Study on the Mechanism of Streptococcal Pyrogenic Exotoxin B-Caused Defective Efferocytosis for Modulating Pro-Inflammatory Responses and Immune Evasion in Group a Streptococcal Infection

Group A Streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases highly associated with severe inflammation. Among the virulence factors, streptococcal pyrogenic exotoxin B (SPE B) is a critical virulence factor in association with the toxic shock syndrome and mortality. Our unpublished results had first revealed that SPE B caused a disruption of macrophage-mediated clearance of apoptotic cells, called efferocytosis, through protein S (a bridging protein) degradation, and mediated further lost bindings of C4b-binding protein (C4BP, an inhibitor of complement) to apoptotic cells. The complex of C4BP-protein S deposited on the surface of apoptotic cells prevents complement-mediated apoptotic cell lysis before efferocytosis, which further initiates anti-inflammatory responses. To date, there are still no well-characterized mechanisms to explore the relationship of efferocytosis to immunopathogenesis of GAS infection. Therefore, the SPE B-caused defective efferocytosis via protein S degradation modulates pro-inflammatory responses and GAS immune evasion that are hypothesized. In Specific Aim 1, we will determine the interaction of protein S and C4BP in GAS infection. Our supporting data showed that SPE B might initially recognize and cleave the C-terminal region of protein S leading to the lost binding of C4BP to apoptotic cells. Accordingly, we will verify the interaction of protein S and C4BP in serum cultured with recombinant SPE B, C192S (SPE B mutant), and SPE A, or with different strains of GAS. In Specific Aim 2, we will verify the role of SPE B involved in a disruption of C4BP binding to apoptotic cells, which causes complement-mediated apoptotic cell lysis. Our results indicated the cleavage of protein S by SPE B decreased the binding ability of C4BP to apoptotic cells, which might destroy the protective effect of C4BP on apoptotic cells from complement attack. Therefore, the presence of SPE B accelerates complement-mediated apoptotic cell lysis that will be studied. In Specific Aim 3, we will investigate the involvement of non-removed apoptotic cells in competition for C4BP binding. C4BP had been demonstrated to bind to GAS surface, and apoptotic cells as well. We then speculate that apoptotic cells may exert the competitive binding ability with C4BP, whereas SPE B can disrupt the binding of apoptotic cells and C4BP that lead more C4BP selectively binding to GAS. In Specific Aim 4, we will study the defective efferocytosis prompts pro-inflammatory responses during GAS infection. Efferocytosis can effectively initiate anti-inflammatory responses (IL-10 stimulation), while the accumulation of non-removed apoptotic cells trigger inflammation resulted from secondary necrosis-associated signaling of damage-associated molecular patterns. Our supporting data showed that SPE B caused inhibitory effects on serum- and protein S-mediated phagocytosis. Accordingly, the effects of SPE B, C192S, and different strains of GAS in regulating efferocytosis and the subsequent cytokines expression will be next investigated. In Specific Aim 5, we will determine the protein S binding motif of SPE B for further potential anti-inflammatory agent development. According to the potential recognition sites of protein S by SPE B, the specific binding motif of protein S to SPE B will be determined. Furthermore, the synthetic peptides that block the interaction of SPE B and protein S will also be verified in SPE B-mediated protein S degradation, efferocytosis disruption, apoptotic cell lysis, and cytokine production. Taken together, this project will help to gain better understanding and provide potential anti-inflammatory strategies in GAS infection.