Mycobacterium smegmatis RoxY is a repressor of oxyS and contributes to resistance to oxidative stress and bactericidal ubiquitin-derived peptides

The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ubpeptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.