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M. Tuberculosis (m + tuberculosis)
Terms modified by M. Tuberculosis Selected AbstractsComparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG CopenhagenELECTROPHORESIS, Issue 19-20 2003Jens Mattow Abstract A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N -terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis- specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793). [source] How B cells shape the immune response against Mycobacterium tuberculosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Paul J. Maglione Abstract Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B-cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non-viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non-viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought. [source] Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37RvFEBS JOURNAL, Issue 1 2009Md. Suhail Alam The whiB -like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV,Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe,2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe,2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe,2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe,2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis. [source] Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidaseFEBS JOURNAL, Issue 4 2008Hua Zhang The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 °C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn2+/Fe2+ -containing protein with a molar ratio of [Mn2+] to [Fe2+] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA. [source] Structural and thermodynamic insights into the binding mode of five novel inhibitors of lumazine synthase from Mycobacterium tuberculosisFEBS JOURNAL, Issue 20 2006Ekaterina Morgunova Recently published genomic investigations of the human pathogen Mycobacterium tuberculosis have revealed that genes coding the proteins involved in riboflavin biosynthesis are essential for the growth of the organism. Because the enzymes involved in cofactor biosynthesis pathways are not present in humans, they appear to be promising candidates for the development of therapeutic drugs. The substituted purinetrione compounds have demonstrated high affinity and specificity to lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis in bacteria and plants. The structure of M. tuberculosis lumazine synthase in complex with five different inhibitor compounds is presented, together with studies of the binding reactions by isothermal titration calorimetry. The inhibitors showed the association constants in the micromolar range. The analysis of the structures demonstrated the specific features of the binding of different inhibitors. The comparison of the structures and binding modes of five different inhibitors allows us to propose the ribitylpurinetrione compounds with C4,C5 alkylphosphate chains as most promising leads for further development of therapeutic drugs against M. tuberculosis. [source] Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L -gulono-1,4-lactone dehydrogenaseFEBS JOURNAL, Issue 19 2006Beata A. Wolucka The last step of the biosynthesis of l -ascorbic acid (vitamin C) in plants and animals is catalyzed by l -gulono-1,4-lactone oxidoreductases, which use both l -gulono-1,4-lactone and l -galactono-1,4-lactone as substrates. l -Gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat l -gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat l -gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes l -gulono-1,4-lactone (Km 5.5 mm) but not l -galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 µm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific l -gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C. [source] Antibody bound to the surface antigen MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challengeFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2004Mark A. Chambers Abstract Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis is a significant disease of man and animals. Whilst cellular immunity is the major immunological component required for protection against these organisms, recent reports have suggested that monoclonal antibodies can modify infection with M. tuberculosis. To test whether the same was true for M. bovis infection, we determined the effect of preincubation of M. bovis with a monoclonal antibody on subsequent intravenous infection of mice. Antibodies bound to the surface of M. bovis increased the survival time of mice infected with M. bovis and changed the morphology of granulomas and the distribution of acid-fast bacilli in the lung. These studies suggest that antibodies directed to the surface of virulent mycobacteria can modulate their virulence in vivo. [source] Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis -infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette,GuérinIMMUNOLOGY, Issue 4 2009Dilara Begum Summary Both CD4+ and CD8+ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette,Guérin (BCG) on the CD8+ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8+ T cells specific to the mycobacterial