michael H; Ngwane, Andile H; Wiid, Ian J; van Helden, Paul D; Betoudji, Fabrice; Nuermberger, Eric L; Thomson, John A

    2015-03-01

    New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 μg/ml and 0.08 to 5.48 μg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 μg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 μg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections. PMID:25534737

  18. Matrix metalloproteinase proteolysis of the mycobacterial HSP65 protein as a potential source of immunogenic peptides in human tuberculosis

    PubMed Central

    Shiryaev, Sergey A.; Cieplak, Piotr; Aleshin, Alexander E.; Sun, Qing; Zhu, Wenhong; Motamedchaboki, Khatereh; Sloutsky, Alexander; Strongin, Alex Y.

    2011-01-01

    Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces MMP-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial HSP65 chaperone is the major target for immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs which cleave HSP65, we used the MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, the membrane associated MMP-14, MMP-15, MMP-16 and MMP-24, and the glycosylphosphatidylinositol-linked MMP-17 and MMP-25 in our studies. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follows. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection. PMID:21752195

  19. Relationship Between Blood Concentrations of Hepcidin and Anemia Severity, Mycobacterial Burden, and Mortality Among Patients With HIV-Associated Tuberculosis

    PubMed Central

    Kerkhoff, Andrew D.; Meintjes, Graeme; Burton, Rosie; Vogt, Monica; Wood, Robin; Lawn, Stephen D.

    2016-01-01

    Background Anemia is very common in patients with human immunodeficiency virus (HIV)–associated tuberculosis, and hepcidin may be key in mediating this. We explored the relationship between blood hepcidin concentrations and anemia severity, mycobacterial burden and mortality in patients with HIV-associated tuberculosis. Methods Consecutive unselected HIV-infected adults in South Africa were systematically investigated for tuberculosis. Three groups were studied: 116 hospitalized inpatients with HIV infection and tuberculosis (hereafter, “hospitalized patients”), 58 ambulatory outpatients with HIV infection and newly diagnosed tuberculosis (hereafter, “ambulatory patients with tuberculosis”), and 58 ambulatory outpatients with HIV infection and without tuberculosis (hereafter, “ambulatory patients without tuberculosis”). Blood hepcidin concentrations were determined for all patients. Vital status at 3 months was determined, and independent predictors of mortality were identified. Results Median hepcidin concentrations were 38.8 ng/mL among hospitalized patients, 19.1 ng/mL among ambulatory patients with tuberculosis, and 5.9 ng/mL among ambulatory patients without tuberculosis (P < .001). In both groups with HIV-associated tuberculosis, hepcidin concentrations were strongly associated with greater anemia severity. Additionally, strong, graded associations were observed between hepcidin and composite indices of mycobacterial burden and dissemination. Patients dying within 3 months had significantly higher hepcidin concentrations, which independently predicted mortality. Conclusions High hepcidin concentrations were strongly associated with disseminated disease, anemia, and poor prognosis in patients with HIV-associated tuberculosis. Hepcidin may be a mechanistically important mediator underlying the high prevalence of severe anemia in these patients. PMID:26136467

  20. A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.

    PubMed

    Mohanty, Soumitra; Jagannathan, Lakshmanan; Ganguli, Geetanjali; Padhi, Avinash; Roy, Debasish; Alaridah, Nader; Saha, Pratip; Nongthomba, Upendra; Godaly, Gabriela; Gopal, Ramesh Kumar; Banerjee, Sulagna; Sonawane, Avinash

    2015-05-22

    Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs. PMID:25825498

  1. A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish*

    PubMed Central

    Mohanty, Soumitra; Jagannathan, Lakshmanan; Ganguli, Geetanjali; Padhi, Avinash; Roy, Debasish; Alaridah, Nader; Saha, Pratip; Nongthomba, Upendra; Godaly, Gabriela; Gopal, Ramesh Kumar; Banerjee, Sulagna; Sonawane, Avinash

    2015-01-01

    Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs. PMID:25825498

  2. Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm: Application to Mycobacterial Zinc Hydrolases.

    PubMed

    Romero, Juan Manuel; Martin, Mariano; Ramirez, Claudia Lilián; Dumas, Victoria Gisel; Marti, Marcelo Adrián

    2015-01-01

    Determination of the free energy profile for an enzyme reaction mechanism is of primordial relevance, paving the way for our understanding of the enzyme's catalytic power at the molecular level. Although hybrid, mostly DFT-based, QM/MM methods have been extensively applied to this type of studies, achieving accurate and statistically converged results at a moderate computational cost is still an open challenge. Recently, we have shown that accurate results can be achieved in less computational time, combining Jarzynski's relationship with a hybrid differential relaxation algorithm (HyDRA), which allows partial relaxation of the solvent during the nonequilibrium steering of the reaction. In this work, we have applied this strategy to study two mycobacterial zinc hydrolases. Mycobacterium tuberculosis infections are still a worldwide problem and thus characterization and validation of new drug targets is an intense field of research. Among possible drug targets, recently two essential zinc hydrolases, MshB (Rv1170) and MA-amidase (Rv3717), have been proposed and structurally characterized. Although possible mechanisms have been proposed by analogy to the widely studied human Zn hydrolases, several key issues, particularly those related to Zn coordination sphere and its role in catalysis, remained unanswered. Our results show that mycobacterial Zn hydrolases share a basic two-step mechanism. First, the attacking water becomes deprotonated by the conserved base and establishes the new C-O bond leading to a tetrahedral intermediate. The intermediate requires moderate reorganization to allow for proton transfer to the amide N and C-N bond breaking to occur in the second step. Zn ion plays a key role in stabilizing the tetrahedral intermediate and balancing the negative charge of the substrate during hydroxide ion attack. Finally, comparative analysis of other Zn hydrolases points to a convergent mechanistic evolution. PMID:26415840

  3. Matrix metalloproteinase proteolysis of the mycobacterial HSP65 protein as a potential source of immunogenic peptides in human tuberculosis.

    PubMed

    Shiryaev, Sergey A; Cieplak, Piotr; Aleshin, Alexander E; Sun, Qing; Zhu, Wenhong; Motamedchaboki, Khatereh; Sloutsky, Alexander; Strongin, Alex Y

    2011-09-01

    Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces matrix metalloproteinase (MMP)-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial 65 kDa heat shock protein (HSP65) chaperone is the major target for the immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs that cleave HSP65, we used MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, membrane-associated MMP-14, MMP-15, MMP-16 and MMP-24, and glycosylphosphatidylinositol-linked MMP-17 and MMP-25. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follow. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection. PMID:21752195

  4. High Mortality of Disseminated Non-Tuberculous Mycobacterial Infection in HIV-Infected Patients in the Antiretroviral Therapy Era

    PubMed Central

    Kobayashi, Tetsuro; Nishijima, Takeshi; Teruya, Katsuji; Aoki, Takahiro; Kikuchi, Yoshimi; Oka, Shinichi; Gatanaga, Hiroyuki

    2016-01-01

    Background Little information is available on the mortality and risk factors associated with death in disseminated non-tuberculous mycobacterial infection (dNTM) in HIV-infected patients in the ART-era. Methods In a single-center study, HIV-infected dNTM with positive NTM culture from sterile sites between 2000 and 2013 were analysed. The clinical characteristics at commencement of anti-mycobacterial treatment (baseline) were compared between those who survived and died. Results Twenty-four patients were analyzed. [The median CD4 27/μL (range 2–185)]. Mycobacterium avium and M. intracellulare accounted for 20 (83%) and 3 (13%) of isolated NTM. NTM bacteremia was diagnosed in 15 (63%) patients. Seven (29%) patients died, and NTM bacteremia was significantly associated with mortality (p = 0.022). The baseline CD4 count was significantly lower in the non-survivors than the survivors (median 7/μL versus 49, p = 0.034). Concomitant AIDS-defining diseases or malignancies were not associated with mortality. Immune-reconstitution syndrome (IRS) occurred to 19 (79%) patients (8 paradoxical and 11 unmasking), and prognosis tended to be better in unmasking-IRS than the other patients (n = 13) (p = 0.078). Patients with paradoxical-IRS had marginally lower CD4 count and higher frequency of bacteremia than those with unmasking-IRS (p = 0.051, and 0.059). Treatment with systemic corticosteroids was applied in 63% and 55% of patients with paradoxical and unmasking-IRS, respectively. Conclusion dNTM in HIV-infected patients resulted in high mortality even in the ART-era. NTM bacteremia and low CD4 count were risk factors for death, whereas patients presented with unmasking-IRS had marginally better prognosis. IRS occurred in 79% of the patients, suggesting difficulty in the management of dNTM. PMID:26985832

  5. Indoleamine 2,3-Dioxygenase, Tryptophan Catabolism, and Mycobacterium avium subsp. paratuberculosis: a Model for Chronic Mycobacterial Infections ▿ †

    PubMed Central

    Plain, Karren M.; de Silva, Kumudika; Earl, John; Begg, Douglas J.; Purdie, Auriol C.; Whittington, Richard J.

    2011-01-01

    Virulent mycobacterial infections progress slowly, with a latent period that leads to clinical disease in a proportion of cases. Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen that causes paratuberculosis or Johne's disease (JD), a chronic intestinal disease of ruminants. Indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates tryptophan metabolism, was originally reported to have a role in intracellular pathogen killing and has since been shown to have an important immunoregulatory role in chronic immune diseases. Here we demonstrate an association between increased IDO levels and progression to clinical mycobacterial disease in a natural host, characterizing gene expression, protein localization, and functional effects. IDO mRNA levels were significantly increased in M. avium subsp. paratuberculosis-infected monocytic cells. Levels of both IDO gene and protein expression were significantly upregulated within the affected tissues of sheep with JD, particularly at the site of primary infection, the ileum, of animals with severe multibacillary disease. Lesion severity was correlated with the level of IDO gene expression. IDO gene expression was also increased in the peripheral blood cells of M. avium subsp. paratuberculosis-exposed sheep and cattle. IDO breaks down tryptophan, and systemic increases were functional, as shown by decreased plasma tryptophan levels, which correlated with the onset of clinical signs, a stage well known to be associated with Th1 immunosuppression. IDO may be involved in downregulating immune responses to M. avium subsp. paratuberculosis and other virulent mycobacteria, which may be an example of the pathogen harnessing host immunoregulatory pathways to aid survival. These findings raise new questions about the host-mycobacterium interactions in the progression from latent to clinical disease. PMID:21730087

  6. Synthesis and evaluation of small libraries of triazolylmethoxy chalcones, flavanones and 2-aminopyrimidines as inhibitors of mycobacterial FAS-II and PknG.

    PubMed

    Anand, Namrata; Singh, Priyanka; Sharma, Anindra; Tiwari, Sameer; Singh, Vandana; Singh, Diwakar K; Srivastava, Kishore K; Singh, B N; Tripathi, Rama Pati

    2012-09-01

    A synthetic strategy to access small libraries of triazolylmethoxy chalcones 4{1-20}, triazolylmethoxy flavanones 5{1-10} and triazolylmethoxy aminopyrimidines 6{1-17} from a common substrate 4-propargyloxy-2-hydroxy acetophenone using a set of different reactions has been developed. The chalcones and flavanones were screened against mycobacterial FAS-II pathway using a recombinant mycobacterial strain, against which the most potent compound showed ∼88% inhibition in bacterial growth and substantially induction of reporter gene activity at 100 μM concentration. The triazolylmethoxy aminopyrimdines were screened against PknG of Mycobaceterium tuberculosis displaying moderate to good activity (23-53% inhibition at 100 μM), comparable to the action of a standard inhibitor. PMID:22854194

  7. Signalling through MyD88 drives surface expression of the mycobacterial receptors MCL (Clecsf8, Clec4d) and Mincle (Clec4e) following microbial stimulation.

    PubMed

    Kerscher, Bernhard; Dambuza, Ivy M; Christofi, Maria; Reid, Delyth M; Yamasaki, Sho; Willment, Janet A; Brown, Gordon D

    2016-01-01

    The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this "two-step" model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity. PMID:27005451

  8. Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases

    PubMed Central

    Takács, Enikő; Nagy, Gergely; Leveles, Ibolya; Harmat, Veronika; Lopata, Anna; Tóth, Judit; Vértessy, Beáta G.

    2010-01-01

    dUTPases are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg2+-coordination. Our results on transient/steady-state kinetics, ligand-binding and a 1.80 Å-resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg2+ accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function. PMID:20493855

  9. Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases.

    PubMed

    Takács, Eniko; Nagy, Gergely; Leveles, Ibolya; Harmat, Veronika; Lopata, Anna; Tóth, Judit; Vértessy, Beáta G

    2010-07-16

    dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 A resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg(2+) accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function. PMID:20493855

  10. Identification of Mycobacterial Antigens in Human Urine by Use of Immunoglobulin G Isolated from Sera of Patients with Active Pulmonary Tuberculosis.

    PubMed

    Kim, Sun Hee; Lee, Nan-Ee; Lee, Jong Seok; Shin, Jeong Hwan; Lee, Ju Yeon; Ko, Jeong-Heon; Chang, Chulhun Ludgerus; Kim, Yong-Sam

    2016-06-01

    Point-of-care (POC) diagnostic testing of tuberculosis (TB) is a tremendous unmet need. In this study, four urinary mycobacterial antigens were identified through two independent approaches using IgG capture and immunodepletion methods. Among these, ModC was validated by a multiple reaction monitoring (MRM) method. As expected, the biomarkers elevated the clinical validity of TB diagnosis when combined with preexisting markers. PMID:26984972

  11. Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

    PubMed Central

    Ufimtseva, Elena

    2016-01-01

    The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells. PMID:27066505

  12. MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis*

    PubMed Central

    Pacheco, Sophia A.; Hsu, Fong-Fu; Powers, Katelyn M.; Purdy, Georgiana E.

