Identification and characterization of ethR and ethA genes impacting the sensitivity of Mycobacterium abscessus to ethionamide

Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium with rising global incidence. This pathogen exhibits intrinsic resistance to most antibiotics, presenting a major public health threat. Ethionamide (ETH) requires bioactivation by monooxygenase EthA to form the active ETH-NAD adduct. We previously identified MAB_3513 (NudC) as a phosphohydrolase that confers intrinsic resistance to M. abscessus by hydrolyzing this adduct. However, deletion of nudC results in only partial susceptibility to ETH, indicating the existence of additional resistance mechanisms. This study identified MAB_0984 as the EthR regulator in M. abscessus. Deletion of ethR in a nudC knockout background ({Delta}{Delta}ethR) significantly enhanced ETH-NAD adduct accumulation, leading to hypersusceptibility to ETH. Notably, the {Delta}ethR mutant exhibited higher susceptibility than {Delta}nudC, demonstrating that EthR is a more dominant mediator of ETH resistance than NudC. Furthermore, MAB_0985 (EthA1) and MAB_0103 (EthA2) were identified as the primary EthAs in M. abscessus. Deletion of either gene alone or in combination in the {Delta}nudC reduced adduct formation and increased resistance, while the triple mutant {Delta}{Delta}ethA1{Delta}ethA2 restored wild-type resistance. Using an intergenic region-eGFP reporter system and quantitative reverse transcription-PCR, we demonstrated that EthR confers resistance by specifically suppressing ethA1 expression in M. abscessus. The Mycobacterium tuberculosis EthR inhibitor BDM31343 could boost the efficacy of ETH against M. abscessus by inhibiting EthR. Collectively, this study identified the ethR and ethA genes in M. abscessus for the first time and elucidated their role in mediating resistance to ETH. Therefore, EthR is a promising target for potentiating the efficacy of ETH against M. abscessus. Impact statementMycobacterium abscessus constitutes an escalating global health threat, primarily due to its intrinsic resistance to most antibiotics. This study identifies MAB_0984 (EthR) as a dominant resistance determinant that exerts a more profound impact on ETH susceptibility than the previously characterized NudC. We demonstrate that EthR mediates resistance by specifically repressing the expression of EthA1 (MAB_0985), one of the primary monooxygenases responsible for ETH bioactivation. The EthRMtb inhibitor BDM31343 potentiated the activity of ETH against M. abscessus through inhibition of the EthR. These findings elucidated the mechanism of ETH resistance in M. abscessus, identifying EthR as a promising target for boosting the efficacy of ETH against M. abscessus.