Mtb32a309,318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8+ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2Db,Mtb32a209,318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8+ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis -infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8+ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8+ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8+ T cells from the BCG-vaccinated M. tuberculosis -infected mice produced interferon-, in response to Mtb32a209,318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8+ T cells in the M. tuberculosis -infected lung is accelerated by subcutaneous vaccination with M. bovis BCG. [source] Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4,2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulationIMMUNOLOGY, Issue 4 2004Helen A. Fletcher Summary Expression of interleukin (IL)-4 is increased in tuberculosis and thought to be detrimental. We show here that in healthy contacts there is increased expression of its naturally occurring antagonist, IL-4delta2 (IL-4,2). We identified contacts by showing that their peripheral blood mononuclear cells (PBMC) released interferon (IFN)-, in response to the Mycobacterium tuberculosis -specific antigen 6 kDa early secretory antigenic target (ESAT-6). Fresh unstimulated PBMC from these contacts contained higher levels of mRNA encoding IL-4,2 (P=0·002) than did cells from ESAT-6 negative donors (noncontacts). These data indicate that contact with M. tuberculosis induces unusual, previously unrecognized, immunological events. We tentatively hypothesize that progression to active disease might depend upon the underlying ratio of IL-4 to IL-4,2. [source] Induction of apoptosis in monocytes by Mycobacterium leprae in vitro: a possible role for tumour necrosis factor-,IMMUNOLOGY, Issue 1 2003M. O. Hernandez Summary A diverse range of infectious organisms, including mycobacteria, have been reported to induce cell death in vivo and in vitro. Although morphological features of apoptosis have been identified in leprosy lesions, it has not yet been determined whether Mycobacterium leprae modulates programmed cell death. For that purpose, peripheral blood mononuclear cells obtained from leprosy patients were stimulated with different concentrations of this pathogen. Following analysis by flow cytometry on 7AAD/CD14+ cells, it was observed that M. leprae induced apoptosis of monocyte-derived macrophages in a dose-dependent manner in both leprosy patients and healthy individuals, but still with lower efficiency as compared to M. tuberculosis. Expression of tumour necrosis factor-, (TNF-,), Bax-,, Bak mRNA and TNF-, protein was also detected in these cultures; in addition, an enhancement in the rate of apoptotic cells (and of TNF-, release) was noted when interferon-, was added to the wells. On the other hand, incubation of the cells with pentoxifylline impaired mycobacterium-induced cell death, the secretion of TNF-,, and gene expression in vitro. In addition, diminished bacterial entry decreased both TNF-, levels and the death of CD14+ cells, albeit to a different extent. When investigating leprosy reactions, an enhanced rate of spontaneous apoptosis was detected as compared to the unreactive lepromatous patients. The results demonstrated that M. leprae can lead to apoptosis of macrophages through a mechanism that could be at least partially related to the expression of pro-apoptotic members of the Bcl-2 protein family and of TNF-,. Moreover, while phagocytosis may be necessary, it seems not to be crucial to the induction of cell death by the mycobacteria. [source] Erythema induratum with pulmonary tuberculosis: histopathologic features resembling true vasculitisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 3 2001Yong Suk Lee MD A 22-year-old South Korean woman presented with a 4-month history of several nodules on both legs. She looked healthy, but suffered from tenderness and swelling of the legs. Physical examination showed multiple, nonulcerating, erythematous nodules occurring on the calves, knee joints, and thighs (Fig. 1). A biopsy specimen of the skin revealed necrotizing vasculitis of medium-sized arteries with fibrinoid necrosis at the border between the dermis and the subcutis. Dense cellular infiltrates, including numerous neutrophils and lymphocytes, presented within and around the vessel walls as in polyarteritis nodosa, with some eosinophils (Fig. 2A,B). There were no other generalized symptoms. She was diagnosed with cutaneous polyarteritis nodosa and was initially treated with systemic steroids. She was given an intravenous injection of Solu-Cortef, 60 mg/6 h for 7 days. This was replaced with oral prednisolone for 2 weeks. The skin lesions and symptoms improved. Figure 1. Small, nut-sized, erythematous, brown-colored nodules and patches on the lower extremities, even above the knee joints Figure 2. (A) Dense infiltration within and around artery (× 40). (B) Slightly expanded lobular panniculitis with vasculitis (× 100) Six months later, she complained of general weakness and recurrent skin lesions. Purified protein derivative (PPD) test gave a moderate positive reaction and chest X-ray examination showed the features of pulmonary tuberculosis: radio-opaque infiltrations in the right lower lung field. A repeated