    2013-01-01

    A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis. PMID:23836904

  13. Nitric Oxide Production Inhibition and Anti-Mycobacterial Activity of Extracts and Halogenated Sesquiterpenes from the Brazilian Red Alga Laurencia Dendroidea J. Agardh

    PubMed Central

    Biá Ventura, Thatiana Lopes; da Silva Machado, Fernanda Lacerda; de Araujo, Marlon Heggdorne; de Souza Gestinari, Lísia Mônica; Kaiser, Carlos Roland; de Assis Esteves, Francisco; Lasunskaia, Elena B.; Soares, Angélica Ribeiro; Muzitano, Michelle Frazão

    2015-01-01

    Background: Red algae of the genus Laurencia J. V. Lamouroux are a rich source of secondary metabolites with important pharmacological activities such as anti-tumoral, anti-inflammatory, anti-fungal, anti-viral, anti-leishmanial, anti-helminthic, anti-malarial, anti-trypanosomal, anti-microbial as well as anti-bacterial against Mycobacterium tuberculosis. Objective: In the present study, we evaluated the inhibition of nitric oxide (NO) and tumor necrosis factor-α production and the anti-mycobacterial activity of crude extracts from the red Alga Laurencia dendroidea (from the South-Eastern coast of Brazil). Halogenated sesquiterpenes elatol (1), obtusol (2) and cartilagineol (3), previously isolated from this Alga by our group, were also studied. Materials and Methods: The lipopolysaccharide-activated macrophage cells (RAW 264.7) were used as inflammation model. Cytotoxic effect was determined using a commercial lactate dehydrogenase (LDH) kit and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The growing Mycobacterium inhibition was verified against Mycobacterium bovis Bacillus Calmette–Guérin and M. tuberculosis H37 Rv strains. Results: The crude extract from Alga collected at Angra dos Reis, RJ, Brazil, was the most active inhibitor of both mycobacterial growth (half maximal inhibitory concentration [IC50] 8.7 ± 1.4 μg/mL) and NO production by activated macrophages (IC50 5.3 ± 1.3 μg/mL). The assays with isolated compounds revealed the anti-mycobacterial activity of obtusol (2), whereas (-)-elatol (1) inhibited the release of inflammatory mediators, especially NO. To our knowledge, this is the first report describing an anti-mycobacterial effect of L. dendroidea extract and demonstrating the association of this activity with obtusol (2). Conclusion: The described effects of active compounds from L. dendroidea are promising for the control of inflammation in infectious diseases and specifically, against mycobacterial infections

  14. Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

    SciTech Connect

    Shen Jianying; Hisaeda, Hajime; Chou Bin; Yu Qingsheng; Tu Liping; Himeno, Kunisuke

    2008-01-25

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8{sup +} T cells. In this study, we exploited UPS to induce CD8{sup +} T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-{gamma}-producing CD8{sup +} T cells compared with those from the latter group of mice.

  15. The mycobacterial cord factor adjuvant analogue trehalose-6,6'-dibehenate (TDB) activates the Nlrp3 inflammasome.

    PubMed

    Schweneker, Katrin; Gorka, Oliver; Schweneker, Marc; Poeck, Hendrik; Tschopp, Jürg; Peschel, Christian; Ruland, Jürgen; Gross, Olaf

    2013-04-01

    The success of a vaccine consists in the induction of an innate immune response and subsequent activation of the adaptive immune system. Because antigens are usually not immunogenic, the addition of adjuvants that activate innate immunity is required. The mycobacterial cord factor trehalose-6,6'-dimycolate (TDM) and its synthetic adjuvant analogue trehalose-6,6'-dibehenate (TDB) rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger innate immunity. In this study, we show that stimulation of bone marrow-derived dendritic cells (BMDCs) with TDB induces Nlrp3 inflammasome-dependent IL-1β secretion. While Card9 is required for NF-κB activation by TDB, it is dispensable for TDB-induced activation of the Nlrp3 inflammasome. Additionally, efflux of intracellular potassium, lysosomal rupture, and oxygen radical (ROS) production are crucial for caspase-1 processing and IL-1β secretion by TDB. In an in vivo inflammation model, we demonstrate that the recruitment of neutrophils by TDB is significantly reduced in the Nlrp3-deficient mice compared to the wild-type mice, while the production of chemokines in vitro is not influenced by the absence of Nlrp3. These results identify the Nlrp3 inflammasome as an essential mediator for the induction of an innate immune response triggered by TDB. PMID:22921586

  16. Platelets Direct Monocyte Differentiation Into Epithelioid-Like Multinucleated Giant Foam Cells With Suppressive Capacity Upon Mycobacterial Stimulation

    PubMed Central

    Feng, Yonghong; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Yin, Hongyun; Dong, Zhengwei; Mao, Ling; Zhou, Jun; Bi, Aixiao; Weber, Stephan; Maertzdorf, Jeroen; Chen, Gang; Chen, Yang; Kaufmann, Stefan H. E.

    2014-01-01

    Background. Epithelioid, foam, and multinucleated giant cells (MNGCs) are characteristics of tuberculosis granulomas, yet the precise genesis and functions of these transformed macrophages are unclear. We evaluated the role of platelets as drivers of macrophage transformation in mycobacterial infection. Methods. We employed flow cytometry and microscopy to assess cellular phenotype and phagocytosis. Immune assays allowed quantification of cytokines and chemokines, whereas gene microarray technology was applied to estimate global transcriptome alterations. Immunohistochemical investigations of tuberculosis granulomas substantiated our findings at the site of infection. Results. Monocytes differentiated in presence of platelets (MP-Macs) acquired a foamy, epithelioid appearance and gave rise to MNGCs (MP-MNGCs). MP-Macs up-regulated activation markers, phagocytosed mycobacteria, and released abundant interleukin 10. Upon extended culture, MP-Macs shared transcriptional features with epithelioid cells and M2 macrophages and up-regulated CXCL5 transcripts. In line with this, CXCL5 concentrations were significantly increased in airways of active tuberculosis patients. The platelet-specific CD42b antigen was detected in MP-Macs, likewise in macrophages, MNGCs, and epithelioid cells within tuberculosis granulomas, along with the platelet aggregation-inducing factor PDPN. Conclusions. Platelets drive macrophage differentiation into MNGCs with characteristics of epithelioid, foam, and giant cells observed in tuberculosis granulomas. Our data define platelets as novel participants in tuberculosis pathogenesis. PMID:24987031

  17. Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate†

    PubMed Central

    Mahapatra, Sebabrata; Yagi, Tetsuya; Belisle, John T.; Espinosa, Benjamin J.; Hill, Preston J.; McNeil, Michael R.; Brennan, Patrick J.; Crick, Dean C.

    2005-01-01

    Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-l-alanine-d-glutamate-meso-diaminopimelic acid-d-alanyl-d-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal d-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan. PMID:15805521

  18. Cationic Liposomes Formulated with Synthetic Mycobacterial Cordfactor (CAF01): A Versatile Adjuvant for Vaccines with Different Immunological Requirements

    PubMed Central

    Agger, Else Marie; Rosenkrands, Ida; Hansen, Jon; Brahimi, Karima; Vandahl, Brian S.; Aagaard, Claus; Werninghaus, Kerstin; Kirschning, Carsten; Lang, Roland; Christensen, Dennis; Theisen, Michael; Follmann, Frank; Andersen, Peter

    2008-01-01

    Background It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. Methods and Findings Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge. Conclusion CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection. PMID:18776936

  19. Elevated expression of T-bet in mycobacterial antigen-specific CD4(+) T cells from patients with tuberculosis.

    PubMed

    Yang, Bingfen; Zhai, Fei; Jiang, Jing; Wang, Xinjing; Cao, Zhihong; Cheng, Xiaoxing

    2015-01-01

    T-bet is a T-box transcriptional factor that controls the differentiation and effector functions of CD4 T cells. In this study, we studied the role of T-bet in regulating CD4(+) T cell immunity against tuberculosis (TB). T-bet expression in Mycobacterium tuberculosis antigen-specific CD4(+) T cells was significantly higher in patients with active TB than in individuals with latent TB infection (p<0.0001). Comparison of T-bet expression in TCM and TEM subsets showed that CD4(+)T-bet(+)M. tuberculosis antigen-specific CD4(+) T cells had significantly lower frequency of TCM (p=0.003) and higher frequency of TEM (p=0.003) than CD4(+)T-bet(-) cells. The expression of PD-1 in antigen-specific CD4(+) T cells was significantly higher in patients with TB than in individuals with latent TB infection (p=0.006). CD4(+)CD154(+)T-bet(+) T cells had significantly higher expression of PD-1 than CD4(+)CD154(+)T-bet(-) T cells (p=0.0028). It is concluded that T-bet expression might be associated with differentiation into effector memory cells and PD-1 expression in mycobacterial antigen-specific CD4(+) T cells. PMID:26302932

  20. In vitro anti-mycobacterial activity of nine medicinal plants used by ethnic groups in Sonora, Mexico

    PubMed Central

    2013-01-01

    Background Sonoran ethnic groups (Yaquis, Mayos, Seris, Guarijíos, Pimas, Kikapúes and Pápagos) use mainly herbal based preparations as their first line of medicinal treatment. Among the plants used are those with anti-tuberculosis properties; however, no formal research is available. Methods Organic extracts were obtained from nine medicinal plants traditionally used by Sonoran ethnic groups to treat different kinds of diseases; three of them are mainly used to treat tuberculosis. All of the extracts were tested against Mycobacterium tuberculosis H37Rv using the Alamar Blue redox bioassay. Results Methanolic extracts from Ambrosia confertiflora, Ambrosia ambrosioides and Guaiacum coulteri showed minimal inhibitory concentration (MIC) values of 200, 790 and 1000 μg/mL, respectively, whereas no effect was observed with the rest of the methanolic extracts at the concentrations tested. Chloroform, dichloromethane, and ethyl acetate extracts from Ambrosia confertiflora showed a MIC of 90, 120 and 160 μg/mL, respectively. Conclusions A. confertiflora and A. ambrosioides showed the best anti-mycobacterial activity in vitro. The activity of Guaiacum coulteri is consistent with the traditional use by Sonoran ethnic groups as anti-tuberculosis agent. For these reasons, it is important to investigate a broader spectrum of medicinal plants in order to find compounds active against Mycobacterium tuberculosis. PMID:24267469

  1. [Implementation of the technical requirements of the UNE-EN-ISO 15189 quality standard in a mycobacterial laboratory].

    PubMed

    Guna Serrano, M del Remedio; Ocete Mochón, M Dolores; Lahiguera, M José; Bresó, M Carmen; Gimeno Cardona, Concepción

    2013-02-01

    The UNE-EN-ISO 15189:2007 standard defines the requirements for quality and competence that must be met by medical laboratories. These laboratories should use this international standard to develop their own quality management systems and to evaluate their own competencies; in turn, this standard will be used by accreditation bodies to confirm or recognize the laboratories' competence. In clinical microbiology laboratories, application of the standard implies the implementation of the technical and specific management requirements that must be met to achieve optimal quality when carrying out microbiological tests. In Spain, accreditation is granted by the Spanish Accreditation Body (Entidad Nacional de Acreditación). This review aims to discuss the practical application of the standard's technical requirements in mycobacterial laboratory. Firstly, we define the scope of accreditation. Secondly, we specify how the items of the standard on personnel management, control of equipment, environmental facilities, method validation, internal controls and customer satisfaction surveys were developed and implemented in our laboratory. PMID:23453231

  2. Evaluation of the humoral response against mycobacterial peptides, homologous to MOG₃₅₋₅₅, in multiple sclerosis patients.

    PubMed

    Cossu, Davide; Mameli, Giuseppe; Masala, Speranza; Cocco, Eleonora; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo Antonio

    2014-12-15

    Bacillus Calmette-Guérin (BCG) and Mycobacterium avium subspecies paratuberculosis (MAP) have been associated with multiple sclerosis (MS). Clinical data indicates that BCG vaccination exerts anti-inflammatory effects in MS; conversely, MAP is thought to be one of the possible infectious factors responsible of MS through a molecular mimicry mechanism. A peptide-based indirect ELISA was used to detect antibodies against the encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35-55 epitope, and two mycobacterial peptides sharing sequence homology with the latter: MAP_2619c352-361/BCG_1224355-364 and BCG_3329c64-74. Among 40 MS patients and 39 healthy volunteers included in the study, only MOG35-55 was capable of inducing a significantly higher humoral response in MS subjects compared to controls. Indeed, 11 out of 40 MS subjects (27.5%) and only 2 out of 39 controls (5%) were antibody-positive for MOG35-55 (p=0.01, AUC=0.65). These findings strengthen the importance of MOG35-55 in MS pathogenesis. The MAP and BCG MOG-homologues epitopes investigated were not recognized in MS patients. Overall, the results allow us concluding that sharing homology of linear epitopes is necessary but not sufficient to induce antibody-mediated cross-reactivity. PMID:25271190

  3. Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-γ immunity.

