Inhibition of the Classical & Lectin Complement Pathways by Staphylococcus aureus Eap

 描述(申请人提供)：在过去的几年里，通过对金黄色葡萄球菌毒力细菌的研究，我们对病原体利用的补体逃避机制的了解急剧增加。通过在人全血炎症模型中筛选分泌型金黄色葡萄球菌蛋白文库，我们已确定细胞外黏附蛋白(EAP)是第一个已知的金黄色葡萄球菌补体(CP)和凝集素(LP)途径的抑制物。EAP以剂量依赖的方式抑制这两种途径，需要与补体成分C4b形成高纳米分子亲和力相互作用。这种相互作用阻止了CP/LP Pro-C3转换酶复合体(C4b/C2)的形成，从而显著降低了活性CP/LP C3转换酶(C4b/C2a)的水平。使用相同的全血模型，我们还发现EAP是中性粒细胞丝氨酸蛋白酶(NSPs)的有效抑制物。与传统的蛇类不同，EAP对NSP的抑制本质上是非共价的。此外，它发生的分子机制不同于它对补体系统的影响，因为两个相关的蛋白质，EapH1和EapH2，也阻断NSP的活性，但对补体没有影响。在这个方案中，我们将通过两个特定的目的来研究EAP对CP/LP影响的特异性的分子基础：(1)我们将表征EAP与补体蛋白C4b结合的生化和结构基础；(2)我们将表征与EAP竞争C4b结合的多肽，并确定它们是否保持对CP/LP的EAP样抑制活性。我们预计，这种综合结构/功能和发现方法将为对CP/LP的监管提供新的见解。反过来，这可能为未来新型补体靶向抗炎疗法的设计和优化提供重要线索。

 DESCRIPTION (provided by applicant): Over the last several years, our understanding of the complement evasion mechanisms utilized by pathogens has increased precipitously through the study of the virulent bacterium Staphylococcus aureus. By screening a library of secreted S. aureus proteins in a human whole-blood model of inflammation, we have identified the Extracellular Adherence Protein (Eap) as the first known S. aureus inhibitor of the Classical (CP) and Lectin (LP) pathways of complement. Eap inhibits both of these pathways in a dose-dependent manner that requires formation of high-nanomolar affinity interaction with complement component C4b. This interaction blocks formation of the CP/LP pro-C3 convertase complex (C4b/C2), which dramatically lowers levels of the active CP/LP C3 convertase (C4b/C2a). Using the same whole-blood model, we have also identified Eap as a potent inhibitor of Neutrophil Serine Proteases (NSPs). Unlike conventional serpins, Eap inhibition of NSPs is non-covalent in nature. Furthermore, it occurs through a molecular mechanism distinct from its effects on the complement system since two related proteins, EapH1 and EapH2, also block NSP activity but have no effect on complement. In this proposal, we will investigate the molecular basis for the specificity of Eap's effects on the CP/LP through two Specific Aims: (1) We will characterize the biochemical and structural basis for Eap binding to complement protein C4b, and (2) We will characterize peptides that compete with Eap for C4b binding and determine whether they retain Eap-like inhibitory activities against the CP/LP. We expect that this integrated structure/function and discovery approach will provide new insight into regulation of the CP/LP. In turn, this may hold important clues into the design and optimization of novel complement-targeted, anti-inflammatory therapeutics in the future.