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

金黄色葡萄球菌是一种主要的人类病原体，可引起局部皮肤或软组织感染，也是危及生命的疾病。在感染过程中，金黄色葡萄球菌必须应对活化的巨噬细胞和中性粒细胞产生的活性氧（ROS）和次氯酸（HOCl）作为主要杀伤机制。金黄色葡萄球菌利用低分子量硫醇杆菌硫醇（BSH）作为抵抗宿主免疫防御的保护机制。BSH有助于金黄色葡萄球菌的毒力，并在活性氧、HOCl、毒素、亲电试剂和抗生素的解毒中发挥作用。在次氯酸盐胁迫下，BSH与蛋白质形成混合二硫化物，称为S-bacillithiolations，是一种广泛的硫醇保护和氧化还原开关机制。利用定量硫醇-氧化还原蛋白质组学方法OxICAT，我们最近在金黄色葡萄球菌中鉴定出58种naocl敏感蛋白，这些蛋白可以对宿主免疫防御发挥保护作用。其中包括5种s -杆菌硫酸化蛋白，其中糖酵解Gap是主要靶点。S-bacillithiolation在H2O2和NaOCl胁迫下具有保护硫醇抗不可逆磺酸过度氧化和氧化还原的功能。结果表明，bacilliredoxins BrxA/B可以催化S-bacillithiolated OhrR、MetE和金黄色葡萄球菌Gap在体外的还原。然而，完整的Brx氧化还原途径尚不清楚。我们进一步构建了首个基因编码的杆菌氧化还毒素融合氧化还原生物传感器（Brx-roGFP2），以监测金黄色葡萄球菌BSH氧化还原电位的动态变化。在这个项目中，我们将描述naocl敏感的硫醇开关在防御氧化应激中的功能。这些硫醇开关包括氧化还原调节剂、杆菌氧化还毒素和其他硫醇二硫还原酶、一氧化氮合酶、毒力因子SsaA2以及代谢酶Gap和AldA。此外，新型Brx-roGFP2和Tpx-roGFP2生物传感器将用于监测金黄色葡萄球菌硫醇开关突变背景下BSH氧化还原电位和细胞内H2O2生成的变化。

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.