    PubMed

    Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Abel, Laurent; Casanova, Jean-Laurent

    2014-12-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria, in otherwise healthy individuals with no overt abnormalities in routine hematological and immunological tests. MSMD designation does not recapitulate all the clinical features, as patients are also prone to salmonellosis, candidiasis and tuberculosis, and more rarely to infections with other intramacrophagic bacteria, fungi, or parasites, and even, perhaps, a few viruses. Since 1996, nine MSMD-causing genes, including seven autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, and IRF8) and two X-linked (NEMO, and CYBB) genes have been discovered. The high level of allelic heterogeneity has already led to the definition of 18 different disorders. The nine gene products are physiologically related, as all are involved in IFN-γ-dependent immunity. These disorders impair the production of (IL12B, IL12RB1, IRF8, ISG15, NEMO) or the response to (IFNGR1, IFNGR2, STAT1, IRF8, CYBB) IFN-γ. These defects account for only about half the known MSMD cases. Patients with MSMD-causing genetic defects may display other infectious diseases, or even remain asymptomatic. Most of these inborn errors do not show complete clinical penetrance for the case-definition phenotype of MSMD. We review here the genetic, immunological, and clinical features of patients with inborn errors of IFN-γ-dependent immunity. PMID:25453225

  4. Immune responses to Mycobacterial heat shock protein 70 accompany self-reactivity to human BiP in rheumatoid arthritis

    PubMed Central

    Shoda, Hirofumi; Hanata, Norio; Sumitomo, Shuji; Okamura, Tomohisa; Fujio, Keishi; Yamamoto, Kazuhiko

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease, and a member of human heat shock protein (HSP) 70 protein family, Binding Immunoglobulin Protein (BiP), has been identified as an important autoantigen for T and B cells. We herein focused on Mycobacterial (Myc) HSPs and immune responses to MycHSPs in RA patients. Serum titers of antibodies against MycHSP70 were significantly elevated in RA patients and correlated with serum anti-BiP antibody titers. A MycHSP70-derived HLA-DR4 major epitope was identified using the proliferative capacity of RA PBMCs as an indicator. The major epitope, MycHSP70287–306, was located at the corresponding position in the major epitope for human BiP336–355, and a strong correlation was found between the proliferation of PBMCs in response to MycHSP70287–306 and BiP336–355. The immunization of HLA-DR4 transgenic mice with MycHSP70 induced the proliferation of T cells and development of anti-BiP antibodies. In contrast, the oral administration of MycHSP70287–306 resulted in the amelioration of collagen-induced arthritis, serum antibody responses, and T cell proliferation. In conclusion, immune responses to MycHSP70 were associated with adaptive immunity against BiP in RA, and could be an important mechanism underlying the development of autoimmunity. PMID:26927756

  5. Menaquinone Synthesis is Critical for Maintaining Mycobacterial Viability During Exponential Growth and Recovery from Non-Replicating Persistence

    PubMed Central

    Dhiman, Rakesh K.; Mahapatra, Sebabrata; Slayden, Richard A.; Boyne, Melissa E.; Lenaerts, Anne; Hinshaw, Jerald C.; Angala, Shiva K.; Chatterjee, Delphi; Biswas, Kallolmay; Narayanasamy, Prabagaran; Kurosu, Michio; Crick, Dean C.

    2016-01-01

    Summary Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low μM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labeling studies and quantitation of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis. PMID:19220750

  6. Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target

    PubMed Central

    Cox, Jonathan A. G.; Fütterer, Klaus; Abrahams, Katherine A.; Bhatt, Apoorva; Alderwick, Luke J.; Reynolds, Robert C.; Loman, Nicholas J.; Nataraj, VijayaShankar; Alemparte, Carlos; Barros, David; Lloyd, Adrian J.; Ballell, Lluis; Hobrath, Judith V.; Besra, Gurdyal S.

    2014-01-01

    The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å. PMID:25409504

  7. Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-γ immunity

    PubMed Central

    Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Abel, Laurent; Casanova, Jean-Laurent

    2014-01-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria, in otherwise healthy individuals with no overt abnormalities in routine hematological and immunological tests. MSMD designation does not recapitulate all the clinical features, as patients are also prone to salmonellosis, candidiasis and tuberculosis, and more rarely to infections with other intramacrophagic bacteria, fungi, or parasites, and even, perhaps, a few viruses. Since 1996, nine MSMD-causing genes, including seven autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, and IRF8) and two X-linked (NEMO, CYBB) genes have been discovered. The high level of allelic heterogeneity has already led to the definition of 18 different disorders. The nine gene products are physiologically related, as all are involved in IFN-γ-dependent immunity. These disorders impair the production of (IL12B, IL12RB1, IRF8, ISG15, NEMO) or the response to (IFNGR1, IFNGR2, STAT1, IRF8, CYBB) IFN-γ. These defects account for only about half the known MSMD cases. Patients with MSMD-causing genetic defects may display other infectious diseases, or even remain asymptomatic. Most of these inborn errors do not show complete clinical penetrance for the case-definition phenotype of MSMD. We review here the genetic, immunological, and clinical features of patients with inborn errors of IFN-γ-dependent immunity. PMID:25453225

  8. Interleukin-1β triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to T cells

    PubMed Central

    Schenk, Mirjam; Fabri, Mario; Krutzik, Stephan R; Lee, Delphine J; Vu, David M; Sieling, Peter A; Montoya, Dennis; Liu, Philip T; Modlin, Robert L

    2014-01-01

    The rapid differentiation of monocytes into macrophages (MΦ) and dendritic cells is a pivotal aspect of the innate immune response. Differentiation is triggered following recognition of microbial ligands that activate pattern recognition receptors or directly by pro-inflammatory cytokines. We demonstrate that interleukin-1β (IL-1β) induces the rapid differentiation of monocytes into CD209+ MΦ, similar to activation via Toll-like receptor 2/1, but with distinct phenotypic and functional characteristics. The IL-1β induced MΦ express higher levels of key markers of phagocytosis, including the Fc-receptors CD16 and CD64, as well as CD36, CD163 and CD206. In addition, IL-1β-induced MΦ exert potent phagocytic activity towards inert particles, oxidized low-density lipoprotein and mycobacteria. Furthermore, IL-1β-induced MΦ express higher levels of HLA-DR and effectively present mycobacterial antigens to T cells. Therefore, the ability of IL-1β to induce monocyte differentiation into MΦ with both phagocytosis and antigen-presenting function is a distinct part of the innate immune response in host defence against microbial infection. PMID:24032597

  9. Clinical features and outcomes of Sweet's syndrome associated with non-tuberculous mycobacterial infection and other associated diseases.

    PubMed

    Chaowattanapanit, Suteeraporn; Choonhakarn, Charoen; Chetchotisakd, Ploenchan; Sawanyawisuth, Kittisak; Julanon, Narachai

    2016-05-01

    Sweet's syndrome (SS) is associated with various diseases including non-tuberculous mycobacterial infection (NTM). Recent reports have shown that SS associated with NTM is increasing. Clinical features of SS associated with NTM may be different from SS associated with other associated diseases. The aim of the present study was to compare clinical parameters and treatment outcomes of SS associated with NTM and other associated diseases. Patients from January 2004 to April 2014 diagnosed with SS were retrospectively enrolled. Clinical variables were compared between SS patients with and without NTM infection. There were 51 SS patients during the study period; 36 patients (70.59%) had NTM. Clinical variables between the NTM and other associated diseases were comparable: age, sex, and pattern and locations of skin lesions. Five laboratory factors were significantly different between the groups including white blood cell counts (NTM 25 800 vs 12 850 cells/mm(3) ), lymphocyte percentages (13.0% vs 18.7%), monocytes (3.0% vs 7.2%), blood urea nitrogen (BUN) (11.7 vs 8.1 mg/dL) and serum creatinine (Cr) (1.0 vs 0.7 mg/dL). The presence of markedly high white blood cell counts, a low percentage of mononuclear cells and high BUN/Cr levels in SS may be a clinical clue to recognize the association with NTM infections; particularly in dissemination. PMID:27109150

  10. Specific interaction between Mycobacterium tuberculosis lipoprotein-derived peptides and target cells inhibits mycobacterial entry in vitro

    PubMed Central

    Ocampo, Marisol; Curtidor, Hernando; Vanegas, Magnolia; Patarroyo, Manuel Alfonso; Patarroyo, Manuel Elkin

    2014-01-01

    Summary Tuberculosis (TB) continues being one of the diseases having the greatest mortality rates around the world, 8.7 million cases having been reported in 2011. An efficient vaccine against TB having a great impact on public health is an urgent need. Usually, selecting antigens for vaccines has been based on proteins having immunogenic properties for patients suffering TB and having had promising results in mice and non-human primates. Our approach has been based on a functional approach involving the pathogen–host interaction in the search for antigens to be included in designing an efficient, minimal, subunit-based anti-tuberculosis vaccine. This means that Mycobacterium tuberculosis has mainly been involved in studies and that lipoproteins represent an important kind of protein on the cell envelope which can also contribute towards this pathogen's virulence. This study has assessed the expression of four lipoproteins from M. tuberculosis H37Rv, i.e. Rv1411c (LprG), Rv1911c (LppC), Rv2270 (LppN) and Rv3763 (LpqH), and the possible biological activity of peptides derived from these. Five peptides were found for these proteins which had high specific binding to both alveolar A549 epithelial cells and U937 monocyte-derived macrophages which were able to significantly inhibit mycobacterial entry to these cells in vitro. PMID:25041568

  11. Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

    PubMed Central

    Carreño, Leandro J.; Batista-Gonzalez, Ana; Baena, Andres; Venkataswamy, Manjunatha M.; Xu, Jiayong; Yu, Xiaobo; Wallstrom, Garrick; Magee, D. Mitchell; LaBaer, Joshua; Achkar, Jacqueline M.; Jacobs, William R.; Chan, John; Porcelli, Steven A.; Casadevall, Arturo

    2014-01-01

    ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. PMID:25271291

  12. Insights into horizontal acquisition patterns of dormancy and reactivation regulon genes in mycobacterial species using a partitioning-based framework.

    PubMed

    Mehra, Varun; Ghosh, Tarini Shankar; Mande, Sharmila S

    2016-09-01

    Horizontal Gene Transfer (HGT) events, initially thought to be rare in Mycobacterium tuberculosis, have recently been shown to be involved in the acquisition of virulence operons in M. tuberculosis. We have developed a new partitioning framework based HGT prediction algorithm, called Grid3M, and applied the same for the prediction of HGTs in Mycobacteria. Validation and testing using simulated and real microbial genomes indicated better performance of Grid3M as compared with other widely used HGT prediction methods. Specific analysis of the genes belonging to dormancy/reactivation regulons across 14 mycobacterial genomes indicated that horizontal acquisition is specifically restricted to important accessory proteins. The results also revealed Burkholderia species to be a probable source of HGT genes belonging to these regulons. The current study provides a basis for similar analyses investigating the functional/evolutionary aspects of HGT genes in other pathogens. A database of Grid3M predicted HGTs in completely sequenced genomes is available at https://metagenomics.atc.tcs.com/Grid3M/. PMID:27581938

  13. Design, synthesis and characterization of novel inhibitors against mycobacterial β-ketoacyl CoA reductase FabG4.

    PubMed

    Banerjee, Deb Ranjan; Dutta, Debajyoti; Saha, Baisakhee; Bhattacharyya, Sudipta; Senapati, Kalyan; Das, Amit K; Basak, Amit

    2014-01-01

    We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1 has shown competitive inhibition with an inhibition constant (Ki) value of 3.97 ± 0.02 μM. On the other hand, compound 2 has been found to be a mixed type inhibitor with a Ki value of 0.88 ± 0.01 μM. Thermodynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis, indicated their good anti-mycobacterial properties. A preliminary structure-activity relationship (SAR) study supports the design of these inhibitors. These compounds may be possible candidates as lead compounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-based inhibitors of other reductase enzymes. PMID:24129589

  14. Nano-Self-Assemblies Based on Synthetic Analogues of Mycobacterial Monomycoloyl Glycerol and DDA: Supramolecular Structure and Adjuvant Efficacy.

    PubMed

    Martin-Bertelsen, Birte; Korsholm, Karen S; Roces, Carla B; Nielsen, Maja H; Christensen, Dennis; Franzyk, Henrik; Yaghmur, Anan; Foged, Camilla

    2016-08-01

    The mycobacterial cell-wall lipid monomycoloyl glycerol (MMG) is a potent immunostimulator, and cationic liposomes composed of a shorter synthetic analogue (MMG-1) and dimethyldioctadecylammonium (DDA) bromide represent a promising adjuvant that induces strong antigen-specific Th1 and Th17 responses. In the present study, we investigated the supramolecular structure and in vivo adjuvant activity of dispersions based on binary mixtures of DDA and an array of synthetic MMG-1 analogues (MMG-2/3/5/6) displaying longer (MMG-2) or shorter (MMG-3) alkyl chain lengths, or variations in stereochemistry of the polar headgroup (MMG-5) or of the hydrophobic moiety (MMG-6). Synchrotron small-angle X-ray scattering experiments and cryo transmission electron microscopy revealed that DDA:MMG-1/2/5/6 dispersions consisted of unilamellar and multilamellar vesicles (ULVs/MLVs), whereas a coexistence of both ULVs and hexosomes was observed for DDA:MMG-3, depending on the DDA:MMG molar ratio. The studies also showed that ULVs were formed, regardless of the structural characteristics of the neat MMG analogues in excess buffer [lamellar (MMG-1/2/5) or inverse hexagonal (MMG-3/6) phases]. Immunization of mice with a chlamydia antigen surface-adsorbed to DDA:MMG-1/3/6 dispersions revealed that all tested adjuvants were immunoactive and induced strong Th1 and Th17 responses with a potential for a central effector memory profile. The MMG-1 and MMG-6 analogues were equally immunoactive in vivo upon incorporation into DDA liposomes, despite the reported highly different immunostimulatory properties of the neat analogues in vitro, which were attributed to the different nanostructural characteristics. This clearly demonstrates that optimal formulation and delivery of MMG analogues to the immune system is of major importance and challenges the use of in vitro screening assays with nondispersed compounds to identify potential new vaccine adjuvants. PMID:27377146

  15. A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components.

    PubMed

    Xia, Mei; Hesser, Danny C; De, Prithwiraj; Sakala, Isaac G; Spencer, Charles T; Kirkwood, Jay S; Abate, Getahun; Chatterjee, Delphi; Dobos, Karen M; Hoft, Daniel F

    2016-09-01

    γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB. PMID:27297390

  16. In vivo gammadelta T cell priming to mycobacterial antigens by primary Mycobacterium tuberculosis infection and exposure to nonpeptidic ligands.

