Abstract

Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus. We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr, helR, and MAB_2807. By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis, we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific {beta}-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies.