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

Mycobacterium abscessus (Mabs) is an emerging non-tuberculosis 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 (MDR-TB) infections. Here, we show that ETH has activity against clinical strains of Mabs in vitro at concentrations that are therapeutically achievable. Using transposon mutagenesis and whole genome sequencing of spontaneous drug-resistant mutants, we identified marR (MAB_2648c) as a genetic determinant of ETH sensitivity in Mabs. The gene marR encodes a transcriptional regulator of the TetR family of regulators. We show that MarR represses expression of MAB_2649 (mmpS5) and MAB_2650 (mmpL5). Further, we show that de-repression of these genes in marR mutants confers resistance to ETH, but not other antibiotics. To identify determinants of resistance that may be shared across antibiotics, 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. ImportanceAntibiotic resistant infections caused by Mycobacterium abscessus (Mabs) have been increasing in prevalence and treatment is often unsuccessful. Success rates range from 30-50%, primarily due to the high intrinsic resistance of Mabs to most clinically useful antibiotics. New therapeutic strategies, including repurposing of existing antibiotics, are urgently needed to improve treatment success rates. Here, we show that the anti-TB antibiotic ethionamide (ETH) has repurposing potential against Mabs, displaying bacteriostatic activity and delaying emergence of drug resistance when combined with clinically relevant antibiotics currently used against Mabs in vitro. We identified genes that modulated susceptibility of Mabs to ETH. marR encodes a transcriptional regulator that when deleted, confers ETH resistance. Our collective findings can be used to further explore the function of other genes that contribute to ETH susceptibility and help design the next generation of antibacterial regimens against Mabs that may potentially include ETH.