Defects in NADH oxidation confer isoniazid resistance

Temperature-sensitive (is) mutants that have thermolabile InhA defects would be particularly useful for defining the function of InhA in fatty acid elongation and/or mycolic acid synthesis. We have tried to obtain an inhA is mutant by isolating isoniazid-, ethionamide-resistant mutants at 30°C that are unable to grow at 42°C, even in the absence of drugs (Miesel et al 1998; Fig. 5). About half of all isoniazid-resistant mutants isolated at 30°C have these traits. Allelic exchange studies showed that these mutants do not have inhA mutations. Most of the mutants require amino acid supplements for growth at 30°C (some are specifically serine/glycine auxotrophs; Fig. 5); this auxotrophic trait is not expected for inhA mutants.

The resistance of the is mutants is caused by defects in NADH dehydrogenase (Ndh), which catalyses the first step in the electron transport chain: NADH oxidation and quinone (Q) reduction (NADH + Q — NAD+ + QH2) (Miesel et al 1998). Assays of NADH dehydrogenase activity indicated that the mutants have reduced activity which can be restored by expression of the wild-type enzyme (Table 1). The mutant phenotypes are also corrected by expression of the wildtype ndh gene. Sequence analysis identified substitutions in ndh in all the is isoniazid-resistant mutants (Fig. 5).

Genetic data indicated that all the phenotypes of the ndh mutants (isoniazid-resistant, ethionamide-resistant, is and auxotrophy) are caused by defects in NADH oxidation (Miesel et al 1998). Expression of NADH-dependent malate dehydrogenase (Mdh) corrects all of the ndh mutant phenotypes, causing thermoresistance, prototrophy, and sensitivity to isoniazid and ethionamide. Mdh catalyses the NADH-dependent interconversion of oxaloacetate and malate (NADH + oxaloacetate — NAD+ + malate) that could provide an additional NADH oxidation system that operates independently from the respiratory chain. Enzyme assays showed that Mdh efficiently catalyses NADH oxidation using oxaloacetate as an electron acceptor (Table 1). mdh was recovered from the M. iuberculosis complex. M. smegmaiis does not express this Mdh enzyme (Prasada Reddyet al 1975).

We have proposed that the ndh defect in the M. smegmaiis mutants lowers the rate of NADH oxidation, which would cause an increase in the NADH/NAD+ ratio (Miesel et al 1998). This imbalance causes the multiple phenotypes: isoniazid and ethionamide resistance; thermosensitivity; and auxotrophy. Mdh expression would correct the phenotypes by oxidizing NADH. The genetic data indicate that Ndh is not a target for isoniazid; instead, the Ndh must potentiate isoniazid and ethionamide by oxidizing NADH. The isoniazid resistance in the ndh mutants could be due to the increased NADH concentration, which may promote dislocation of the isonicotinic acyl-NADH from InhA. Another possibility is

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FIG. 5. Phenotypes of a representative ndb mutant (ndh-4) of Mycobacterium smegmatis that is resistant to isoniazid and ethionamide; and complementation with the ndb and mdh genes (Miesel et al 1998). The isoniazid-sensitive parent strain and the ndh-4 mutant carry pMV26'l, which confers kanamycin resistance (top half of each plate). The lower half of each plate shows the complementation by the Mycobacterium tuberculosis ndb gene expressed from pMV26'l (left) and complementation by the Mycobacterium bovis Bacillus Calmette—Guérin mdh gene (right). Isoniazid and ethionamide were used at 50 /ig/ml, and kanamycin (10 /ig/ml) was included into all medium to select for maintenance of plasmids.

FIG. 5. Phenotypes of a representative ndb mutant (ndh-4) of Mycobacterium smegmatis that is resistant to isoniazid and ethionamide; and complementation with the ndb and mdh genes (Miesel et al 1998). The isoniazid-sensitive parent strain and the ndh-4 mutant carry pMV26'l, which confers kanamycin resistance (top half of each plate). The lower half of each plate shows the complementation by the Mycobacterium tuberculosis ndb gene expressed from pMV26'l (left) and complementation by the Mycobacterium bovis Bacillus Calmette—Guérin mdh gene (right). Isoniazid and ethionamide were used at 50 /ig/ml, and kanamycin (10 /ig/ml) was included into all medium to select for maintenance of plasmids.

that NADH may competitively inhibit KatG-mediated oxidation reactions that activate isoniazid.

Results reported in early literature by Middlebrook & Cohn (1953) suggest that isoniazid resistance in M. tuberculosis can occur by metabolic defects which may be similar to the ndh defects found in M. smegmatis. These investigators attempted to culture isoniazid-resistant tubercle bacilli from patients treated with isoniazid monotherapy and found that eight out of 21 patients produced tubercle bacilli that have growth defects. Of these eight isolates, five were auxotrophs, and three were not cultureable in rich or minimal medium. Perhaps these resistant tubercle bacilli had NADH oxidation defects that prevented their growth on certain types of medium.

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