The <i>MAB_3513c</i> gene plays a key role in intrinsic resistance of <i>Mycobacterium abscessus</i> to isoniazid and ethionamide

Isoniazid (INH) and ethionamide (ETH) are important antitubercular analogs that inhibit mycolic acid synthesis. However, <i>Mycobacterium abscessus</i> (<i>M. abscessus</i>) exhibits intrinsic resistance to both drugs. The molecular mechanisms underlying this resistance remain poorly understood. We identified <i>MAB_3513</i>c (<i>nudC</i>), encoding a functional NADH pyrophosphatase NudC that hydrolyzes drug-NAD adducts, as being crucial for intrinsic resistance to INH and ETH in <i>M. abscessus</i>. Susceptibility to both antibiotics was markedly enhanced upon either deleting <i>nudC</i> or introducing the P226Q point mutation in this conserved residue. High-performance liquid chromatography-mass spectrometry analysis further demonstrated that the deletion of <i>nudC</i> resulted in accumulation of the ETH-NAD adduct and a decrease in its hydrolysis rate in ETH-treated <i>M. abscessus</i>. Size-exclusion chromatography analysis revealed that wild-type NudC predominantly forms dimers, whereas the P226Q mutant exists mainly as monomers, consistent with a loss of enzymatic function. A pivotal proline at position 226 governs its dimerization and enzymatic activity. To complement the <i>nudC</i> knockout <i>M. abscessus</i> strain, we introduced seven different versions of the gene separately, each producing a protein with a point mutation at different key residues: D133A, N148D, F156A, F193A, E207Q, E211Q, and R231Q. None of them restore the resistance to INH and ETH, illustrating that these sites are also critical for the catalytic activity of NudC. This discovery elucidates a previously unrecognized mechanism of drug resistance in <i>M. abscessus</i> and highlights NudC as a potential target for therapeutic intervention.IMPORTANCE<i>Mycobacterium abscessus</i> poses a serious therapeutic challenge due to its extensive drug resistance. This study elucidates the molecular mechanism behind its intrinsic resistance to the critical antitubercular drugs isoniazid and ethionamide. We identify the NudC (MAB_3513c) enzyme as a central resistance factor that functions by hydrolyzing the active drug-NAD adducts. Crucially, we demonstrate that NudC's enzymatic activity, dependent on its dimerization and key catalytic residues, is essential for this resistance mechanism. This finding establishes NudC as a promising therapeutic target for combating this highly drug-resistant pathogen.