    PubMed Central

    Poccia, F.; Malkovsky, M.; Pollak, A.; Colizzi, V.; Sireci, G.; Salerno, A.; Dieli, F.

    1999-01-01

    BACKGROUND: The recognition of phosphorylated nonpeptidic microbial metabolites by Vgamma9Vdelta2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vgamma9Vdelta2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vgamma9Vdelta2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of gammadelta T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured. RESULTS: The Vgamma9Vdelta2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vgamma9Vdelta2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of gammadelta T cell activation in vivo. The in vivo reactivity of Vgamma9Vdelta2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vgamma9Vdelta2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children. CONCLUSIONS: The increased reactivity of Vgamma9Vdelta2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vgamma9Vdelta2 responses may be useful for monitoring the efficacy of antimycobacterial therapies. PMID:10449808

  17. Eukaryotic-Type Ser/Thr Protein Kinase Mediated Phosphorylation of Mycobacterial Phosphodiesterase Affects its Localization to the Cell Wall

    PubMed Central

    Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE) utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB, and PknL), Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behavior. On the other hand, phosphomimics of mPDE (T309D or T309E), exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue. PMID:26904001

  18. Predicting results of mycobacterial culture on sputum smear reversion after anti-tuberculous treatment: a case control study

    PubMed Central

    2010-01-01

    Background Little is currently known regarding sputum smear reversion (acid-fast smear becomes positive again after negative conversion) during anti-tuberculous treatment. This study aimed to evaluate its occurrence in patients with pulmonary tuberculosis (TB) and identify factors predicting results of mycobacterial culture for smear-reversion of sputum samples. Methods The retrospective review was performed in a tertiary referral center and a local teaching hospital in Taiwan. From 2000 to 2007, patients with smear-positive culture-confirmed pulmonary TB experiencing smear reversion after 14 days of anti-tuberculous treatment were identified. Results The 739 patients with smear-positive pulmonary TB had 74 (10%) episodes of sputum smear reversion that grew Mycobacterium tuberculosis in 22 (30%) (Mtb group). The remaining 52 episodes of culture-negative sputum samples were classified as the non-Mtb group. The anti-tuberculous regimen was modified after confirming smear reversion in 15 (20%). Fourteen episodes in the Mtb group and 15 in the non-Mtb group occurred during hospitalization. All were admitted to the negative-pressure rooms at the time of smear reversion. Statistical analysis showed that any TB drug resistance, smear reversion within the first two months of treatment or before culture conversion, and the absence of radiographic improvement before smear reversion were associated with the Mtb group. None of the smear reversion was due to viable M. tuberculosis if none of the four factors were present. Conclusions Sputum smear reversion develops in 10% of patients with smear-positive pulmonary TB, with 30% due to viable M. tuberculosis bacilli. Isolation and regimen modification may not be necessary for all drug-susceptible patients who already have radiographic improvement and develop smear reversion after two months of treatment or after sputum culture conversion. PMID:20205743

  19. Health-related quality of life, comorbidities and mortality in pulmonary nontuberculous mycobacterial infections: A systematic review.

    PubMed

    Yeung, Man Wah; Khoo, Edwin; Brode, Sarah K; Jamieson, Frances B; Kamiya, Hiroyuki; Kwong, Jeffrey C; Macdonald, Liane; Marras, Theodore K; Morimoto, Kozo; Sander, Beate

    2016-08-01

    Nontuberculous mycobacterial (NTM) infections are increasing in disease frequency worldwide. This systematic review examines health-related quality of life (HRQOL), comorbidities and mortality associated with pulmonary NTM disease. We searched MEDLINE, EMBASE, CINAHL, Scopus Life Sciences, conference proceedings and Google (earliest date available to February 2015) for primary studies. Eligible studies compared populations with and without pulmonary NTM disease in high-income jurisdictions. We excluded studies on HIV/AIDS. All languages were accepted. Two reviewers followed MOOSE and PRISMA reporting guidelines and independently appraised quality using STROBE. All studies were summarized qualitatively regardless of quality. Of 3193 citations screened, we included 17 studies mostly from Taiwan (n = 5) and the USA (n = 4). Two studies assessed HRQOL; one assessed comorbidities, 11 assessed mortality, and three assessed multiple outcomes. Populations with pulmonary NTM reported significantly worse or similar HRQOL than the general population, depending on the instruments used. Some suggested greater prevalence of having bronchiectasis (n = 2) and greater risk of developing pulmonary tuberculosis (n = 1). Most (n = 7) suggested no difference in mortality, although only one was age-matched and gender-matched to the general population. Four suggested NTM populations had higher mortality-two of which compared with the general population and were deemed of high quality, while two compared with non-NTM patients from hospital. High clinical heterogeneity in study design may explain discordant results. Bias assessments and controlling for confounding were carried out poorly. No consistent trends were observed although there is suggestion of an increased health burden from respiratory diseases and increased mortality associated with pulmonary NTM disease. PMID:27009804

  20. A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components

    PubMed Central

    Xia, Mei; Hesser, Danny C.; De, Prithwiraj; Sakala, Isaac G.; Spencer, Charles T.; Kirkwood, Jay S.; Abate, Getahun; Chatterjee, Delphi

    2016-01-01

    γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB. PMID:27297390

  1. Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients.

    PubMed

    Vemula, Mani H; Ganji, Rakesh; Sivangala, Ramya; Jakkala, Kiran; Gaddam, Sumanlatha; Penmetsa, Sitaramaraju; Banerjee, Sharmistha

    2016-01-01

    Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins

  2. Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients

    PubMed Central

    Vemula, Mani H.; Ganji, Rakesh; Sivangala, Ramya; Jakkala, Kiran; Gaddam, Sumanlatha; Penmetsa, Sitaramaraju; Banerjee, Sharmistha

    2016-01-01

    Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4+ T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can

  3. Benzo[d]thiazol-2-yl(piperazin-1-yl)methanones as new anti-mycobacterial chemotypes: Design, synthesis, biological evaluation and 3D-QSAR studies.

    PubMed

    Pancholia, Sahaj; Dhameliya, Tejas M; Shah, Parth; Jadhavar, Pradeep S; Sridevi, Jonnalagadda Padma; Yogeshwari, Perumal; Sriram, Dharmarajan; Chakraborti, Asit K

    2016-06-30

    The benzo[d]thiazol-2-yl(piperazin-1-yl)methanones scaffold has been identified as new anti-mycobacterial chemotypes. Thirty-six structurally diverse benzo[d]thiazole-2-carboxamides have been prepared and subjected to assessment of their potential anti-tubercular activity through in vitro testing against Mycobacterium tuberculosis H37Rv strain and evaluation of cytotoxicity against RAW 264.7 cell lines. Seventeen compounds showed anti-mycobacterial potential having MICs in the low (1-10) μM range. The 5-trifluoromethyl benzo[d]thiazol-2-yl(piperazin-1-yl)methanones emerged to be the most promising resulting in six positive hits (2.35-7.94 μM) and showed low-cytotoxicity (<50% inhibition at 50 μg/mL). The therapeutic index of these hits is 8-64. The quantitative structure activity relationship has been established adopting a statistically reliable CoMFA model showing high prediction (rpred(2)=0.718,rncv(2)=0.995). PMID:27061982

  4. Mycobacterial receptor, Clec4d (CLECSF8, MCL), is coregulated with Mincle and upregulated on mouse myeloid cells following microbial challenge

    PubMed Central

    Kerscher, Bernhard; Wilson, Gillian J.; Reid, Delyth M.; Mori, Daiki; Taylor, Julie A.; Besra, Gurdyal S.; Yamasaki, Sho; Brown, Gordon D.

    2015-01-01

    The C‐type lectin receptor (CTLR), Clec4d (MCL, CLECSF8), is a member of the Dectin‐2 cluster of CTLRs, which also includes the related receptors Mincle and Dectin‐2. Like Mincle, Clec4d recognizes mycobacterial cord factor, trehalose dimycolate, and we recently demonstrated its key role in anti‐mycobacterial immunity in mouse and man. Here, we characterized receptor expression in naïve mice, under inflammatory conditions, and during Mycobacterium bovis BCG infection using newly generated monoclonal antibodies. In naïve mice, Clec4d was predominantly expressed on myeloid cells within the peritoneal cavity, blood, and bone marrow. Unexpectedly, basal expression of Clec4d was very low on leukocytes in the lung. However, receptor expression was significantly upregulated on pulmonary myeloid cells during M. bovis BCG infection. Moreover, Clec4d expression could be strongly induced in vitro and in vivo by various microbial stimuli, including TLR agonists, but not exogenous cytokines. Notably, we show that Clec4d requires association with the signaling adaptor FcRγ and Mincle, but not Dectin‐2, for surface expression. In addition, we provide evidence that Clec4d and Mincle, but not Dectin‐2, are interdependently coregulated during inflammation and infection. These data show that Clec4d is an inducible myeloid‐expressed CTLR in mice, whose expression is tightly linked to that of Mincle. PMID:26558717

  5. A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease.

    PubMed

    Kong, Xiao-Fei; Bousfiha, Aziz; Rouissi, Abdelfettah; Itan, Yuval; Abhyankar, Avinash; Bryant, Vanessa; Okada, Satoshi; Ailal, Fatima; Bustamante, Jacinta; Casanova, Jean-Laurent; Hirst, Jennifer; Boisson-Dupuis, Stéphanie

    2013-01-01

    We report identical twins with intellectual disability, progressive spastic paraplegia and short stature, born to a consanguineous family. Intriguingly, both children presented with lymphadenitis caused by the live Bacillus Calmette-Guérin (BCG) vaccine. Two syndromes - hereditary spastic paraplegia (HSP) and mycobacterial disease - thus occurred simultaneously. Whole-exome sequencing (WES) revealed a homozygous nonsense mutation (p.R1105X) of the AP4E1 gene, which was confirmed by Sanger sequencing. The p.R1105X mutation has no effect on AP4E1 mRNA levels, but results in lower levels of AP-4ε protein and of the other components of the AP-4 complex, as shown by western blotting, immunoprecipitation and immunofluorescence. Thus, the C-terminal part of the AP-4ε subunit plays an important role in maintaining the integrity of the AP-4 complex. No abnormalities of the IL-12/IFN-γ axis or oxidative burst pathways were identified. In conclusion, we identified twins with autosomal recessive AP-4 deficiency associated with HSP and mycobacterial disease, suggesting that AP-4 may play important role in the neurological and immunological systems. PMID:23472171

  6. Mycobacterial HBHA induces endoplasmic reticulum stress-mediated apoptosis through the generation of reactive oxygen species and cytosolic Ca2+ in murine macrophage RAW 264.7 cells

    PubMed Central

    Choi, J-A; Lim, Y-J; Cho, S-N; Lee, J-H; Jeong, J A; Kim, E J; Park, J B; Kim, S H; Park, H S; Kim, H-J; Song, C-H

    2013-01-01

    Mycobacterial heparin-binding haemagglutinin antigen (HBHA) is a virulence factor that induces apoptosis of macrophages. Endoplasmic reticulum (ER) stress-mediated apoptosis is an important regulatory response that can be utilised to study the pathogenesis of tuberculosis. In the present study, HBHA stimulation induced ER stress sensor molecules in a caspase-dependent manner. Pre-treatment of RAW 264.7 cells with an IκB kinase 2 inhibitor reduced not only C/EBP homology protein expression but also IL-6 and monocyte chemotactic protein-1 (MCP-1) production. BAPTA-AM reduced both ER stress responses and caspase activation and strongly suppressed HBHA-induced IL-6 and MCP-1 production in RAW 264.7 cells. Enhanced reactive oxygen species (ROS) production and elevated cytosolic [Ca2+]i levels were essential for HBHA-induced ER stress responses. Collectively, our data suggest that HBHA induces cytosolic [Ca2+]i, which influences the generation of ROS associated with the production of proinflammatory cytokines. These concerted and complex cellular responses induce ER stress-associated apoptosis during HBHA stimulation in macrophages. These results indicate that the ER stress pathway has an important role in the HBHA-induced apoptosis during mycobacterial infection. PMID:24336077

  7. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5

    PubMed Central

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  8. Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

    PubMed Central

    Schmitz, Karl R.; Sauer, Robert T.

