Role of the bacillithiol redox buffer for redox control in Firmicutes bacteria

原核和真核细胞会遇到活性氧（ROS）、活性氮（RNS）和活性亲电物质（RES），这些物质是在呼吸和代谢过程中产生的，或者是由抗生素和外源药物等有毒化合物提供的。致病菌必须特别应对在感染过程中被激活的巨噬细胞释放的强氧化剂次氯酸。在最近的一项研究中，我们研究了NaOCl引起枯草芽孢杆菌转录组和氧化还原蛋白质组的变化。我们发现枯草芽孢杆菌中的芽孢杆菌硫醇（Cys- glcn -苹果酸盐，BSH）氧化还原缓冲液在二硫胁迫条件下对必需酶活性位点Cys残基的氧化还原调控和保护中起重要作用。NaOCl胁迫导致氧化还原敏感的marr型抑制因子OhrR和蛋氨酸生物合成途径中保护细胞免受NaOCl毒性的四种酶（MetE, YxjG， PpaC和SerA）的S-bacillithiolation。在真核生物和大肠杆菌中，蛋白质s -谷胱甘肽化已成为一种主要的细胞调节机制，在氧化应激反应中，s -谷胱甘肽化引起了几种代谢酶的失活。因此，我们在这项资助中旨在研究BSH在产生BSH的厚壁菌门细菌中通过s -杆菌硫酸化对细胞质和调节蛋白的氧化还原调节中的全球生理作用，包括工业上重要的芽孢杆菌物种（嗜盐芽孢杆菌、克劳氏芽孢杆菌、巨芽孢杆菌、解淀粉芽孢杆菌）和肉葡萄球菌、耐辐射的耐辐射球菌以及致病性蜡样芽孢杆菌。我们将使用基于2D凝胶的氧化还原蛋白质组学方法，霰弹枪- lc -MS/MS分析和基于MS的稳定同位素代谢标记（14N/ 15n -铵），结合bsh修饰的肽和蛋白质的免疫沉淀，来鉴定和量化s -杆菌硫代酸在ROS响应中的程度。我们的第一个蛋白质组研究已经确定了新的杆菌还毒素（YphP， YqiW和YtxJ）作为s -杆菌硫酰化的靶标，可以作为硫醇二硫氧化还原酶（bacilliredoxins, Brx）在s -杆菌硫酰化蛋白的还原中起作用。因此，我们将利用brx突变株的表型分析和新的基于凝胶和无凝胶的氧化还原蛋白质组学方法来分析新型bsh相关杆菌还毒素的功能和底物。

Prokaryotic and eukaryotic cells are encountered by reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive electrophilic species (RES) which are generated during respiration and metabolism or supplied externally by toxic compounds, such as antibiotics and xenobiotics. Pathogenic bacteria have to cope especially with the strong oxidant hypochloric acid that is released by activated macrophages during the infection process. In a recent study, we have studied changes in the transcriptome and redox proteome caused by NaOCl in Bacillus subtilis. We discovered important roles of the bacillithiol (Cys-GlcN-Malate, BSH) redox buffer in B. subtilis in redox regulation and protection of active site Cys residues of essential enzymes against irreversible oxidations under disulfide stress conditions. NaOCl stress caused S-bacillithiolation of the redox-sensing MarR-type repressor OhrR and of four enzymes of the methionine biosynthesis pathway (MetE, YxjG, PpaC and SerA) that protect cells against NaOCl toxicity. In eukaryotes and Escherichia coli, protein S-glutathionylation has emerged as a major cellular regulatory mechanism and the inactivation of several metabolic enzymes is caused by S-glutathionylation in response to oxidative stress. Thus, we aim in this grant to investigate the global physiological role of BSH in redox regulation of cytoplasmic and regulatory proteins by S-bacillithiolation among BSH-producing Firmicutes bacteria, including industrial important Bacillus species (Bacillus halodurans, Bacillus clausii, Bacillus megaterium, Bacillus amyloliquefaciens,) and Staphylococcus carnosus, the radioresistant Deinococcus radiodurans as well as the pathogenic Bacillus cereus. We will use 2D gel-based redox proteomics methods, shotgun-LC-MS/MS analysis and MS-based stable isotop metabolic labelling with 14N/15N-ammonium coupled to immunoprecipitation of BSH-modified peptides and proteins to identify and quantify the extent of S-bacillithiolations in response to ROS. Our first proteome-wide studies have identified novel bacilliredoxins (YphP, YqiW and YtxJ) as target for S-bacillithiolation that could function as thiol-disulfide oxidoreductases (bacilliredoxins, Brx) in reduction of S-bacillithiolated proteins. Thus, the functions and substrates of novel BSH-related bacilliredoxins will be analyzed using phenotype analyses of brx mutant strains and novel gel-based and gel-free redox proteomics methods.