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.
The complement system is recognized as a valuable pharmacological target for treating both chronic and acute inflammatory diseases. However, only an exceedingly limited panel of FDA approved drugs and indications exist for specifically targeting the complement system. Understanding the structure/function relationships of naturally occurring staphylococcal inhibitors of the alternative pathway C3 convertase, such as members of the SCIN and Efb protein families, has provided a basic science foundation for capitalizing upon these proteins' activities toward future development of new types of complement targeted therapeutics. By analogy, we have recently identified the staphylococcal protein Eap as a specific inhibitor of the classical and lectin complement pathways, and our preliminary efforts indicate that Eap acts on the C3 convertase shared by these pathways. The purpose of this investigation is to provide information on Eap structure/function and to identify peptide mimics of Eap so that we might leverage this toward future design of complement-targeted therapeutics.
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