    2014-01-01

    Summary Mycobacterial Clp-family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologs in other bacteria. Both ClpX and ClpC1 catalyze ATP-dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein-substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators. PMID:24976069

  9. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

    PubMed

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  10. A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens

    PubMed Central

    Baumann, Ralf; Kaempfer, Susanne; Chegou, Novel N.; Oehlmann, Wulf; Spallek, Ralf; Loxton, André G.; van Helden, Paul D.; Black, Gillian F.; Singh, Mahavir; Walzl, Gerhard

    2015-01-01

    Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%—with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases. PMID:26347586

  11. Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis.

    PubMed

    Kremer, L; Douglas, J D; Baulard, A R; Morehouse, C; Guy, M R; Alland, D; Dover, L G; Lakey, J H; Jacobs, W R; Brennan, P J; Minnikin, D E; Besra, G S

    2000-06-01

    Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs. PMID:10747933

  12. The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene

    PubMed Central

    Li, Yuqing; He, Zheng-Guo

    2012-01-01

    Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N11-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H2O2, but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H2O2.Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress. PMID:22272299

  13. Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines.

    PubMed

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-01-22

    Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme. PMID:19920138

  14. The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach

    PubMed Central

    Leisching, Gina; Pietersen, Ray-Dean; Mpongoshe, Vuyiseka; van Heerden, Carel; van Helden, Paul; Wiid, Ian; Baker, Bienyameen

    2016-01-01

    During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in

  15. A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

    PubMed Central

    Ghosh, Shreya; Samaddar, Sourabh; Kirtania, Prithwiraj

    2015-01-01

    ABSTRACT Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc2155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc2155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCE Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves

  16. Nontuberculous mycobacterial pulmonary infections

    PubMed Central

    Odell, John A.

    2014-01-01

    Pulmonary infections due to nontuberculous mycobacteria (NTM) are increasingly recognized worldwide. Although over 150 different species of NTM have been described, pulmonary infections are most commonly due to Mycobacterium avium complex (MAC), Mycobacterium kansasii, and Mycobacterium abscessus. The identification of these organisms in pulmonary specimens does not always equate with active infection; supportive radiographic and clinical findings are needed to establish the diagnosis. It is difficult to eradicate NTM infections. A prolonged course of therapy with a combination of drugs is required. Unfortunately, recurrent infection with new strains of mycobacteria or a relapse of infection caused by the original organism is not uncommon. Surgical resection is appropriate in selected cases of localized disease or in cases in which the infecting organism is resistant to medical therapy. Additionally, surgery may be required for infections complicated by hemoptysis or abscess formation. This review will summarize the practical aspects of the diagnosis and management of NTM thoracic infections, with emphasis on the indications for surgery and the results of surgical intervention. The management of NTM disease in patients with human immunodeficiency virus (HIV) infections is beyond the scope of this article and, unless otherwise noted, comments apply to hosts without HIV infection PMID:24624285

  17. Genotype heterogeneity of Mycobacterium tuberculosis within geospatial hotspots suggests foci of imported infection in Sydney, Australia.

    PubMed

    Gurjav, Ulziijargal; Jelfs, Peter; Hill-Cawthorne, Grant A; Marais, Ben J; Sintchenko, Vitali

    2016-06-01

    In recent years the State of New South Wales (NSW), Australia, has maintained a low tuberculosis incidence rate with little evidence of local transmission. Nearly 90% of notified tuberculosis cases occurred in people born in tuberculosis-endemic countries. We analyzed geographic, epidemiological and genotypic data of all culture-confirmed tuberculosis cases to identify the bacterial and demographic determinants of tuberculosis hotspot areas in NSW. Standard 24-loci mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-24) typing was performed on all isolates recovered between 2009 and 2013. In total 1692/1841 (91.9%) cases with confirmed Mycobacterium tuberculosis infection had complete MIRU-24 and demographic data and were included in the study. Despite some year-to-year variability, spatio-temporal analysis identified four tuberculosis hotspots. The incidence rate and the relative risk of tuberculosis in these hotspots were 2- to 10-fold and 4- to 8-fold higher than the state average, respectively. MIRU-24 profiles of M. tuberculosis isolates associated with these hotspots revealed high levels of heterogeneity. This suggests that these spatio-temporal hotspots, within this low incidence setting, can represent areas of predominantly imported infection rather than clusters of cases due to local transmission. These findings provide important epidemiological insight and demonstrate the value of combining tuberculosis genotyping and spatiotemporal data to guide better-targeted public health interventions. PMID:26187743

  18. Closely Related Mycobacterial Strains Demonstrate Contrasting Levels of Efficacy as Antitumor Vaccines and Are Processed for Major Histocompatibility Complex Class I Presentation by Multiple Routes in Dendritic Cells

    PubMed Central

    Cheadle, Eleanor J.; O'Donnell, Dearbhaile; Selby, Peter J.; Jackson, Andrew M.

    2005-01-01

    Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growing Mycobacterium smegmatis and the slow-growing M. bovis M. bovis BCG, were engineered to express a model tumor antigen, the Kb-restricted dominant cytotoxic T-lymphocyte epitope OVA257-264. Recombinant M. bovis BCG but not recombinant M. smegmatis conferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinant M. smegmatis infection led to a greater degree of dendritic cell maturation than recombinant M. bovis BCG infection. Antigen from recombinant M. smegmatis was processed and presented as OVA257-264 on Kb molecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinant M. bovis BCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growing M. bovis BCG but not that vectored by fast-growing, readily destroyed M. smegmatis is processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development. PMID:15664917

  19. Disseminated Bacillus Calmette-Guérin and Susceptibility to Mycobacterial Infections-Implications on Bacillus Calmette-Guérin Vaccinations.

    PubMed

    Lee, Pamela Pw

    2015-08-01

    Bacillus Calmette-Guérin (BCG) is a live vaccine and has the potential to cause local disease and systemic dissemination in immunocompromised hosts, including infants who are infected with human immunodeficiency virus (HIV) through vertical transmission, and patients with primary immunodeficiencies (PID) such as severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), hyper-IgM syndrome, and defects of the IL12- IFNγ axis (Mendelian susceptibility to mycobacterial diseases, MSMD). Disseminated BCG is extremely difficult to treat. The chance of complete eradication is low unless functional immune response is restored by haematopoietic stem cell transplant. Prolonged use of anti-mycobacterial drugs often causes organ toxicities and drug resistance. Inflammatory complications which develop upon immunoreconstitution post-transplant may necessitate immunosuppressive treatment, which adversely affect immune recovery and increases risks of opportunistic infections. Multiple BCG reactivations can occur in patients with CGD and MSMD, and BCG can remain latent until reactivations take place in adulthood and manifest as disease. It is important for neonatologists, general practitioners, primary care clinicians and nurses working in maternal and child care centres to be aware of BCG-related complications, which may be the first sign of an underlying immunodeficiency. As neonatal BCG is included in standard vaccination schedule in many countries, it is a challenge to identify and avoid administration of BCG to infants who potentially have PIDs. Deferring BCG vaccination is recently advocated to protect highly vulnerable populations, but the appropriate strategy is yet to be determined. Newborn screening for SCID offers a potential to avoid this complication, if an integrated system of screening and vaccination can be organised. PMID:26477962

  20. Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

    PubMed

    Marinho, Fábio V; Fahel, Júlia S; Scanga, Charles A; Gomes, Marco Tulio R; Guimarães, Gabriela; Carvalho, Gabrielle R M; Morales, Stefanny V; Báfica, André; Oliveira, Sergio Costa

    2016-09-01

    The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections. PMID:27439514

  1. Rapid Rebound of the Treg Compartment in DEREG Mice Limits the Impact of Treg Depletion on Mycobacterial Burden, but Prevents Autoimmunity

    PubMed Central

    Varela, Filipa; Behrends, Jochen; Swallow, Maxine; Kruse, Friederike; Krull, Freyja; Ghorbani, Peyman; Mayer, Christian T.

    2014-01-01

    The development of an effective vaccine against tuberculosis (Tb) represents one of the major medical challenges of this century. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the only vaccine available at present, is mostly effective at preventing disseminated Tb in children, but shows variable protection against pulmonary Tb, the most common form in adults. The reasons for this poor efficacy are not completely understood, but there is evidence that T regulatory cells (Tregs) might be involved. Similarly, Tregs have been associated with the immunosuppression observed in patients infected with Tb and are therefore believed to play a role in pathogen persistence. Thus, Treg depletion has been postulated as a novel strategy to potentiate M. bovis BCG vaccination on one side, while on the other, employed as a therapeutic approach during chronic Tb infection. Yet since Tregs are critically involved in controlling autoimmune inflammation, elimination of Tregs may therefore also incur the danger of an excessive inflammatory immune response. Thus, understanding the dynamics and function of Tregs during mycobacterial infection is crucial to evaluate the potential of Treg depletion as a medical option. To address this, we depleted Tregs after infection with M. bovis BCG or Mycobacterium tuberculosis (Mtb) using DEREG mice, which express the diphtheria toxin (DT) receptor under the control of the FoxP3 locus, thereby allowing the selective depletion of FoxP3+ Tregs. Our results show that after depletion, the Treg niche is rapidly refilled by a population of DT-insensitive Tregs (diTregs) and bacterial load remains unchanged. On the contrary, impaired rebound of Tregs in DEREG × FoxP3GFP mice improves pathogen burden, but is accompanied by detrimental autoimmune inflammation. Therefore, our study provides the proof-of-principle that, although a high degree of Treg depletion may contribute to the control of mycobacterial infection, it carries the risk of autoimmunity

  2. Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

    PubMed Central

    Harris, Liam; Kuo, Emmeline; Zhou, Tina T.; Ahn, Christine J.; Nguyen, Lin; He, Qixin; Lu, Jamie; Menchavez, Phuong T.; Shin, Annie; Holton, Thomas; Sawaya, Michael R.; Cascio, Duilio; Eisenberg, David

    2013-01-01

    The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes. PMID:24312350

  3. Rapid rebound of the Treg compartment in DEREG mice limits the impact of Treg depletion on mycobacterial burden, but prevents autoimmunity.

    PubMed

    Berod, Luciana; Stüve, Philipp; Varela, Filipa; Behrends, Jochen; Swallow, Maxine; Kruse, Friederike; Krull, Freyja; Ghorbani, Peyman; Mayer, Christian T; Hölscher, Christoph; Sparwasser, Tim

    2014-01-01

    The development of an effective vaccine against tuberculosis (Tb) represents one of the major medical challenges of this century. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the only vaccine available at present, is mostly effective at preventing disseminated Tb in children, but shows variable protection against pulmonary Tb, the most common form in adults. The reasons for this poor efficacy are not completely understood, but there is evidence that T regulatory cells (Tregs) might be involved. Similarly, Tregs have been associated with the immunosuppression observed in patients infected with Tb and are therefore believed to play a role in pathogen persistence. Thus, Treg depletion has been postulated as a novel strategy to potentiate M. bovis BCG vaccination on one side, while on the other, employed as a therapeutic approach during chronic Tb infection. Yet since Tregs are critically involved in controlling autoimmune inflammation, elimination of Tregs may therefore also incur the danger of an excessive inflammatory immune response. Thus, understanding the dynamics and function of Tregs during mycobacterial infection is crucial to evaluate the potential of Treg depletion as a medical option. To address this, we depleted Tregs after infection with M. bovis BCG or Mycobacterium tuberculosis (Mtb) using DEREG mice, which express the diphtheria toxin (DT) receptor under the control of the FoxP3 locus, thereby allowing the selective depletion of FoxP3+ Tregs. Our results show that after depletion, the Treg niche is rapidly refilled by a population of DT-insensitive Tregs (diTregs) and bacterial load remains unchanged. On the contrary, impaired rebound of Tregs in DEREG × FoxP3GFP mice improves pathogen burden, but is accompanied by detrimental autoimmune inflammation. Therefore, our study provides the proof-of-principle that, although a high degree of Treg depletion may contribute to the control of mycobacterial infection, it carries the risk of autoimmunity

  4. IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-α) secreted by mycobacterial antigen-stimulated adherent cells

    PubMed Central

    THAKURDAS, S M; HASAN, Z; HUSSAIN, R

    2004-01-01

    Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-α. Mycobacterial components directly stimulate adherent cells to secrete TNF-α. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-α in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-α expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-α blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation. PMID:15086397

  5. Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions.

    PubMed

    Bińczak-Kuleta, Agnieszka; Szwed, Aleksander; Walter, Mark R; Kołban, Maciej; Ciechanowicz, Andrzej; Clark, Jeremy S C

    2016-08-01

    Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity. PMID:27356097

  6. Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions.

    PubMed

    Bińczak-Kuleta, Agnieszka; Szwed, Aleksander; Walter, Mark R; Kołban, Maciej; Ciechanowicz, Andrzej; Clark, Jeremy S C

    2016-08-01

    Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity. PMID:27483180

  7. Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions

    PubMed Central

    Bińczak-Kuleta, Agnieszka; Szwed, Aleksander; Walter, Mark R.; Kołban, Maciej; Ciechanowicz, Andrzej; Clark, Jeremy S. C.

    2016-01-01

    Previously, dominant partial interferon-gamma receptor 1 (IFN-γ-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-γ-R1/IFN-γ-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity.

  8. Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay▿

    PubMed Central

    Shin, Sung Jae; Anklam, Kelly; Manning, Elizabeth J. B.; Collins, Michael T.

