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

原核细胞和真核细胞都会遇到呼吸代谢过程中产生的活性氧(ROS)、活性氮(RNS)和活性亲电活性(RES)，或者是由抗生素和外源化合物等有毒化合物提供的。病原菌必须应对，特别是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对枯草芽孢杆菌转录组和氧化还原蛋白质组的影响。我们发现枯草杆菌中的杆菌硫醇(Cys-GlcN-Malate，BSH)氧化还原缓冲液在氧化还原调节和保护必需酶活性部位Cys残基免受二硫化物胁迫条件下的不可逆氧化中起着重要作用。NaOCl胁迫导致氧化还原敏感型MAR型抑制子OhrR和蛋氨酸生物合成途径的四种酶(Mete、YxjG、PPAC和血清)发生S-杆菌硫基化，从而保护细胞免受NaOCl毒害。在真核生物和大肠杆菌中，蛋白质S谷胱甘肽基化已成为一种主要的细胞调控机制，几种代谢酶的失活是由S谷胱甘肽基化对氧化应激的反应引起的。因此，在这项拨款中，我们的目的是研究bsh在通过S-杆菌硫醇化作用在产生bsh的Firmicuts细菌(包括工业上重要的芽孢杆菌(嗜盐芽孢杆菌、克劳斯芽孢杆菌、巨大芽孢杆菌、淀粉液化芽孢杆菌)和肉质葡萄球菌、耐辐射耐辐射球菌以及致病蜡样芽孢杆菌)之间氧化还原调节细胞质和调节蛋白中的全球生理作用。我们将使用基于二维凝胶的氧化还原蛋白质组学方法、鸟枪-LC-MS/MS分析和基于MS的稳定的14N/15N-铵结合免疫沉淀修饰的多肽和蛋白质来鉴定和定量S-杆菌硫基化对ROS的响应程度。我们的首次全蛋白质组学研究确定了新的杆状氧化还蛋白(YphP、YQiW和YtxJ)作为S-杆状硫化作用的靶标，它可以作为硫醇-二硫键氧化还原酶(杆状氧化还原酶，BRx)来还原S-杆状硫化蛋白。因此，将利用BRX突变菌株的表型分析和基于凝胶和无凝胶的氧化还原蛋白质组学新方法来分析与BSH相关的新型杆菌氧化还蛋白的功能和底物。

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.