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，S.金黄色葡萄球菌必须科普由活化的巨噬细胞和嗜中性粒细胞产生的活性氧（ROS）和次氯酸（HOCl）作为主要的杀伤机制。S.金黄色葡萄球菌使用低分子量硫醇杆菌硫醇（BSH）作为针对宿主免疫防御的保护机制。BSH对S.在ROS、HOCl、毒素、亲电试剂和抗生素的解毒中起作用。在次氯酸盐胁迫下，BSH与蛋白质形成混合二硫化物，称为S-杆菌锂化，作为广泛的巯基保护和氧化还原开关机制。利用定量巯基-氧化还原蛋白质组学方法OxICAT，我们最近在S.金黄色葡萄球菌，可以发挥保护作用，对宿主的免疫防御。其中有五种S-杆菌硫醇化蛋白，包括糖酵解间隙作为主要靶标。Gap的S-杆菌锂硫醇化在巯基保护中起作用，防止过氧化为不可逆的磺酸，并在H2 O2和NaOCl胁迫下起氧化还原调节作用。显示杆菌氧还蛋白BrxA/B催化B中S-杆菌硫醇化的OhrR、MetE的还原。subtilis和S.金黄色葡萄球菌。然而，完整的Brx氧化还原途径是未知的。我们进一步构建了第一个基因编码的融合bacilliredoxin的氧化还原生物传感器（Brx-roGFP 2），以监测BSH氧化还原电位的动态变化。金黄色。在这个项目中，我们将描述感兴趣的NaOCl敏感的巯基开关在防御氧化应激中的功能。这些巯基开关包括氧化还原调节剂、杆菌氧还蛋白和其他巯基-二硫化物还原酶、一氧化氮合酶、毒力因子SsaA 2和代谢酶Gap和AldA。此外，新型Brx-roGFP 2和Tpx-roGFP 2生物传感器将被应用于监测BSH氧化还原电位和细胞内H2 O2产生的变化。金黄色葡萄球菌巯基转换突变体背景。

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.