结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

The genetics and biochemistry of isoniazid resistance in mycobacterium tuberculosis

Although the primary targets of activated isoniazid (INH) are proteins involved in the biosynthesis of cell wall mycolic acids, clinical resistance is dominated by specific point mutations in katG. Mutations associated with target mutations contribute to, but still cannot completely explain, resistance to INH. Despite the wealth of genetic information currently available, the molecular mechanism of cell death induced by INH remains elusive.     

结核分枝杆菌异烟肼耐药性的分子遗传和生物化学机理

枝菌酸是结核分枝杆菌细胞壁组分之一。异烟肼作用的主要靶标是参与枝菌酸生物合成的蛋白质。临床分离耐药株多数在KatG蛋白发生点突变,该蛋白也是INH作用靶标之一。但是,现有数据还不能解释INH耐药和INH杀灭细菌的全部机理。我们研究结核分枝杆菌耐药机理应该高起点,综合利用生物信息学、基因芯片、蛋白质组学、结构生物学等技术进行研究。     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis

Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have emerged worldwide. In many countries and regions, these resistant strains constitute a serious threat to the efficacy of tuberculosis control programs. An important element in gaining control of this epidemic is developing an understanding of the molecular basis of resistance to the most important antituberculosis drugs: isoniazid, rifampin, and pyrazinamide. On the basis of this information, more exacting laboratory testing, and ultimately more appropriate and timely treatment regimens, can be developed.     

Detection of mutations associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis by polymerase chain reaction-ligase detection reaction

Mycobacterium tuberculosis （MTB） infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB （MDR-TB）, defined as being resistant to at least rifampicin （RMP） and isoniazid （1NH）,     

ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC , the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of β-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.     

Iron enhances the susceptibility of pathogenic mycobacteria to isoniazid, an antitubercular drug

The catalase-peroxidase KatG of Mycobacterium tuberculosis plays a central role in the mechanism of action of the anti-tubercular drug isoniazid (INH). Like other bacterial catalases, mycobacterial catalase-peroxidases are dual active enzymes with both catalase and peroxidase activities in the same protein molecule. In our previous study, we showed that iron deprivation resulted in the loss of peroxidase activity in several non-pathogenic mycobacterial species. Here we extended the study to pathogenic mycobacteria and showed that the peroxidase activity, associated with iron-sufficient (4 μg Fe/ml) conditions of growth was responsible for INH activation. Upon iron deprivation (0.02 μg Fe/ml), peroxidase activity was abolished and there was no activation of INH, as demonstrated both by INH-mediated NBT reduction (spectrophotometrically and activity staining in gels) and by viability studies as assayed by the microplate Alamar Blue assay (MABA). In the viability assay, iron-sufficient M. tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG were susceptible to INH and iron-deficient organisms expressing negligible peroxidase survived high concentrations of the drug. It␣is well known that M. tuberculosis is sensitive to low concentrations of INH while the minimum inhibitory concentration of the drug is quite high for other mycobacteria, especially the non-pathogenic species. We showed this difference to be due to the specificity of the peroxidase for the drug. As withholding of iron is one of the host’s mechanisms of controlling an invading pathogen, the implications of these observations on the efficacy of the anti-tubercular drug INH are discussed with reference to the iron status within the human host.