Inactivation of cAMP receptor protein (MSMEG_6189) increases isoniazid susceptibility in Mycobacterium smegmatis via altered oxidative phosphorylation, elevated ROS production, and loss of ahpC expression.

Isoniazid (INH) is a first-line drug for treating drug-susceptible tuberculosis. The genome of Mycobacterium smegmatis encodes two cAMP receptor proteins (Crp), MSMEG_6189 (Crp1) and MSMEG_0539 (Crp2). The deletion of crp1 markedly increased susceptibility to INH, whereas the deletion of crp2 had no effect. In contrast, susceptibility to rifampicin was unchanged in the Δcrp1 mutant. Gene expression analysis revealed strong upregulation of katG1, which encodes the INH-activating catalase-peroxidase, and an almost complete abolishment of ahpC expression, which encodes alkyl hydroperoxide reductase and contributes to INH resistance, in the Δcrp1 mutant. The mutant also exhibited elevated intracellular reactive oxygen species (ROS) levels and reduced respiration. These elevated ROS levels likely resulted from alterations in the respiratory electron transport chain, including reduced levels of the cytochrome bcc1 complex, increased expression of the nuo and sdh1 operons (encoding type I NADH dehydrogenase and succinate dehydrogenase, respectively), and the loss of bd quinol oxidase induction under conditions of diminished bcc1 complex levels. ROS accumulation, in turn, appears to inactivate the FurA repressor, leading to the induction of katG1 expression. Collectively, these findings suggest that the loss of Crp1 increases INH susceptibility through multiple mechanisms that promote INH activation, including ROS-mediated induction of katG1 and increased intracellular ROS levels. Furthermore, the absence of ahpC expression in the Δcrp1 mutant further contributes to its increased INH susceptibility. IMPORTANCE: This study revealed that the cAMP receptor protein, Crp1, plays a role in controlling key genes involved in both INH activation (katG1) and resistance (ahpC) in Mycobacterium smegmatis. Inactivation of Crp1 was shown to cause an increase in cellular ROS levels, which likely results from a bottleneck of electron flow at the cytochrome bcc1 complex in the electron transport chain and the abolishment of bd quinol oxidase induction under conditions of reduced bcc1 complex levels. The resulting increase in ROS, combined with the simultaneous loss of ahpC expression, renders the bacterium significantly more vulnerable to INH. These findings provide deeper insights into the interplay among oxidative phosphorylation, ROS generation, and INH susceptibility.