Toward the quantitative characterization of arginine phosphorylations in Staphylococcus aureus

The Gram positive bacterium Staphylococcus aureus plays an important role as an opportunistic pathogen and causative agent of nosocomial infections. As research gained insight into host specific adaptation and a broad range of virulence mechanisms, S. aureus evolved as a model organism for human pathogens. Hence, the investigation of staphylococcal proteome expression and regulation supports the understanding of the pathogenicity and relevant physiology of this organism. This study focused on the analysis of protein regulation by reversible protein phosphorylation, in particular on arginine residues. Therefore, both proteome and phosphoproteome of S. aureus COL wild type were compared with the arginine phosphatase deletion mutant S. aureus COL ΔptpB under control and stress conditions in a quantitative manner. A gel-free approach, adapted to the special challenges of arginine phosphorylation, was applied to analyze the phosphoproteome of exponential growing cells after oxidative stress caused by sublethal concentrations of H2O2. Together with phenotypic characterization of S. aureus COL ΔptpB, this study disclosed first insights into the physiological role of arginine phosphorylations in Gram positive pathogens. The spectral library based quantification of phosphopeptides finally allowed to link arginine phosphorylation to staphylococcal oxidative stress response, amino acid metabolism and virulence.

革兰氏阳性菌（Gram-positive bacterium）金黄色葡萄球菌（Staphylococcus aureus）是一类重要的机会致病菌，同时也是医院获得性感染（nosocomial infections）的病原菌。随着研究对宿主特异性适应与多样毒力机制的认知不断深化，金黄色葡萄球菌已成为人类病原菌研究的模式生物。因此，针对葡萄球菌蛋白质组（staphylococcal proteome）的表达与调控开展研究，有助于深化对该病原菌致病性与相关生理学特征的认知。本研究聚焦于可逆蛋白质磷酸化（reversible protein phosphorylation，尤其是精氨酸残基（arginine residues）的磷酸化）对蛋白质调控的分析。为此，研究人员在对照与应激条件下，以定量方式比较了金黄色葡萄球菌COL野生型（S. aureus COL wild type）与其精氨酸磷酸酶缺失突变体S. aureus COL ΔptpB的蛋白质组与磷酸化蛋白质组（phosphoproteome）。本研究采用适配精氨酸磷酸化特殊研究难点的无凝胶方法（gel-free approach），对经亚致死浓度过氧化氢（H₂O₂）诱导氧化应激后的指数生长期细胞的磷酸化蛋白质组进行了分析。结合对S. aureus COL ΔptpB的表型表征（phenotypic characterization），本研究首次揭示了精氨酸磷酸化在革兰氏阳性病原菌中的生理学功能。基于光谱库的磷酸肽（phosphopeptides）定量分析最终将精氨酸磷酸化与葡萄球菌的氧化应激响应（oxidative stress response）、氨基酸代谢（amino acid metabolism）及毒力（virulence）建立了关联。