An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes. 相似文献
Arylamine N-acetyltransferase (NAT) enzymes are found in a broad range of eukaryotes and prokaryotes. There is increasing evidence that NAT enzymes could contribute to antibiotic resistance in pathogenic bacteria such as Mycobacterium tuberculosis. Nocardia farcinica is an opportunistic human pathogen that causes pulmonary infections (nocardiosis) with clinical manifestations that resemble tuberculosis. While the genomic sequence of this prokaryote has been determined, studies of N. farcinica proteins remain almost nonexistent. In particular, N. farcinica proteins putatively involved in antibiotic resistance mechanisms have not been described structurally or functionally. Here, we have characterized a new NAT enzyme (NfNAT) from N. farcinica at the structural and functional level. NfNAT is the first N. farcinica protein for which a 3D structure is reported. We showed that this novel prokaryotic isoform is structurally and functionally related to the mycobacterial NAT enzymes. In particular, NfNAT was found to display high N-acetyltransferase activity towards several known NAT substrates including the antitubercular drug isoniazid. Interestingly, isoniazid is not used for the treatment of nocardiosis and has been shown to be poorly active against several nocardial species. On the contrary, NfNAT was found to be poorly active towards sulfamethoxazole, a sulfonamide drug considered as the treatment of choice for the treatment of nocardiosis. The functional and structural data reported in this study will help to develop our understanding of the role of NAT enzymes in nocardia and mycobacteria and may help in the rational design of NAT antagonists for a range of clinical applications. 相似文献
Early detection of drug resistance in Mycobacterium tuberculosis
isolates allows for earlier and more effective treatment of patients. The aim of this
study was to investigate the performance of the malachite green decolourisation assay
(MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M.
tuberculosis clinical isolates. Fifty M. tuberculosis
isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and
RIF-susceptible samples, were tested. The sensitivity, specificity, positive
predictive value (PPV), negative predictive value (NPV) and agreement of the assay
for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the
sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%,
100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests
of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the
reference method. There was a minor discrepancy in the tests of two additional
isolates, as they were sensitive to INH by the reference method, but resistant by the
MGDA test. The drug susceptibility test results were obtained within eight-nine days.
In conclusion, the MGDA test is a reliable and accurate method for the rapid
detection of INH and RIF resistance compared with the reference method and the MGDA
test additionally requires less time to obtain results. 相似文献
Isoniazid is a frontline drug used in the treatment of tuberculosis (TB). Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase/peroxidase activity of the katG gene product. TB kills two million people every year and the situation is getting worse due to the increase in prevalence of HIV/AIDS and emergence of multidrug-resistant strains of TB. Arylamine N-acetyltransferase (NAT) is a drug-metabolizing enzyme (E.C. 2.1.3.5). NAT can acetylate isoniazid, transferring an acetyl group from acetyl coenzyme A onto the terminal nitrogen of the drug, which in its N-acetylated form is therapeutically inactive. The bacterium responsible for TB, Mycobacterium tuberculosis, contains and expresses the gene encoding the NAT protein. Isoniazid binds to the NAT protein from Salmonella typhimurium and we report here the mode of binding of isoniazid in the NAT enzyme from Mycobacterium smegmatis, closely related to the M. tuberculosis and S. typhimurium NAT enzymes. The mode of binding of isoniazid to M. smegmatis NAT has been determined using data collected from two distinct crystal forms. We can say with confidence that the observed mode of binding of isoniazid is not an artifact of the crystallization conditions used. The NAT enzyme is active in mycobacterial cells and we propose that isoniazid binds to the NAT enzyme in these cells. NAT activity in M. tuberculosis is likely therefore to modulate the degree of activation of isoniazid by other enzymes within the mycobacterial cell. The structure of NAT with isoniazid bound will facilitate rational drug design for anti-tubercular therapy. 相似文献
The “Microtitre” Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an “S9-mix” metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens.
The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and β-naphthoflavone (benzoflavone). as a safe substitute for Aroclor-1254. The post-mitochondrial preparations from rats induced with the combined inducers had a similar “metabolic competence” to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening.
It was found that the level of “S9” present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening. 相似文献
A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations. 相似文献
A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes. 相似文献