Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase

The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidal function requires activation by Mycobacterium tuberculosis catalase-peroxidase (KatG) to produce an acyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. The binding of INH and a series of hydrazide analogues to resting KatG was examined using optical and calorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants (on and off rates). This work revealed high-affinity binding of these substrates to a small fraction of ferric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound water molecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated with a large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05-1.6 microM, and optical stopped-flow measurements demonstrated kon values in the range of 0.5-27 x 10(3) M-1 s-1 with very small koff rates. Binding parameters did not depend on pH in the range 5-8. High-affinity binding of INH is disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] and KatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for association with the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated in good yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a better understanding of the action mechanism of INH.     

Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil

We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb) complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72%) clinical isolates were clustered and 32/114 (28%) were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3%) (the ill-defined T superfamily) (12/114, 10.5%), Haarlem (8/114, 7%), X clade (6/114, 5.3%), S clade (3/114, 2.6%) and the East-African Indian and Manu types, each with 1/114 (0.9%) isolates. A considerable number of strains (n = 20, 17.5%) showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB) (92/114, 80.7%) in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.     

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 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.     

Binding of activated isoniazid with acetyl-CoA carboxylase from Mycobacterium tuberculosis

AccD6 (acetyl coenzyme A (CoA) carboxylase), plays an important role in mycolic acid synthesis of Mycobacterium tuberculosis (Mtb). Induced gene expression by isoniazid (isonicotinylhydrazine - INH), anti-tuberculosis drug) shows the expression of accD6. It is our interest to study the binding of activated INH with the AccD6 model using molecular docking procedures. The study predicts a primary binding site for activated INH (isonicotinyl acyl radical) in AccD6 as a potential target.     

Molecular characterization of rpoB gene mutations in rifampicine-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.     

Genomic stability over 9 years of an isoniazid resistant Mycobacterium tuberculosis outbreak strain in Sweden

In molecular epidemiological studies of drug resistant Mycobacterium tuberculosis (TB) in Sweden a large outbreak of an isoniazid resistant strain was identified, involving 115 patients, mainly from the Horn of Africa. During the outbreak period, the genomic pattern of the outbreak strain has stayed virtually unchanged with regard to drug resistance, IS6110 restriction fragment length polymorphism and spoligotyping patterns. Here we present the complete genome sequence analyses of the index isolate and two isolates sampled nine years after the index case as well as experimental data on the virulence of this outbreak strain. Even though the strain has been present in the community for nine years and passaged between patients at least five times in-between the isolates, we only found four single nucleotide polymorphisms in one of the later isolates and a small (4 amino acids) deletion in the other compared to the index isolate. In contrast to many other evolutionarily successful outbreak lineages (e.g. the Beijing lineage) this outbreak strain appears to be genetically very stable yet evolutionarily successful in a low endemic country such as Sweden. These findings further illustrate that the rate of genomic variation in TB can be highly strain dependent, something that can have important implications for epidemiological studies as well as development of resistance.     

Characterization of mRNA interferases from Mycobacterium tuberculosis

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.     

Molecular characterization of Mycobacterium tuberculosis isolates in a region of Brazil with a high incidence of tuberculosis

One hundred and seventy Mycobacterium tuberculosis clinical isolates were characterized by spoligotyping to evaluate the biodiversity of tubercle bacilli in a region of Brazil with a high incidence of tuberculosis (Pelotas and Rio Grande cities - Rio Grande do Sul State). The spoligotyping results were compared to the World Spoligotyping Database (Institut Pasteur de Guadeloupe), which contains data from >14,000 worldwide isolates of M. tuberculosis. The isolates clustered by spoligotyping were further characterized by IS6110-RFLP to confirm the clonal relationship. Sixty-six different spoligotypes were identified, grouping 125 of the isolates (74%). Approximately half of the isolates belonged to seven of the most frequently occurring spoligotypes in the database. Three shared types (with two or more isolates) not previously identified were given the type numbers 826, 827 and 863. An additional 45 spoligotypes were identified that did not match any existing database pattern. RFLP characterization reduced the number of isolates in most of the clusters, thereby showing a higher differentiation capacity than spoligotyping. These results highlight the importance of molecular epidemiology studies of tuberculosis in insufficiently studied regions with a high TB burden, in order to uncover the true extent of genetic diversity of the pathogen.     

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.     

Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Background

Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods

This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results

Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion

We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.     

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.     

Detection of resistance to isoniazid by denaturing gradient-gel electrophoresis DNA sequencing in Mycobacterium tuberculosis clinical isolates

Isoniazid (INH) resistance was genotypically assessed in 104 (37 INH-susceptible, 67 INH-resistant) genetically unrelated Mycobacterium tuberculosis strains cultured in North Italy. The PCR products of selected regions of the katG gene, the oxyR-ahpC intergenic region, and the inhA regulatory region were analyzed utilizing the double gradient-denaturing gradient gel electrophoresis (DG-DGGE) technique and confirmed by DNA sequencing. Mutations were detected in 61 (91%) of the INH-resistant strains, the relative frequency of the mutations being 65.7% in katG, 23.9% in oxyR-ahpC, and 13.4% in inhA. Previously described alterations, invariably associated with drug resistance, accounted for 95.1% of the mutations. No alterations were found in the INH-susceptible strains. DG-DGGE analysis and DNA sequencing were equally sensitive, but the former is cheaper, easier and more robust. Rapid genotypic assessment of INH resistance by means of the methodology described here could reasonably be used in clinical mycobacteriology laboratories.     

Genetic diversity among Mycobacterium tuberculosis isolates from Mexican patients

Forty-six isolates of the Mycobacterium tuberculosis complex were typified by PCR of the IS6110 region and by Mycobacterium bovis specific primers JB21/JB22. Isolate MVG01 was typified as M. bovis, being the first record of a case of human tuberculosis caused by this species in Mexico. RAPD-PCR was used to describe the genetic diversity of the remaining 45 M. tuberculosis complex isolates. The corrected genotypic diversity value calculated for the analyzed population was 0.96, the estimated mean gene diversity was 0.235, and the corrected Shannon-Weiner index was 2.15. All allele-loci combinations generated showed significant linkage disequilibria. The distribution of genetic variation was analyzed both by the unweighted pair group method with arithmetic averages clustering and by principal coordinates analysis. Unweighted pair group method with arithmetic averages clustering resulted in a tree with four main clusters and one unclustered strain (MVG20), the principal coordinates analysis strain distribution pattern being consistent with this grouping. The obtained results suggest that the studied isolates belong to a clonal population having significant genetic diversity. Our genetic diversity results are comparable with those reported for other populations of M. tuberculosis, although only three RAPD primers were used.     

Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis

Molecular Biology - Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role...     

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.     

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.     

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.