Functional characterization of NaOCl-sensitive thiol-switches and their impact on the BSH redox potential in Staphylococcus aureus

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Staphylococcus aureus is a major human pathogen that can cause local skin or soft tissue infections, but also life-threatening diseases. During infections, S. aureus has to cope with reactive oxygen species (ROS) and hypochloric acid (HOCl) that are produced by activated macrophages and neutrophils as major killing mechanism. S. aureus uses the low molecular weight thiol bacillithiol (BSH) as protection mechanism against the host immune defense. BSH contributes to virulence of S. aureus and functions in detoxification of ROS, HOCl, toxins, electrophiles and antibiotics. Under hypochlorite stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations as a widespread thiol-protection and redox-switch mechanism. Using the quantitative thiol-redox proteomics approach OxICAT, we recently identified 58 NaOCl-sensitive proteins in S. aureus that could play protective roles against the host immune defense. Among these are five S-bacillithiolated proteins including the glycolytic Gap as the major target. S-bacillithiolation of Gap functions in thiol-protection against overoxidation to irreversible sulfonic acids and redox-regulation under H2O2 and NaOCl stress. The bacilliredoxins BrxA/B were shown to catalyze the reduction of S-bacillithiolated OhrR, MetE in B. subtilis and Gap in S. aureus in vitro. However, the complete Brx redox pathway is unknown. We have further constructed the first genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus. In this project, we will characterize the functions of the interesting NaOCl-sensitive thiol-switches in the defense against oxidative stress. These thiol-switches include redox regulators, bacilliredoxins and other thiol-disulfide reductases, the nitric oxide synthase, the virulence factor SsaA2 and the metabolic enzymes Gap and AldA. In addition, novel Brx-roGFP2 and Tpx-roGFP2 biosensors will be applied to monitor the changes in the BSH redox potential and intracellular H2O2 generation in the S. aureus thiol-switch mutant backgrounds.