A proteomic view on the cell biology of Staphylococcus aureus proteome. Staphylococcus aureus

The genome sequence is the “blue-print of life”, but proteomics is required to relate this blue-print of life to the actual physiology of living cells. Because of their low complexity – only about 2.000 proteins make a Staphylococcus aureus cell viable – bacteria are excellent model systems to identify the entire protein assembly of a living organism. Many of the studies on physiological proteomics of bacteria, however, still rely on 2D gel-based technologies that visualize only a minor part of the proteome. In this study it will be shown that the majority of proteins expressed in the Gram-positive human pathogen S. aureus can be identified and even quantified by a combination of gel-based and gel-free approaches, the latter having undergone formidable improvements over the last 10 years. S. aureus has been the model of choice for our proteomic studies, because it combines high pathogenicity with increasing antibiotic resistance, which calls for new leads in the development of anti-staphylococcal therapy. On the way towards the entire proteome of S. aureus, we analysed four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell-surface associated and extracellular proteins, the two latter fractions containing most of the virulence factors. To obtain quantitative results, we compared growing cells and non-growing/stationary phase cells using the 15N metabolic labelling approach. With almost 2.000 proteins, 80 % of the expressed proteome was identified and even quantified in growing and non-growing cells. A comprehensive inventory of the entire protein assembly during the two physiologically distinct states is presented, which integrates data ranging from gene expression to subcellular localization. Three major protein classes were distinguished: (i) Proteins induced in the stationary phase only (ii) Vegetative proteins, no longer synthesized but stable in the stationary phase (iii) Vegetative proteins, no longer required in non-growing cells and finally degraded. This quantitative proteomics approach for growing/non-growing cells, which represents a proof-of-principle for whole-cell physiological proteomics, can now be extended to address particular infection-relevant physiological states. Importantly, the present model study represents the highest coverage of a bacterial proteome so far (except low complexity bacteria). It thus paves the way towards a new understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening a new era in infection-related research on this crucial pathogen. Overall design: For the DNA-microarray experiment RNA was extracted from two independently grown cultures. One was grown in 14N labelled BioExpress medium and the other in 15N labelled BioExpress medium. Samples of exponentially growing cells (OD600=0.5) and of cell at 5h after entry into stationary phase were harvested from every culture. Equal amounts of all four RNAs were pooled and the pool was used as common reference (Cy3-labeld) for the four sample RNAs (Cy3). Thus, in total four hybridizations, one for each sample versus the common reference, were performed.

基因组序列堪称“生命蓝图”，但要将这份生命蓝图与活细胞的真实生理机能相联系，则必须借助蛋白质组学（proteomics）研究。由于细菌的蛋白质组复杂度较低——仅需约2000种蛋白质即可维持金黄色葡萄球菌（Staphylococcus aureus）细胞存活，因此细菌是鉴定完整生物体蛋白质组的绝佳模型系统。然而，当前诸多细菌生理蛋白质组学研究仍依赖二维凝胶（2D gel）技术，这类方法仅能检测到蛋白质组中极小一部分组分。本研究证实，借助凝胶技术与无凝胶（gel-free）技术的联合策略，即可鉴定甚至定量革兰氏阳性人类病原菌金黄色葡萄球菌中表达的绝大多数蛋白质；其中无凝胶技术在过去十年间已取得长足进步。选择金黄色葡萄球菌（下文简称S. aureus）作为本蛋白质组学研究的模型菌株，是因为其兼具高致病性与日益严峻的抗生素耐药性，这使得开发抗葡萄球菌治疗策略亟需新的研究方向。为解析S. aureus的完整蛋白质组，我们分析了四个亚蛋白质组组分：胞质蛋白、膜结合蛋白、细胞表面相关蛋白以及胞外蛋白；后两类组分包含绝大多数毒力因子。为获取定量结果，我们采用15N代谢标记（15N metabolic labelling）方法，对比了增殖期细胞与非增殖/静止期细胞的蛋白质组。在增殖期与非增殖期细胞中，我们共鉴定并定量了近2000种蛋白质，覆盖了约80%的表达蛋白质组。本研究呈现了这两种生理状态下完整蛋白质组的全面编目，整合了从基因表达到亚细胞定位的多维度数据。我们划分出三大类蛋白质：（i）仅在静止期诱导表达的蛋白质；（ii）营养生长期合成、但在静止期仍保持稳定的蛋白质；（iii）营养生长期合成、但在非增殖细胞中不再需要并最终被降解的蛋白质。这种针对增殖/非增殖细胞的定量蛋白质组学方法，已验证了全细胞生理蛋白质组学的可行性，未来可进一步拓展至与感染相关的特定生理状态研究。值得注意的是，本模型研究是目前已报道的细菌蛋白质组覆盖度最高的案例（低复杂度细菌除外）。这一成果为我们重新理解S. aureus及其相关病原菌的细胞生理与病理生理机制铺平了道路，为针对这类关键病原菌的感染相关研究开启了全新纪元。整体实验设计：在DNA微阵列（DNA-microarray）实验中，我们从两组独立培养的菌液中提取总RNA。一组菌液培养于14N标记的BioExpress培养基，另一组培养于15N标记的BioExpress培养基。从每一组菌液中分别收取指数增殖期细胞（OD600=0.5）以及进入静止期5小时后的菌体样本。将四份RNA样品等量混合，以该混合池作为通用参考样品（标记为Cy3），并与四份待测RNA样品（标记为Cy3）进行杂交。最终共完成四次杂交实验，每份待测样品分别与通用参考样品进行一次杂交。