Altered NADH/NAD ⁺ Ratio Mediates Coresistance to Isoniazid and Ethionamide in Mycobacteria

<jats:title>ABSTRACT</jats:title> <jats:p> The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant mutations in the target gene <jats:italic>inhA</jats:italic> , encoding an enoyl-ACP reductase, or by recessive mutations in <jats:italic>ndh</jats:italic> , encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated novel <jats:italic>ndh</jats:italic> mutants of <jats:italic>Mycobacterium smegmatis</jats:italic> and <jats:italic>Mycobacterium bovis</jats:italic> BCG. The <jats:italic>M. smegmatis ndh</jats:italic> mutants were highly resistant to INH and ETH, while the <jats:italic>M. bovis</jats:italic> BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in intracellular NADH/NAD <jats:sup>+</jats:sup> ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation. We conclude that <jats:italic>ndh</jats:italic> mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits the binding of INH-NAD or ETH-NAD adduct to InhA. </jats:p>