Ethionamide (ETA) is an important component of second-line therapy for the treatment of multidrug-resistant tuberculosis. Synthesis of radiolabeled ETA and an examination of drug metabolites formed by whole cells of Mycobacterium tuberculosis (MTb) have allowed us to demonstrate that ETA is activated by Soxidation before interacting with its cellular target. ETA is metabolized by MTb to a 4-pyridylmethanol product remarkably similar in structure to that formed by the activation of isoniazid by the catalase-peroxidase KatG. We have demonstrated that overproduction of Rv3855 (EtaR), a putative regulatory protein from MTb, confers ETA resistance whereas overproduction of an adjacent, clustered monooxygenase (Rv3854c, EtaA) confers ETA hypersensitivity. Production of EtaA appears to be negatively regulated by EtaR and correlates directly with [ 14 C]ETA metabolism, suggesting that EtaA is the activating enzyme responsible for thioamide oxidation and subsequent toxicity. Coding sequence mutations in EtaA were found in 11 of 11 multidrug-resistant MTb patient isolates from Cape Town, South Africa. These isolates showed broad cross-resistance to thiocarbonyl containing drugs including ETA, thiacetazone, and thiocarlide.

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.