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Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance.

ABSTRACT: The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the Escherichia coli oxyR mutant, which is relatively sensitive to INH, with a Mycobacterium tuberculosis plasmid library and selected for INH-resistant clones. Three genes were identified and called ceo for their ability to complement the Escherichia coli oxyR mutant. ceoA was the previously identified M. tuberculosis glf gene, which encodes a 399-amino-acid NAD+- and flavin adenine dinucleotide-requiring enzyme responsible for catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The proteins encoded by the ceoBC pair were homologous with one another and with the N terminus of the potassium uptake regulatory protein TrkA. Each of the three Ceo proteins contains a motif common to NAD+ binding pockets. Overexpression of the M. tuberculosis glf gene by placing it under the control of the hsp60 promoter on a multicopy plasmid in Mycobacterium bovis BCG produced a strain for which the INH MIC was increased 50% compared to that for the control strains, while similar overexpression of the ceoBC pair had no effect on INH susceptibility in BCG. Mycobacterial extracts containing the overexpressed Glf protein did not bind radiolabeled INH directly, suggesting a more complex mechanism than the binding of unmodified INH. Our results support the hypothesis that upregulated mycobacterial proteins such as Glf may contribute to INH resistance in M. tuberculosis by binding a modified form of INH or by sequestering a factor such as NAD+ required for INH activity.

SUBMITTER: Chen P

PROVIDER: S-EPMC108635 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance.

Chen P P Bishai W R WR

Infection and immunity 19981101 11

The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the Escherichia coli oxyR mutant, which is relatively sensitive to INH, with a Mycobacterium tuberculosis plasmid library and selected for INH-resistant clones. Three genes were identified and called ceo for their ability to complement the Escherichia coli oxyR mutant. ceoA was the pre ...[more]

PMID: 9784509

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Project description:Drug resistant tuberculosis is increasing world-wide. Resistance against isoniazid (INH), rifampicin (RIF), or both (multi-drug resistant TB, MDR-TB) is of particular concern, since INH and RIF form part of the standard regimen for TB disease. While it is known that suboptimal treatment can lead to resistance, it remains unclear how host immune responses and antibiotic dynamics within granulomas (sites of infection) affect emergence and selection of drug-resistant bacteria. We take a systems pharmacology approach to explore resistance dynamics within granulomas. We integrate spatio-temporal host immunity, INH and RIF dynamics, and bacterial dynamics (including fitness costs and compensatory mutations) in a computational framework. We simulate resistance emergence in the absence of treatment, as well as resistance selection during INH and/or RIF treatment. There are four main findings. First, in the absence of treatment, the percentage of granulomas containing resistant bacteria mirrors the non-monotonic bacterial dynamics within granulomas. Second, drug-resistant bacteria are less frequently found in non-replicating states in caseum, compared to drug-sensitive bacteria. Third, due to a steeper dose response curve and faster plasma clearance of INH compared to RIF, INH-resistant bacteria have a stronger influence on treatment outcomes than RIF-resistant bacteria. Finally, under combination therapy with INH and RIF, few MDR bacteria are able to significantly affect treatment outcomes. Overall, our approach allows drug-specific prediction of drug resistance emergence and selection in the complex granuloma context. Since our predictions are based on pre-clinical data, our approach can be implemented relatively early in the treatment development process, thereby enabling pro-active rather than reactive responses to emerging drug resistance for new drugs. Furthermore, this quantitative and drug-specific approach can help identify drug-specific properties that influence resistance and use this information to design treatment regimens that minimize resistance selection and expand the useful life-span of new antibiotics.

Dataset Information

Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance.

Publications

Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance.

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