Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium . This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M . avium . There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M . tuberculosis was found to be four times greater. Examination of in vitro -generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M . avium , however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M . avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

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.     

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.     

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10⁻⁸). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.     

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.     

Genomic arrangement of a putative operon involved in maltose transport in the Mycobacterium tuberculosis complex and Mycobacterium leprae

A Mycobacterium bovis gene coding for a putative MalE maltose binding protein was cloned and its full-length sequence determined. Database searches revealed 99.9% identity with IpqY, encoding a putative sugar uptake protein from Mycobacterium tuberculosis strain H37Rv. The deduced protein product showed high sequence similarity to MalE-like proteins from a variety of bacterial species, including Mycobacterium leprae. Analysis of flanking database sequences from M. tuberculosis and M. leprae revealed the presence of malF-, malG- and malK-like genes. Comparison of these mycobacterial sequences with other maltose operons has allowed us to deduce a unique genomic arrangement of the genes involved in the uptake of maltose in members of the Mycobacterium tuberculosis complex and M. leprae.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.     

Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.     

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.     

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.     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.