biopsy revealed mild vasculitis with more diffuse lobular infiltrations of the subcutaneous tissue compared with the former specimen. Polymerase chain reaction (PCR) and tissue culture for Mycobacterium tuberculosis were performed from a biopsy specimen. DNA was extracted from skin tissue with an AplisystemTM DNA/RNA detection kit using the resin-mediated boiling method (Stargene, Seoul, South Korea). The primers were designed on the basis of the M. tuberculosis gene IS6110 target (sense primer, 5,-CCA GAT GCA CCG TCG AAC GGC TGA T-3, antisense primer, 5,-CGC TCG CTG AAC CGG ATC GAT GTG T-3,). The amplification was performed with uracil- N -glycosylase (UNG), to prevent carry-over contamination, and internal control primers, to correct for false-negative reaction (Kox LF, Rhienthong D, Miranda AM et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1994; 32: 672,678; Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990; 93: 125,128). According to the manufacturer's instructions, amplification was carried out for 40 cycles with denaturation at 94 °C for 40 s, annealing at 70 °C for 1 min, and extension at 72 °C for 1 min in a thermal cycler (Perkin,Elmer Cetus, Norwalk, CT, USA). The results of PCR and tissue culture for M. tuberculosis using the biopsy specimen were all negative (Fig. 3). Figure 3. Negative result in PCR for M. tuberculosis (negative control is not shown; M, marker; P, positive control; I, internal control; S, specimen) The patient was finally diagnosed with erythema induratum with pulmonary tuberculosis and was started on antituberculosis medication (isoniazid 400 mg, rifampicin 600 mg, ethambutol 800 mg, and pyrazinamide 1500 mg daily). She showed prompt improvement after 2 weeks of medication. After 9 months of antituberculosis therapy, her skin lesions and chest X-ray had cleared. She was followed up for 4 months with no recurrence of skin and pulmonary lesions. [source] Pathological and immunological profiles of rat tuberculosisINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2004Isamu Sugawara Summary To investigate the pathological and immunological profiles of rat tuberculosis, Lewis female rats were infected aerially with Mycobacterium tuberculosis. Histopathology, immunological profiles of mononuclear cells from M. tuberculosis -infected rat lung tissue, and the expression patterns of cytokine and iNOS mRNAs were examined over time. M. tuberculosis induced granulomatous lesions in the lungs, spleen, lymph nodes and liver, but these lesions lacked central necrosis. Multinucleate giant cells were observed in late-phase tuberculosis. CD4+ and CD8+ T cells increased with time and reached a peak 5 weeks after infection, decreasing gradually thereafter. ED1 antigen, suggestive of alveolar macrophages, was expressed at a high level in early phase tuberculosis and remained at the same level even in the late phase. OX62 antigen increased gradually and reached a peak 5 weeks after infection. Interferon-,, tumour necrosis factor-, and iNOS mRNAs were expressed strongly over time, but their expression decreased 12 weeks after infection. Because rat tuberculosis is very similar to murine tuberculosis and it is easy to obtain mononuclear cells from M. tuberculosis -infected rat lung tissue, the rat tuberculosis model appears to be suitable for immunological studies in vivo. [source] Miliary tuberculosis and necrotizing tuberculous fasciitis , An unusual coexistence in a rheumatoid arthritis patientINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 2 2010Hyun-Hee KWON Abstract We report a case of a 65-year-old Korean female patient with rheumatoid arthritis, who presented with extensive necrotizing fasciitis of the gluteus muscles, as an unusual initial manifestation of miliary tuberculosis. The patient had been previously treated with conventional disease-modifying antirheumatic drugs and low-dose steroids for 7 years. However, she recently developed fever, warmth and painful swelling in her right buttock. Magnetic resonance imaging indicated necrotizing fasciitis of the gluteus muscles and a fasciectomy specimen revealed a Mycobacterium tuberculosis infection. Two weeks after a fasciectomy, miliary tuberculosis of the lung was diagnosed by high resolution chest computed tomography. Soft tissue infection due to M. tuberculosis should be included as a differential diagnosis in the immunocompromised host. Clinicians should be alert to the possibility of miliary tuberculosis even in the absence of respiratory symptoms and normal chest radiograph. [source] Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolorIUBMB LIFE, Issue 10 2008Catriona Harper Abstract The mechanisms governing the regulation of nitrogen metabolism in Corynebacterium glutamicum and Streptomyces coelicolor have been extensively studied. These Actinomycetales are closely related to the Mycobacterium genus and may therefore serve as a models to elucidate the cascade of nitrogen signalling in other mycobacteria. Some factors involved in nitrogen metabolism in Mycobacterium tuberculosis have been described, including glutamine synthetase and its adenylyltransferase, but not much data concerning the other components involved in the signalling cascade is available. In this