MarR-Dependent Transcriptional Regulation of mmpSL5 Induces Ethionamide Resistance in Mycobacterium abscessus

Mycobacterium abscessus (Mabs) is an emerging nontuberculosis mycobacterial (NTM) pathogen responsible for a wide variety of respiratory and cutaneous infections that are difficult to treat with standard antibacterial therapy. Mabs has a high degree of both innate and acquired antibiotic resistance to most clinically relevant drugs, including standard anti-mycobacterial agents. Ethionamide (ETH), an inhibitor of mycolic acid biosynthesis, is currently utilized as a second-line agent for treating multidrug-resistant tuberculosis infections. Here, we show that ETH displays activity against clinical strains of Mabs in vitro at concentrations that are >100× lower than other mycolic acid targeting drugs. Using transposon mutagenesis followed by transposon sequencing (Tn-Seq) and whole-genome sequencing of spontaneous ETH-resistant mutants, we identified MAB_2648c as a genetic determinant of ETH sensitivity in Mabs. MAB_2648c encodes a MarR family transcriptional regulator of the TetR class of regulators. We show that MAB_2648c represses expression of MAB_2649 (mmpS5) and MAB_2650 (mmpL5). Further, we show that derepression of these genes in MAB_2648c mutants confers resistance to ETH, but not other antibiotics. To identify determinants of resistance that may be shared across antibiotics with distinct mechanisms of action, we also performed Tn-Seq during treatment with amikacin and clarithromycin, drugs currently used clinically to treat Mabs. We found very little overlap in genes that modulate the sensitivity of Mabs to all three antibiotics, suggesting a high degree of specificity for resistance mechanisms in this emerging pathogen.