Role of sialoglycan binding in the pathogenesis of streptococcal endocarditis

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PROJECT SUMMARY The oral streptococci are a major cause of infective endocarditis (IE). Several of these species express Siglec- like, serine-rich repeat (SRR) adhesins that bind a variety of O-linked sialoglycans on human glycoproteins and cells. Expression of the SRR adhesin GspB of Streptococcus gordonii M99 results in increased virulence in animal models of IE. This enhanced pathogenicity is thought to be due the interaction of GspB with the trisaccharide sialyl-T antigen (sTa, a core 1 sialoglycan) on the platelet receptor GPIb, leading to increased attachment of bacteria in the blood stream to cardiac valve surfaces and vegetation formation. Isogenic mutants of M99 expressing GspB variants that preferentially bind core 2 sialoglycans,(e.g., sialyl-lactosamine) are less virulent, as compared with the WT strain. These findings indicate that differences in the type of sialoglycan bound ("selectivity") affect the ability of organisms to both initiate and propagate endocardial infection, with some interactions enhancing pathogenesis, while other interactions reducing disease. We hypothesize that in vivo, the binding of streptococci in the bloodstream to sTa on platelets or cardiac surfaces helps initiate infection, and that this binding is enhanced by hemodynamic effects created by high shear flow. In contrast, binding to core 2 sialoglycans on blood cells may lead to increased bacterial clearance, thereby attenuating virulence. Subsequent interactions of bacteria on valves with core 1 versus core 2 sialoglycans may affect disease progression via different effects on platelet activation, clotting, or endothelial healing. To further address the roles of sialoglycan binding selectivity in the pathogenesis of IE, we will compare by flow the above isogenic strains for their binding to platelets, RBCs and WBCs in vitro. We will also examine whether these strains bind platelets that differ in GPIb sialoglycan content, and whether binding to sTa versus core 2 structures affects platelet activation. In addition, we will assess how selectivity impacts the attachment of bacteria to damaged cardiac surfaces, as modeled by microfluidic chambers lined with immobilized platelets, von Willebrand Factor, collagen or activated human vascular endothelial cells, and the effect of hemodynamic forces on binding. We will also explore the impact of selectivity on vegetation formation by assessing strain differences in triggering platelet contractility and effects on platelet-dependent endothelial healing. We will also compare vegetations produced in vivo by these strains for key features linked to virulence, including co- localization with platelet and biofilm production. These studies will provide novel insights as to how sialoglycan selectivity and hemodynamic forces affect the initiation and propagation of endocardial infection. By defining the mechanisms for these events, this research will redefine our understanding of the key steps in the pathogenesis of streptococcal IE. These concepts and findings will be broadly applicable to other endovascular pathogens, and will provide novel insights into microbial interactions with host glycans.