review a comparative study of factors involved in nitrogen metabolism in C. glutamicum and S. coelicolor is made to identify similarities with M. tuberculosis on both a genomic and proteomic level. This may provide insight into a potential global mechanism of nitrogen control in Mycobacterium tuberculosis. © 2008 IUBMB IUBMB Life, 60(10): 643,650, 2008 [source] Effect of recombinant Rv1009 protein on promoting the growth of Mycobacterium tuberculosisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008X. Wu Abstract Aims:, To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time. Method and Results:, We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium. Conclusions:, The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples. Significance and Impact of the Study:, rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture. [source] A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe arrayJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007S.-L. Zhang Abstract Aims:, To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. Methods and Results:, A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC -6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88·5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR - ahpC intergenic region (86·5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88·5% and 100% for rifampicin resistance, and 86·5% and 100% for isoniazid resistance, respectively. Conclusion:, A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. Significance and Impact of the Study:, This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated. [source] Novel method for clearing red blood cell debris from BacT/ALERT® blood culture medium for improved microscopic and antimycobacterial drug susceptibility test resultsJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2007Krishnamoorthy Gopinath Abstract Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH4Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH4Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150,mM of NH4Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37°C for 15,min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5±39.4 to 53.2±4.2,mg/mL in the M. tuberculosis -spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp. J. Clin. Lab. Anal. 21:220,226, 2007. © 2007 Wiley-Liss, Inc. [source] Diagnosis of tuberculosis: Available technologies, limitations, and possibilitiesJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2003Sanjay K. Garg Abstract Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections. J. Clin. Lab. Anal. 17: 155,163, 2003. © 2003 Wiley-Liss, Inc. [source] Is susceptibility to tuberculosis acquired or inherited?JOURNAL OF INTERNAL MEDICINE, Issue 2 2007E. Schurr Abstract. Tuberculosis is an ongoing major public health problem on a global scale. One of the striking features of the disease is that only an estimated 10% of immunocompetent persons infected by the causative pathogen Mycobacterium tuberculosis will develop clinical signs of disease. This well-established epidemiological observation has prompted an intense search for the factors that trigger advancement of infection to disease in the small proportion of susceptible individuals. Central to this search is the questions if tuberculosis patients are inherently susceptible to the disease or if disease development is promoted by specific environmental factors. It is known that genetic and non-genetic factors of both the bacterium and the host have impact on the host response to M. tuberculosis. Yet, little is known about the interaction of these different factors and the resulting impact on disease development. Recent work suggests that in addition to common host susceptibility genes a second group of susceptibility loci exists the action of which strongly depends on the individual's clinical and exposure history. The latter genes may have a very strong effect on promoting advancement from infection to disease only in specific epidemiological settings. These findings suggest that a more detailed knowledge of gene,environment interactions in tuberculosis is necessary to understand why a small proportion of individuals are susceptible to the disease whilst the majority of humans are naturally resistant to tuberculosis. [source] Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. aviumLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2008A. Amaro Abstract Aims:, To compare three methods for DNA extraction from Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium. Methods and Results:, The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS6110 in M. bovis and M. tuberculosis, and of a 1700 bp fragment of FR300 region in M. avium avium. Conclusions:, Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex. Significance and Impact of the Study:, The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis. [source] A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cellsMICROBIOLOGY AND IMMUNOLOGY, Issue 10 2009Mari Nomoto ABSTRACT The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc2155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes. [source] Molecular Modeling and Receptor-Dependent (RD) 3D-QSAR Approach to a Set of Antituberculosis DerivativesMOLECULAR