    2009-01-01

    Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1

  9. Differentiation of Mycobacterial Species by PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA Polymerase Gene (rpoB)

    PubMed Central

    Kim, Bum-Joon; Lee, Keun-Hwa; Park, Bo-Na; Kim, Seo-Jeong; Bai, Gill-Han; Kim, Sang-Jae; Kook, Yoon-Hoh

    2001-01-01

    PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rifr region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714–1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens. PMID:11376042

  10. Anti-dormant mycobacterial activity and target molecule of melophlins, tetramic acid derivatives isolated from a marine sponge of Melophlus sp.

    PubMed

    Arai, Masayoshi; Yamano, Yoshi; Kamiya, Kentaro; Setiawan, Andi; Kobayashi, Motomasa

    2016-07-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is a major world health problem that is responsible for the deaths of 1.5 million people each year. In addition, the requirement for long-term therapy to cure TB complicates treatment of the disease. One of the major reasons for the extended chemotherapeutic regimens and wide epidemicity of TB is that M. tuberculosis has the ability to persist in a dormant state. We therefore established a new screening system to search for substances with activity against dormant mycobacteria using M. smegmatis and M. bovis BCG cultivated in medium containing propionate as sole carbon source to induce dormancy. Subsequently, melophlins A (1), G (2), H (3), and I (4), tetramic acid derivatives, were re-discovered from the Indonesian marine sponge of Melophlus sp. as anti-dormant mycobacterial substances. Moreover, target analysis of melophlin A indicated that it targeted the BCG1083 protein of putative exopolyphosphatase and the BCG1321c protein of diadenosine 5',5‴-P(1),P(4)-tetraphosphate phosphorylase. PMID:27193014

  11. Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification.

    PubMed Central

    Badak, F Z; Kiska, D L; O'Connell, M; Nycz, C M; Hartley, C; Setterquist, S; Hopfer, R L

    1997-01-01

    Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures. PMID:9114414

  12. Cell wall lipids from Mycobacterium bovis BCG are inflammatory when inoculated within a gel matrix: characterization of a new model of the granulomatous response to mycobacterial components.

    PubMed

    Rhoades, Elizabeth R; Geisel, Rachel E; Butcher, Barbara A; McDonough, Sean; Russell, David G

    2005-05-01

    The chronic inflammatory response to Mycobacterium generates complex granulomatous lesions that balance containment with destruction of infected tissues. To study the contributing factors from host and pathogen, we developed a model wherein defined mycobacterial components and leukocytes are delivered in a gel, eliciting a localized response that can be retrieved and analysed. We validated the model by comparing responses to the cell wall lipids from Mycobacterium bovis bacillus Calmette-Guerin (BCG) to reported activities in other models. BCG lipid-coated beads and bone marrow-derived macrophages (input macrophages) were injected intraperitoneally into BALB/c mice. Input macrophages and recruited peritoneal exudate cells took up fluorescently tagged BCG lipids, and matrix-associated macrophages and neutrophils produced tumor necrosis factor, interleukin-1alpha, and interleukin-6. Leukocyte numbers and cytokine levels were greater in BCG lipid-bearing matrices than matrices containing non-coated or phosphatidylglycerol-coated beads. Leukocytes arrived in successive waves of neutrophils, macrophages and eosinophils, followed by NK and T cells (CD4(+), CD8(+), or gammadelta) at 7 days and B cells within 12 days. BCG lipids also predisposed matrices for adherence and vascularization, enhancing cellular recruitment. We submit that the matrix model presents pertinent features of the murine granulomatous response that will prove to be an adaptable method for study of this complex response. PMID:15850754

  13. New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level.

    PubMed

    Nigou, J; Vercellone, A; Puzo, G

    2000-06-23

    Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins. PMID:10873458

  14. Exposure to a Mycobacterial Antigen, ESAT-6, Exacerbates Granulomatous and Fibrotic Changes in a Multiwall Carbon Nanotube Model of Chronic Pulmonary Disease

    PubMed Central

    Malur, Anagha; Barna, Barbara P; Patel, Janki; McPeek, Matthew; Wingard, Christopher J; Dobbs, Larry; Thomassen, Mary Jane

    2016-01-01

    Recent studies suggest additive effects of environmental pollutants and microbial antigens on respiratory disease. We established a granuloma model in which instilled multiwall carbon nanotubes (MWCNT) elicit granulomatous pathology. We hypothesized that mycobacterial antigen ESAT-6, a T cell activator associated with tuberculosis and sarcoidosis, might alter pathology. Wild-type C57Bl/6 mice received MWCNT with or without ESAT-6 peptide. Controls received vehicle (surfactant-PBS) or ESAT-6 alone. Mice were evaluated 60 days later for granulomas, fibrosis, and bronchoalveolar lavage (BAL) cell expression of inflammatory mediators (CCL2, MMP-12, and Osteopontin). Results indicated increased granulomas, fibrosis, and inflammatory mediators in mice receiving the combination of MWCNT+ESAT-6 compared to MWCNT or vehicle alone. ESAT-6 alone showed no significant effect on these pathological endpoints. However, CD3 (+) lymphocyte infiltration of lung tissue increased with MWCNT+ESAT-6 versus MWCNT alone. Findings suggest that concurrent exposure to microbial antigen and MWCNT exacerbates chronic pulmonary disease. PMID:27019768

  15. Dimerization of an immunoactivating peptide derived from mycobacterial hsp65 using N-hydroxysuccinimide based bifunctional reagents is critical for its antitumor properties.

    PubMed

    Bezouška, Karel; Kubínková, Zuzana; Stříbný, Jiří; Volfová, Barbora; Pompach, Petr; Kuzma, Marek; Šírová, Milada; Říhová, Blanka

    2012-10-17

    We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization. PMID:22988810

  16. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

    PubMed Central

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M.; Nandicoori, Vinay K.; Khosla, Sanjeev

    2015-01-01

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation. PMID:25824946

  17. RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection.

    PubMed

    Yu, Chien-Hsiung; Micaroni, Massimo; Puyskens, Andreas; Schultz, Thomas E; Yeo, Jeremy Changyu; Stanley, Amanda C; Lucas, Megan; Kurihara, Jade; Dobos, Karen M; Stow, Jennifer L; Blumenthal, Antje

    2015-10-15

    Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105(-/-) macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105(-/-) macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105(-/-) macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA-mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105(-/-) macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105(-/-) macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton's tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria. PMID:26371254

  18. Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin-binding hemagglutinin adhesin antigen.

    PubMed

    Fukui, Masayuki; Shinjo, Kikuko; Umemura, Masayuki; Shigeno, Satoko; Harakuni, Tetsuya; Arakawa, Takeshi; Matsuzaki, Goro

    2015-12-01

    Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin-binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN-γ and anti-HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB. PMID:26577130

  19. Comparison of Mycobacterial Growth Indicator Tube with Culture on RGM Selective Agar for Detection of Mycobacteria in Sputum Samples from Patients with Cystic Fibrosis.

    PubMed

    Eltringham, Ian; Pickering, Julie; Gough, Helen; Preece, Clair L; Perry, John D

    2016-08-01

    Nontuberculous mycobacteria (NTM) are an important cause of pulmonary disease in patients with cystic fibrosis (CF). A new culture medium (RGM medium) for the isolation of rapidly growing mycobacteria from the sputum of cystic fibrosis patients has recently been reported. The aim of this study was to compare culture of sputum samples on RGM medium with culture using a standard automated liquid culture method. Sputum samples were obtained from 187 distinct patients with CF attending King's College Hospital, London, United Kingdom. Each sample was decontaminated with 3% oxalic acid and inoculated into a mycobacterial growth indicator tube (MGIT) that was monitored for 42 days using the Bactec MGIT 960 instrument. Each sample was also cultured, without decontamination, onto RGM medium, which was incubated for 10 days at 30°C. Mycobacteria were isolated from 28 patients (prevalence, 15%). Mycobacteria were detected in 24 samples (86%) using the MGIT and in 23 samples (82%) using RGM medium (P = 1.00). In this setting, RGM medium showed sensitivity equivalent to that of the MGIT for isolation of NTM from the sputum of patients with CF. RGM medium offers a simple, convenient tool that can be embedded into routine culture methods, allowing the culture of all sputum samples that are submitted from patients with CF. PMID:27225412

  20. Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages.

    PubMed

    McCarthy, Travis R; Torrelles, Jordi B; MacFarlane, Amanda Shearer; Katawczik, Melanie; Kutzbach, Beth; Desjardin, Lucy E; Clegg, Steven; Goldberg, Joanna B; Schlesinger, Larry S

    2005-11-01

    Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors. PMID:16238626

  1. Factors associated with tuberculosis infection, and with anti-mycobacterial immune responses, among five year olds BCG-immunised at birth in Entebbe, Uganda

    PubMed Central

    Lule, Swaib Abubaker; Mawa, Patrice A.; Nkurunungi, Gyaviira; Nampijja, Margaret; Kizito, Dennison; Akello, Florence; Muhangi, Lawrence; Elliott, Alison M.; Webb, Emily L.

    2015-01-01

    Background BCG is used widely as the sole licensed vaccine against tuberculosis, but it has variable efficacy and the reasons for this are still unclear. No reliable biomarkers to predict future protection against, or acquisition of, TB infection following immunisation have been identified. Lessons from BCG could be valuable in the development of effective tuberculosis vaccines. Objectives Within the Entebbe Mother and Baby Study birth cohort in Uganda, infants received BCG at birth. We investigated factors associated with latent tuberculosis infection (LTBI) and with cytokine response to mycobacterial antigen at age five years. We also investigated whether cytokine responses at one year were associated with LTBI at five years of age. Methods Blood samples from age one and five years were stimulated using crude culture filtrates of Mycobacterium tuberculosis in a six-day whole blood assay. IFN-γ, IL-5, IL-13 and IL-10 production was measured. LTBI at five years was determined using T-SPOT.TB® assay. Associations with LTBI at five years were assessed using multivariable logistic regression. Multiple linear regression with bootstrapping was used to determine factors associated with cytokine responses at age five years. Results LTBI prevalence was 9% at age five years. Only urban residence and history of TB contact/disease were positively associated with LTBI. BCG vaccine strain, LTBI, HIV infection, asymptomatic malaria, growth z-scores, childhood anthelminthic treatment and maternal BCG scar were associated with cytokine responses at age five. Cytokine responses at one year were not associated with acquisition of LTBI by five years of age. Conclusion Although multiple factors influenced anti-myocbacterial immune responses at age five, factors likely to be associated with exposure to infectious cases (history of household contact, and urban residence) dominated the risk of LTBI. PMID:25529292

  2. Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase

    PubMed Central

    Zhang, Wenli; Jones, Victoria C.; Scherman, Michael S.; Mahapatra, Sebabrata; Crick, Dean; Bhamidi, Suresh; Xin, Yi; McNeil, Michael R.; Ma, Yufang

    2008-01-01

    UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDP-GlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phosphate, and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. Since inhibition of peptidoglycan synthesis often results in cell lysis, M. tuberculosis GlmU is a potential anti-tuberculosis drug target. In this study we cloned M. tuberculosis Rv1018c (glmU gene) and expressed soluble GlmU protein in E. coli BL21(DE3). Enzymatic assays showed that M. tuberculosis GlmU protein exhibits both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme. As the amount of functional GlmU enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubby-rounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD340 read out was developed. These studies therefore support the further development of M. tuberculosis GlmU enzyme as a target for new anti-tuberculosis drugs. PMID:18573680

  3. Contribution of MINCLE-SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB.

    PubMed

    Ostrop, Jenny; Jozefowski, Katrin; Zimmermann, Stephanie; Hofmann, Katharina; Strasser, Erwin; Lepenies, Bernd; Lang, Roland

    2015-09-01

    Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE. PMID:26202982

  4. Immunotherapy of Epstein-Barr Virus Associated Malignancies Using Mycobacterial HSP70 and LMP2A356–364 Epitope Fusion Protein

    PubMed Central

    Liu, Genyan; Yao, Kun; Wang, Bing; Chen, Yun; Zhou, Feng; Guo, Yidi; Xu, Jian; Shi, Hongzhen

    2009-01-01

    Epstein-Barr virus infection is strongly associated with a number of malignancies. The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies. In previous studies, the T cell epitopes of LMP2A have been identified systematically. However, the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly, which could be overcome by combination with appropriate adjuvants. Heat shock protein is a natural chaperon, which is able to activate the classical major histocompatibility complex class I antigen-processing pathway (cross-presentation). In this study, a minigene encoding LMP2A356–364 (FLYALALLL) was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70. The epitope fusion protein was expressed and purified, and the cross-presentation of LMP2A356–364 by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated. Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation. Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A356–364 generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant (IFA). Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups. Our results suggested that MtHsp70-LMP2A356–364 fusion protein was more effective than the CD8+ T cell epitope alone on anti-tumor immunity. As a result, the MtHsp70-LMP2A356–364 fusion protein is considered to be a promising candidate vaccine for EBV related malignancies. PMID:20003818

  5. DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection.

    PubMed

    Dalmia, Neha; Klimstra, William B; Mason, Carol; Ramsay, Alistair J

    2015-01-01

    There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters. PMID:26317509

  6. Incidence of active mycobacterial infections in Brazilian patients with chronic inflammatory arthritis and negative evaluation for latent tuberculosis infection at baseline - A longitudinal analysis after using TNFα blockers

