Mycobacterium smegmatis RoxY Is a Repressor ofoxySand Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides

Abstract

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.Despite the availability of antibiotics to combat tuberculosis, it is one of the leading causes of death due to infectious disease. The World Health Organization estimates that Mycobacterium tuberculosis infects one-third of the world's population, and 8 million new cases of tuberculosis are reported annually (http://www.who.int/mediacentre/factsheets/fs104/en/). M. tuberculosis infections persist for decades despite the host immune response that exposes the bacterium to a number of stressful environments during the course of infection. The nature of these host defenses and the repertoire of M. tuberculosis resistance mechanisms are still being elucidated. The ability of M. tuberculosis to survive within the host macrophage is key to its pathogenesis. While the pathogen is well-equipped to establish a niche in resting macrophages, activation of the host macrophage shifts the balance toward mycobacterial clearance (26,38). The mycobactericidal activity of activated macrophages includes both oxidative and nonoxidative mechanisms. Studies with mice indicate that reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) function in control of tuberculosis infection: M. tuberculosis infection of NADPH phagosome oxidase (NOX2)-deficient mice indicates a role for ROI in the lung, and Nos2 Ϫ/Ϫ mice are quickly killed upon infection with M. tuberculosis (1,5,17). Enhanced fusion of the bacterium-containing vacuole with the macrophage lysosome also contributes to the mycobactericidal activity of activated macrophages. The induction of autophagy in Mycobacterium bovis BCG-and M. tuberculosis-infected macrophages by serum starvation, rapamy...

Molecular basis of mycobacterial survival in macrophages

“…Overexpression of OxyS in M. smegmatis reduced transcription of the AhpCD genes, which encode a peroxide detoxification system. Thus, RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response, and are important for resistance to host antimicrobial peptides [204].…”

Roles of three FurA paralogs in the regulation of genes pertaining to peroxide defense in Mycobacterium smegmatis mc2155

“…Alkyl hydroperoxide reductase C (AhpC) is a member of the peroxiredoxin family that catalyzes reduction of organic peroxides and H 2 O 2 into corresponding alcohols and water respectively (Wood et al ., ). The ahpC gene and its downstream gene ahpD encoding peroxiredoxin reductase form an operon in Mycobacterium smegmatis (Daugherty et al ., ). Since AhpC is capable of detoxifying peroxide species and peroxynitrite (ONOO − ) through its enzymatic activity catalyzing the reduction of the peroxide linkage (OO), it has been suggested that this enzyme plays an important role in protection of mycobacterial cells from harmful effects of ONOO − (Master et al ., ).…”

Regulation of the ahpC Gene Encoding Alkyl Hydroperoxide Reductase in Mycobacterium smegmatis

“…It was reported that AhpC can be also reduced by thioredoxin C (TrxC) and NADPH-dependent thioredoxin reductase [10] . The ahpC gene forms an operon with its downstream gene, ahpD [11] . Genes encoding the OxyR homologs, which are LysR family regulators and involved in peroxide stress response, are divergently located upstream of the ahpCD operons in most mycobacteria [12] .…”