INFORMATICS, Issue 11-12 2009Fernanda, Kerly, Mesquita Pasqualoto Abstract In this study, receptor-dependent (RD) 3D-QSAR models were built for a set of thirty-seven isoniazid derivatives bound to the enoyl-acp reductase from M. tuberculosis, called InhA (PDB entry code 1zid). Ligand-receptor (L-R) molecular dynamics (MD) simulations [500,000 steps; the step size was 0.001,ps (1,fs)] were carried out at 310,K (biological assay temperature). The hypothesized active conformations resulting from a previously reported receptor-independent (IR) 4D-QSAR analysis were used as the molecular geometries of each ligand in this structure-based L-R binding research. The dependent variable is the reported MIC values against M. tuberculosis var. bovis. The independent variables (descriptors) are energy terms of a modified first-generation AMBER force field combined with a hydration shell aqueous solvation model. Genetic function approximation (GFA) formalism and partial least squares (PLS) regression were employed as the fitting functions to develop 3D-QSAR models. The bound ligand solvation energy, the sum of electrostatic and hydrogen bonding energies of the unbound ligand, the bending energy of the unbound ligand, the electrostatic intermolecular L-R energy, and the change in hydrogen bonding energy upon binding were found as important energy contributions to the binding process. The 3D-QSAR model at 310,K has good internal and external predictability and may be regarded as representative of the binding process of ligands to InhA. [source] Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosisMOLECULAR MICROBIOLOGY, Issue 5 2008Catherine Vilchèze Summary Spontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized. Precise null deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M. tuberculosis. The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation. In vivo, a mycothiol-deficient strain grew normally in immunodeficient mice, but was slightly defective for growth in immunocompetent mice. Mutations in mshA demonstrate the non-essentiality of mycothiol for growth in vitro and in vivo, and provide a novel mechanism of ethionamide resistance in M. tuberculosis. [source] Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulatorMOLECULAR MICROBIOLOGY, Issue 3 2006Marek Fol Summary Paired two-component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP-1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome,lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation-defective MtrA (MtrAD53N) was partially replicative in macrophages, but was attenuated in mice. Quantitative real-time PCR analyses revealed that expression of dnaA, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation-dependent manner. Chromatin immunoprecipitation using anti-MtrA antibodies provided direct evidence that MtrA regulator binds to dnaA promoter in vivo indicating that dnaA promoter is a MtrA target. Simultaneous overexpression of mtrA regulator and its cognate mtrB kinase neither inhibited growth nor sharply increased the expression levels of dnaA in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to non-phosphorylated MtrA response regulator. [source] The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriCMOLECULAR MICROBIOLOGY, Issue 6 2006Murty V. V. S. Madiraju Summary Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaATB) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaATB promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaATB proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC,DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaATB bound to DnaA boxes similarly with ATP or ADP. DnaATB binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation. [source] A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretionMOLECULAR MICROBIOLOGY, Issue 6 2004Lian-Yong Gao Summary Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871,Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host. [source] Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibioticsMOLECULAR MICROBIOLOGY, Issue 6 2003Nancy A. Buchmeier Summary Mycothiol, MSH or 1d - myo -inosityl 2-(N -acetyl- l -cysteinyl)amido-2-deoxy- , - d -glucopyranoside, is an unusual conjugate of N -acetylcysteine (AcCys) with 1d - myo -inosityl 2-acetamido-2-deoxy-,- d -glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced , 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs. [source] DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexAMOLECULAR MICROBIOLOGY, Issue 3 2002Elaine O. Davis Summary The ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA. [source] Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 5 2002Ranjana Pathania Summary Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis. [source] Regulation of catalase,peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosisMOLECULAR MICROBIOLOGY, Issue 4 2001Alexander S. Pym Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase,peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus ,10 and ,35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a ,(furA,katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the ,(furA,katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production. [source] | ||||||||||||||||||||||||||||||||||