Project Summary: Adhesion is a critical step in development of infective endocarditis (IE). However, despite Streptococcus oralis being a common cause of sub-acute IE there are no defined adhesion mechanisms for this species. Ultimately, filling this gap in knowledge may lead to strategies to reduce the burden of IE. Other bacterial species bind terminal carbohydrates or glycosidase exposed cryptic carbohydrates. Preliminary data suggest that in order to maintain adhesion to host surfaces, despite production of a glycosidase that cleaves the terminal receptor, S. oralis has adopted a novel strategy that simultaneously uses both terminal and cryptic receptors. These data show that S. oralis binds platelets via terminal sialic acid and underlying β-1,4 linked galactose, which is only exposed upon cleavage of terminal sialic acid by S. oralis neuraminidase. Given the broad distribution of these carbohydrates, it is likely that this adhesion mechanism contributes to S. oralis binding to multiple IE-relevant host components and that these interactions are critical to disease development. This strategy of simultaneously binding terminal and cryptic receptors is likely used by other glycosidase positive species and by glycosidase negative species during polymicrobial infections. Adhesion to both carbohydrates requires a serine rich repeat protein Fap1. This is the first example of a serine rich repeat protein required to bind β-1,4 linked galactose and two distinct carbohydrate receptors. These data lead to the hypothesis that in order for neuraminidase producing S. oralis to maintain adhesion to host surfaces during development of IE, this species employs a novel strategy of simultaneously binding terminal sialic acid and neuraminidase exposed β-1,4 linked galactose and that the SRRP Fap1 directly binds these carbohydrates. Three Aims will test this hypothesis. Aim 1: Determine the ability of Fap1 to directly bind sialic acid and β-1,4 linked galactose. Preliminary data lead us to hypothesize that Fap1 directly binds sialic acid and β-1,4 linked galactose. Studies will further define the role of Fap1 in adhesion by determining the ability of this adhesin to directly bind these carbohydrates, the specific carbohydrate structures bound and the strength of these interactions. Aim 2: Determine the role of Fap1 in binding other IE relevant host components. Most preliminary data was generated using platelets; however, adhesion to other host components on the damaged valve is also likely important in pathogenesis. Given that glycan structures containing sialic acid and β-1,4 linked galactose are common on host cells and proteins, this aim will test the hypothesis that Fap1-dependent adhesion to these carbohydrates is a conserved mechanism for binding host components present on damaged valves. Aim 3: Determine the contribution of Fap1 to IE using a 3D microvessel model. A cutting edge 3D microvessel model will be adapted to study the contribution of Fap1- mediated adhesion to pathogenesis. Vegetation development of the parent and fap1 mutant will be compared in real-time and the impact of modifying the glycan present on different components of the system determined.

Project Narrative The proposed research is relevant to public health because defining pathogenic mechanisms of Streptococcus oralis is ultimately expected to lead to the development of more effective prognostic, diagnostic, preventative or therapeutic measures. Hence, the proposed research is relevant to the part of the NIH’s mission which pertains to gaining fundamental knowledge that will enhance health and reduce illness.