    PubMed Central

    Gomes, Carina Mori Frade; Terreri, Maria Teresa; de Moraes-Pinto, Maria Isabel; Barbosa, Cássia; Machado, Natália Pereira; Melo, Maria Roberta; Pinheiro, Marcelo Medeiros

    2015-01-01

    Several studies point to the increased risk of reactivation of latent tuberculosis infection (LTBI) in patients with chronic inflammatory arthritis (CIAs) after using tumour necrosis factor (TNF)α blockers. To study the incidence of active mycobacterial infections (aMI) in patients starting TNF α blockers, 262 patients were included in this study: 109 with rheumatoid arthritis (RA), 93 with ankylosing spondylitis (AS), 44 with juvenile idiopathic arthritis (JIA) and 16 with psoriatic arthritis (PsA). All patients had indication for anti-TNF α therapy. Epidemiologic and clinical data were evaluated and a simple X-ray and tuberculin skin test (TST) were performed. The control group included 215 healthy individuals. The follow-up was 48 months to identify cases of aMI. TST positivity was higher in patients with AS (37.6%) than in RA (12.8%), PsA (18.8%) and JIA (6.8%) (p < 0.001). In the control group, TST positivity was 32.7%. Nine (3.43%) patients were diagnosed with aMI. The overall incidence rate of aMI was 86.93/100,000 person-years [95% confidence interval (CI) 23.6-217.9] for patients and 35.79/100,000 person-years (95% CI 12.4-69.6) for control group (p < 0.001). All patients who developed aMI had no evidence of LTBI at the baseline evaluation. Patients with CIA starting TNF α blockers and no evidence of LTBI at baseline, particularly with nonreactive TST, may have higher risk of aMI. PMID:26560983

  7. Optimization of Pyrrolamides as Mycobacterial GyrB ATPase Inhibitors: Structure-Activity Relationship and In Vivo Efficacy in a Mouse Model of Tuberculosis

    PubMed Central

    P, Shahul Hameed; Mukherjee, Kakoli; Nandi, Vrinda; Waterson, David; Shandil, Radha; Balganesh, Meenakshi; Sambandamurthy, Vasan K.; Raichurkar, Anand Kumar; Deshpande, Abhijeet; Ghosh, Anirban; Awasthy, Disha; Shanbhag, Gajanan; Sheikh, Gulebahar; McMiken, Helen; Puttur, Jayashree; Reddy, Jitendar; Werngren, Jim; Read, Jon; Kumar, Mahesh; R, Manjunatha; Chinnapattu, Murugan; Madhavapeddi, Prashanti; Manjrekar, Praveena; Basu, Reetobrata; Gaonkar, Sheshagiri; Sharma, Sreevalli; Hoffner, Sven; Humnabadkar, Vaishali; Subbulakshmi, Venkita; Panduga, Vijender

    2014-01-01

    Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10−6 to 10−8, and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents. PMID:24126580

  8. Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection

    PubMed Central

    Du, George; Qiu, Liyou; Shen, Ling; Sehgal, Probhat; Shen, Yun; Huang, Dan; Letvin, Norman L.; Chen, Zheng W.

    2010-01-01

    Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vβ families and 13 Jβ segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNγ-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10−5 to 10−6 antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by ≥2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNγ-producing T cell clones using as few as 2×106 PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases. PMID:16403511

  9. Use of molecular methods to identify the Mycobacterium tuberculosis complex (MTBC) and other mycobacterial species and to detect rifampin resistance in MTBC isolates following growth detection with the BACTEC MGIT 960 system.

    PubMed

    Somoskovi, Akos; Song, Qunfeng; Mester, Judit; Tanner, Charise; Hale, Yvonne M; Parsons, Linda M; Salfinger, Max

    2003-07-01

    A prospective study was organized by using a total of 1,585 consecutive clinical specimens to determine whether biomass obtained from positive growth in the MGIT 960 system could be used directly in AccuProbe DNA hybridization tests, the PCR-based Inno-LiPA Rif.TB (LiPA) assay, and a PCR-based DNA sequencing of the rpoB gene for the rapid identification of the Mycobacterium tuberculosis complex (MTBC) and other mycobacterial species and for the determination of rifampin (RIF) resistance in MTBC strains. The results were compared to routine culture, identification, and susceptibility testing techniques performed on the same samples. The study results revealed that the DNA AccuProbe assay (on the day of growth positivity) readily identified 95.7%, the LiPA assay readily identified 98.6%, and rpoB sequencing readily identified 97.1% of the 70 MTBC isolates from mycobacterial growth indicator tubes (MGIT). In addition, application of the LiPA for the identification and RIF susceptibility testing of the MTBC in growth-positive MGIT resulted in a turnaround time of less than 2 weeks after specimen receipt. Although DNA sequencing of rpoB required a slightly longer (16 days) turnaround time, this method was capable of identifying several species of nontuberculous mycobacteria in addition to identifying MTBC and determining RIF susceptibility or resistance. The molecular methods were also found to rapidly identify RIF-susceptible and -resistant MTBC in two of the three mixed mycobacterial cultures weeks earlier than conventional methods. In conclusion, the biomass obtained in MGIT at the time of growth positivity in the 960 system is sufficient for use in all three molecular tests, and this approach can reduce the turnaround time for reporting results. PMID:12843007

  10. A peptide fragment from the human COX3 protein disrupts association of Mycobacterium tuberculosis virulence proteins ESAT-6 and CFP10, inhibits mycobacterial growth and mounts protective immune response

    PubMed Central

    2014-01-01

    Background Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions worldwide. The currently available anti-TB drugs and vaccines have proved insufficient to contain this scourge, necessitating an urgent need for identification of novel drug targets and therapeutic strategies. The disruption of crucial protein-protein interactions, especially those that are responsible for virulence in Mycobacterium tuberculosis – for example the ESAT-6:CFP10 complex – are a worthy pursuit in this direction. Methods We therefore sought to improvise a method to attenuate M. tuberculosis while retaining the latter’s antigenic properties. We screened peptide libraries for potent ESAT-6 binders capable of dissociating CFP10 from ESAT-6. We assessed the disruption by a peptide named HCL2, of the ESAT-6:CFP10 complex and studied its effects on mycobacterial survival and virulence. Results We found that HCL2, derived from the human cytochrome c oxidase subunit 3 (COX3) protein, disrupts ESAT-6:CFP10 complex, binds ESAT-6 potently, disintegrates bacterial cell wall and inhibits extracellular as well as intracellular mycobacterial growth. In addition, an HCL2 expressing M. tuberculosis strain induces both Th1 and Th17 host protective responses. Conclusions Disruption of ESAT-6:CFP10 association could, therefore, be an alternate method for attenuating M. tuberculosis, and a possible route towards future vaccine generation. PMID:24985537

  11. Defining dormancy in mycobacterial disease.

    PubMed

    Lipworth, S; Hammond, R J H; Baron, V O; Hu, Yanmin; Coates, A; Gillespie, S H

    2016-07-01

    Tuberculosis remains a threat to global health and recent attempts to shorten therapy have not succeeded mainly due to cases of clinical relapse. This has focussed attention on the importance of "dormancy" in tuberculosis. There are a number of different definitions of the term and a similar multiplicity of different in vitro and in vivo models. The danger with this is the implicit assumption of equivalence between the terms and models, which will make even more difficult to unravel this complex conundrum. In this review we summarise the main models and definitions and their impact on susceptibility of Mycobacterium tuberculosis. We also suggest a potential nomenclature for debate. Dormancy researchers agree that factors underpinning this phenomenon are complex and nuanced. If we are to make progress we must agree the terms to be used and be consistent in using them. PMID:27450015

  12. Recent TB transmission, clustering and predictors of large clusters in London, 2010–2012: results from first 3 years of universal MIRU-VNTR strain typing

    PubMed Central

    Hamblion, Esther L; Le Menach, Arnaud; Anderson, Laura F; Lalor, Maeve K; Brown, Tim; Abubakar, Ibrahim; Anderson, Charlotte; Maguire, Helen; Anderson, Sarah R

    2016-01-01

    Background The incidence of TB has doubled in the last 20 years in London. A better understanding of risk groups for recent transmission is required to effectively target interventions. We investigated the molecular epidemiological characteristics of TB cases to estimate the proportion of cases due to recent transmission, and identify predictors for belonging to a cluster. Methods The study population included all culture-positive TB cases in London residents, notified between January 2010 and December 2012, strain typed using 24-loci multiple interspersed repetitive units-variable number tandem repeats. Multivariable logistic regression analysis was performed to assess the risk factors for clustering using sociodemographic and clinical characteristics of cases and for cluster size based on the characteristics of the first two cases. Results There were 10 147 cases of which 5728 (57%) were culture confirmed and 4790 isolates (84%) were typed. 2194 (46%) were clustered in 570 clusters, and the estimated proportion attributable to recent transmission was 34%. Clustered cases were more likely to be UK born, have pulmonary TB, a previous diagnosis, a history of substance abuse or alcohol abuse and imprisonment, be of white, Indian, black-African or Caribbean ethnicity. The time between notification of the first two cases was more likely to be <90 days in large clusters. Conclusions Up to a third of TB cases in London may be due to recent transmission. Resources should be directed to the timely investigation of clusters involving cases with risk factors, particularly those with a short period between the first two cases, to interrupt onward transmission of TB. PMID:27417280

  13. Mycobacterial virulence. Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity.

    PubMed

    North, R J; Izzo, A A

    1993-06-01

    The kinetics of growth of two virulent strains of mycobacteria (M. tuberculosis Erdman and M. tuberculosis H37Rv) and two attenuated strains (M. tuberculosis H37Ra and M. bovis Bacillus Calmette-Guerin [BCG]) were studied in the lungs, livers, spleens, and kidneys of severe combined immunodeficient (SCID) mice and of their coisogenic CB-17 immunocompetent counterparts. It was found, in keeping with the findings of earlier investigators (Pierce, C. H., R. J. Dubos, and W. B. Schaefer. 1953. J. Exp. Med. 97:189.), that in immunocompetent mice, virulent organisms grew progressively only in the lungs, whereas the growth of attenuated organisms was controlled in all organs. In SCID mice, in contrast, virulent mycobacteria grew rapidly and progressively in all organs, as did BCG, although at a slower rate. However, H37Ra failed to grow progressively in any organs of SCID mice, unless the mice were treated with hydrocortisone. In fact, hydrocortisone treatment enabled virulent, as well as attenuated, organisms to grow strikingly more rapidly in all organs of SCID mice and in all organs of CB-17 mice. A histological study showed that in SCID mice, multiplication of mycobacteria in the liver occurs in the cytoplasm of macrophages in granulomas and presumably in macrophages in other organs. It is suggested, therefore, that the macrophages of SCID mice possess a glucocorticoid-sensitive mycobacterial mechanism that prevents virulent and avirulent mycobacteria from expressing their true minimal doubling times. In the absence of this mechanism in the lungs of hydrocortisone-treated SCID mice, the doubling times of Erdman, H37Rv, BCG, and H37Ra were 17.7, 17.4, 44.6, and 98.6 h, respectively. The possible importance of a rapid multiplication rate for mycobacterial virulence is discussed. PMID:8496688

  14. A role for tumour necrosis factor-alpha, complement C5 and interleukin-6 in the initiation and development of the mycobacterial cord factor trehalose 6,6'-dimycolate induced granulomatous response.

    PubMed

    Welsh, Kerry J; Abbott, April N; Hwang, Shen-An; Indrigo, Jessica; Armitige, Lisa Y; Blackburn, Michael R; Hunter, Robert L; Actor, Jeffrey K

    2008-06-01

    Trehalose 6,6'-dimycolate (TDM) is a glycolipid component of the mycobacterial cell wall that causes immune responses in mice similar to Mycobacterium tuberculosis (MTB) infection, including granuloma formation with production of proinflammatory cytokines. The precise roles of tumour necrosis factor (TNF)-alpha, complement C5 and interleukin (IL)-6 in the molecular events that lead to the initiation and maintenance of the granulomatous response to TDM have not been fully elucidated. Macrophage proinflammatory responses from wild-type and complement-deficient mice after infection with MTB were assessed, and compared to responses from organisms in which surface TDM had been removed. Removal of TDM abolished proinflammatory responses, markedly so in the complement-deficient macrophages. Mice deficient in TNF-alpha, C5a and IL-6, along with wild-type C57BL/6 controls, were intravenously injected with TDM in a water-in-oil emulsion, and analysed for histological response and cytokine production in lungs. Wild-type C57BL/6 mice formed granulomas with increased production of IL-1beta, IL-6, TNF-alpha, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-12p40, interferon-gamma (IFN-gamma), and IL-10 protein and mRNA. TNF-alpha-deficient mice failed to produce a histological response to TDM, with no increases in cytokine production following TDM administration. While C5a-deficient mice exhibited inflammation, they did not form structured granulomas and initially had decreased production of proinflammatory mediators. IL-6-deficient mice initiated granuloma formation, but failed to maintain the granulomas through day 7 and demonstrated decreased early production of proinflammatory mediators in comparison to wild-type mice. These data suggest that TNF-alpha is critical for initiation of the granulomatous response, C5a is necessary for formation of cohesive granulomas, and IL-6 plays a key role in the granuloma maintenance response to mycobacterial TDM. PMID:18524936

  15. Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors.

