Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
Mol Cell Proteomics. 2012 Nov;11(11):1123-39. doi: 10.1074/mcp.M111.014191. Epub 2012 Jul 25.
Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.
金黄色葡萄球菌感染涉及多种黏附素和毒素，其表达取决于复杂的调控网络。黏附素包括一组通过保守的 LPXTG 基序共价连接到肽聚糖的表面蛋白。在这里，我们在时间过程实验中确定了 S. aureus Newman 的 LPXTG-蛋白的蛋白质和 mRNA 表达，以及它们与体外纤维蛋白原黏附的关系。实验是在全局辅助基因调节剂 (agr)、表面蛋白 A (Spa) 和纤维蛋白原结合蛋白 A (ClfA) 的突变体以及在富含铁或贫铁介质中生长时进行的。通过对活细菌进行胰蛋白酶刮削回收表面蛋白。释放的肽通过液相色谱与串联质谱分析。为了明确鉴定 LPXTG-蛋白特有的肽，使用在替代乳球菌中异源表达的 S. aureus LPXTG-蛋白参考文库，对分析条件进行了优化。通过微阵列确定转录组。通过蛋白质组学检测到 S. aureus Newman 中存在的 18 个 LPXTG-蛋白中的 16 个。9 个 LPXTG-蛋白表现出类似 agr 的钟形表达，在 agr 阴性突变体中被阻断，包括 Spa、纤维连接蛋白结合蛋白 A (FnBPA)、ClfA、铁结合 IsdA 和 IsdB、免疫调节剂 SasH、功能未知的 SasD、生物膜相关的 SasG 和耐甲氧西林相关的 FmtB。然而，只有 Spa 和 SasH 在亲本及其 agr-突变体中平行修饰了它们的蛋白质组学和 mRNA 谱，而所有其他 LPXTG-蛋白独立于它们的 mRNA 修饰了它们的蛋白质组学谱。此外，ClfA 在生长后期（24 小时）在纤维蛋白原黏附试验中高度转录和活跃，而在蛋白质组学中仍未被检测到。另一方面，铁调节的 IsdA-B-C 在缺铁条件下其蛋白表达增加了 10 多倍。因此，金黄色葡萄球菌的蛋白质组学、转录组学和黏附表型显示出不同的谱。此外，胰蛋白酶肽特征表明在不同环境中存在不同的蛋白结构域暴露，这可能与抗黏附素疫苗有关。对金黄色葡萄球菌生理学的全面理解应整合这三种方法。