    PubMed

    Davodeau, F; Peyrat, M A; Hallet, M M; Gaschet, J; Houde, I; Vivien, R; Vie, H; Bonneville, M

    1993-08-01

    Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells. The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones. Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related. Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones. However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive. Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag. Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag. PMID:8393042

  16. Evaluation of the GenoType Mycobacteria Direct assay for the simultaneous detection of the Mycobacterium tuberculosis complex and four atypical mycobacterial species in smear-positive respiratory specimens.

    PubMed

    Seagar, A-Louise; Prendergast, Carmel; Emmanuel, F Xavier; Rayner, Alan; Thomson, Susan; Laurenson, Ian F

    2008-05-01

    A novel, commercially available reverse hybridization assay [GenoType Mycobacteria Direct (GTMD), version 2.0; Hain Lifescience] was evaluated for the direct detection of five clinically relevant mycobacterial species [Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, Mycobacterium malmoense, Mycobacterium kansasii and Mycobacterium intracellulare] from 54 smear-positive respiratory specimens and the findings were compared with culture results. Three approaches were used for specimen preparation using either whole or 'split' sample volumes and N-acetyl-l-cysteine/3 % NaOH or 4 % NaOH as decontamination chemicals. Forty-three out of 52 samples in which RNA amplification was successful gave GTMD results that concurred with the identification of the cultured isolate. All cases of MTBC were detected. Twenty-two samples contained M. tuberculosis complex, seven had M. kansasii, four had M. malmoense, seven contained atypical mycobacteria other than those detectable using the GTMD assay and three specimens contained no viable mycobacteria. The assay is easy to use and can be completed in one working day. Results interpretation is facilitated by the inclusion of an internal amplification control with each sample to allow identification of specimens containing amplification inhibitors. A positive GTMD result will quickly identify patients with MTBC infection or provide specific identification of four other atypical mycobacteria from the same specimen. This allows more rapid drug susceptibility testing, treatment, and public health and infection control decisions. PMID:18436594

  17. Enhanced protection against pulmonary mycobacterial challenge by chitosan-formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma-interferon(+) T cell responses.

    PubMed

    Ai, Wenqing; Yue, Yan; Xiong, Sidong; Xu, Wei

    2013-03-01

    Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi-epitope gene vaccine, which induces splenic gamma-interferon (IFN-γ)(+) T helper cell 1 responses. The resultant chitosan-pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4(+) and CD8(+) IFN-γ(+) T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG) challenge was consistently achieved by the chitosan-DNA formulation both as the vaccine alone or in a BCG prime-vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN-γ(+) T cell response, which correlated positively with immunological protection. PMID:23489083

  18. Prime-boost BCG vaccination with DNA vaccines based in β-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.

    PubMed

    Cervantes-Villagrana, Alberto R; Hernández-Pando, Rogelio; Biragyn, Arya; Castañeda-Delgado, Julio; Bodogai, Monica; Martínez-Fierro, Margarita; Sada, Eduardo; Trujillo, Valentin; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

    2013-01-11

    The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that β-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding β-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB. PMID:23196205

  19. Ten tandem repeats of {beta}-hCG 109-118 enhance immunogenicity and anti-tumor effects of {beta}-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

    SciTech Connect

    Zhang Yankai; Yan Rong; He Yi; Liu Wentao; Cao Rongyue; Yan Ming; Li Taiming; Liu Jingjing; Wu Jie . E-mail: wu_jie97@yahoo.com.cn

    2006-07-14

    The {beta}-subunit of human chorionic gonadotropin ({beta}-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of {beta}-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with {beta}-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-{beta}hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-{beta}hCGCTP37 and HSP65-{beta}hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-{beta}hCGCTP37 elicited much higher levels of specific anti-{beta}-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-{beta}hCGCTP37, which should suggest that HSP65-X10-{beta}hCGCTP37 may be an effective protein vaccine for the treatment of {beta}-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.

  20. Mycobacterial antigen-induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non-diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)-vaccinated healthy subjects

    PubMed Central

    Al-Attiyah, R J; Mustafa, A S

    2009-01-01

    Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non-diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell-mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non-diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)-vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen-induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-β], and Th2 (IL-4, IL-5, IL-10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1pool in healthy subjects. In response to complex mycobacterial antigens, both IFN-γ and TNF-β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL-10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN-γ and IL-10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions. PMID:19737232

  1. Biochemical characterization of three mycobacterial ribosomal fractions.

    PubMed

    Portelance, V; Beaudet, R

    1983-02-01

    The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. PMID:6189570

  2. Three types of response to mycobacterial antigens.

    PubMed

    Lockwood, D N; McManus, I C; Stanford, J L; Thomas, A; Abeyagunawardana, D V

    1987-11-01

    Responses to pathogenic and environmental mycobacteria were assessed in 2680 children in India and Sri Lanka using quadruple skin-testing with new tuberculins. Statistical analysis of the results, by fitting a log-linear mixture model, confirmed the presence of three different categories of response: category 2 non-responders (about 55%) did not react to any component of the mycobacteria; category 3 responders (about 40%) were sensitive to the species-specific group iv antigens; and category 1 responders (about 5%) were sensitive to the group i antigens which are common to all mycobacteria. The proportions of the three response categories vary with age and with BCG status. BCG vaccination and increasing age act independently to decrease the proportion of category 2 non-responders and increase the proportion of category 3 individuals. BCG vaccination and increasing age interact to increase the proportion of category 1 responders. PMID:3443158

  3. The treatment of rapidly growing mycobacterial infections.

    PubMed

    Kasperbauer, Shannon H; De Groote, Mary Ann

    2015-03-01

    Rapidly growing mycobacteria (RGM) include a diverse group of species. We address the treatment of the most commonly isolated RGM-M abscessus complex, M fortuitum, and M chelonae. The M abscessus complex is composed of 3 closely related species: M abscessus senso stricto (hereafter M abscessus), M massiliense, and M bolletii. Most studies address treatment of M abscessus complex, which accounts for 80% of lung disease caused by RGM and is the second most common RGM to cause extrapulmonary disease (after M fortuitum). The M abscessus complex represent the most drug-resistant nontuberculous mycobacteria and are the most difficult to treat. PMID:25676520

  4. Innate Immune Effectors in Mycobacterial Infection

    PubMed Central

    Saiga, Hiroyuki; Shimada, Yosuke; Takeda, Kiyoshi

    2011-01-01

    Tuberculosis, which is caused by infection with Mycobacterium tuberculosis (Mtb), remains one of the major bacterial infections worldwide. Host defense against Mtb is mediated by a combination of innate and adaptive immune responses. In the last 15 years, the mechanisms for activation of innate immunity have been elucidated. Toll-like receptors (TLRs) have been revealed to be critical for the recognition of pathogenic microorganisms including mycobacteria. Subsequent studies further revealed that NOD-like receptors and C-type lectin receptors are responsible for the TLR-independent recognition of mycobacteria. Several molecules, such as active vitamin D3, secretary leukocyte protease inhibitor, and lipocalin 2, all of which are induced by TLR stimulation, have been shown to direct innate immune responses to mycobacteria. In addition, Irgm1-dependent autophagy has recently been demonstrated to eliminate intracellular mycobacteria. Thus, our understanding of the mechanisms for the innate immune response to mycobacteria is developing. PMID:21274449

  5. Seals, seal trainers, and mycobacterial infection.

    PubMed

    Thompson, P J; Cousins, D V; Gow, B L; Collins, D M; Williamson, B H; Dagnia, H T

    1993-01-01

    In 1986, three seals died in a marine park in Western Australia; culture of postmortem tissue suggested infection with Mycobacterium bovis. In 1988, a seal trainer who had been employed at the Western Australian marine park until 1985 developed pulmonary tuberculosis caused by M. bovis while working in a zoo 3,000 km away on the east coast of Australia. Culture characteristics, biochemical behavior, sodium dodecyl sulphate polyacrylamide gel electrophoresis, and restriction endonuclease analysis suggested that the strains of M. bovis infecting the seals and trainer were identical but unique and differed from reference strains and local cattle strains of M. bovis. The infection in both the seals and the trainer had a destructive but indolent course. This is the first time that M. bovis has been observed in seals and the first time that tuberculous infection has been documented to be transmitted from seals to humans. Further investigation of the extent of tuberculous infection in seal populations elsewhere in the world seems warranted, and those working with seals and other marine animals should be monitored for infection. PMID:8420412

  6. A highly conserved mycobacterial cholesterol catabolic pathway

    PubMed Central

    García-Fernández, Esther; Frank, Daniel J.; Galán, Beatriz; Kells, Petrea M.; Podust, Larissa M.; García, José L.; Ortiz de Montellano, Paul R.

    2013-01-01

    Summary Degradation of the cholesterol side-chain in M. tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from M. smegmatis mc2 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion of cyp125A3 or cyp142A2 does not impair growth of M. smegmatis mc2 155 on cholesterol. However, deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome. PMID:23489718

  7. Nontuberculous Mycobacterial Infections in Cystic Fibrosis.

    PubMed

    Martiniano, Stacey L; Nick, Jerry A; Daley, Charles L

    2016-03-01

    Nontuberculous mycobacteria (NTM) are important emerging cystic fibrosis (CF) pathogens, with estimates of prevalence ranging from 6% to 13%. Diagnosis of NTM disease in patients with CF is challenging, as the infection may remain indolent in some, without evidence of clinical consequence, whereas other patients suffer significant morbidity and mortality. Treatment requires prolonged periods of multiple drugs and varies depending on NTM species, resistance pattern, and extent of disease. The development of a disease-specific approach to the diagnosis and treatment of NTM infection in CF patients is a research priority, as a lifelong strategy is needed for this high-risk population. PMID:26857770

  8. Cutaneous amelanotic signet-ring cell malignant melanoma with interspersed myofibroblastic differentiation in a young cat.

    PubMed

    Hirz, Manuela; Herden, Christiane

    2016-07-01

    The diagnosis of malignant melanoma can be difficult because these tumors can be amelanotic and may contain diverse variants and divergent differentiations, of which the signet-ring cell subtype is very rare and has only been described in humans, dogs, cats, and a hamster. We describe herein histopathologic and immunohistochemical approaches taken to diagnose a case of signet-ring cell malignant melanoma with myofibroblastic differentiation in a cat. A tumor within the abdominal skin of a 2-year-old cat was composed of signet-ring cells and irregularly interwoven streams of spindle cells. Both neoplastic cell types were periodic-acid-Schiff, Fontana, and Sudan black B negative. Signet-ring cells strongly expressed vimentin and S100 protein. Spindle cells strongly expressed vimentin and smooth muscle actin; some cells expressed S100, moderately neuron-specific enolase, and others variably actin and desmin. A few round cells expressed melan A, and a few plump spindle cells expressed melan A and PNL2, confirming the diagnosis of amelanotic signet-ring cell malignant melanoma with myofibroblastic differentiation in a cat. Differential diagnoses were excluded, including signet-ring cell forms of adenocarcinomas, lymphomas, liposarcomas, leiomyosarcomas, squamous cell carcinomas, basal cell carcinomas, and adnexal tumors. PMID:27154314

  9. An Empirical Analysis of Interspersal Research Evidence, Implications, and Applications of the Discrete Task Completion Hypothesis.

    ERIC Educational Resources Information Center

    Skinner, Christopher H.

    2002-01-01

    Researchers have posited that when students work on assignments with many discrete tasks, that each completed discrete task may be a conditioned reinforcer. If the discrete task completion hypothesis is accurate, then relative task completion rates should influence choice behavior in the same manner as relative rates of reinforcement. Results of a…

  10. The Effects of Interspersal and Reinforcement on Math Fact Accuracy and Learning Rate

    ERIC Educational Resources Information Center

    Rumberger, Jessica L.

    2013-01-01

    Mathematics skill acquisition is a crucial component of education and ongoing research is needed to determine quality instructional techniques. A ubiquitous instructional question is how to manage time. This study investigated several flashcard presentation methods to determine the one that would provide the most learning in a set amount of time.…

  11. Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs.

    PubMed

    Cheng, Chaoyang; Motohashi, Reiko; Tsuchimoto, Suguru; Fukuta, Yoshimichi; Ohtsubo, Hisako; Ohtsubo, Eiichi

    2003-01-01

    The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains. PMID:12519908

  12. Interspersion of sequences in avian myeloblastosis virus rna that rapidly hybridize with leukemic chicken cell DNA.

    PubMed Central

    Drohan, W N; Shoyab, M; Wall, R; Baluda, M A

    1975-01-01

    Liquid hybridization of progressively smaller fragments (35S, 27S, 15.5S, 12.5S, and 8S) of poly(A)-selected avian myeloblastosis virus RNA with excess DNA from leukemic chicken myeloblasts revealed that all sizes of RNA contained sequences complementary to both slowly and rapidly hybridizing cellular DNA sequences. Apparently, the RNA sequences which hybridize rapidly with excesses of cellular DNA are not restricted to any one region of the avian myeloblastosis virus 35S RNA. Instead, they appear to be randomly distributed over the entire 35S avian myeloblastosis virus RNA molecule with some positioned within 200 nucleotides of the poly(A) tract at the 3' end of the RNA. PMID:163372

  13. Functional Analyses of Mycobacterial Lipoprotein Diacylglyceryl Transferase and Comparative Secretome Analysis of a Mycobacterial lgt Mutant

    PubMed Central

    Tschumi, Andreas; Grau, Thomas; Albrecht, Dirk; Rezwan, Mandana; Antelmann, Haike

    2012-01-01

    Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Usi