Previous grant support has led to the discovery of a novel Streptococcus parasanguis fimbriae associated adhesion, Fapl. Fapl-like molecules and genes involved in Fapl glycosylation are strikingly conserved throughout oral streptococci and are also found in important pathogens, such as Streptococcus pneumoniae and Staphylococcus aureus. We will use S. parasanguis as a model system to study the function of Fapl-like proteins in these pathogenic bacteria. Fapl is required for the formation of a S. parasanguis biofilm. We have determined that the N-terminal polypeptide of Fapl (rFapl) is essential for initial attachment of bacteria to the biofilm surface. Fapl is glycosylated, and glycosylation of this protein is involved in maturation of biofilm. The overall goal of this application is highlighted by two Specific Aims: (1) to determine the structure/function relationship of the rFapl adhesin that is required for initial bacterial attachment and (2) to define the functions of the fapl flanking genes in Fapl glycosylation and bacterial biofilm formation. We will bring genetic, structural biology (NMR spectroscopy and X-ray crystallography), in vitro binding biochemical assays and in vivo animal model studies together to address how the structure of the Fapl polypeptide influences its function. These comprehensive approaches should shed light on the structural basis for binding of Fapl to salivary receptors, and for predicted Fapl-like molecules of other medically important pathogens. Proteins of the fapl flanking region may constitute a coupled secretion and glycosylation pathway for Fapl. To begin to understand the mechanisms involved, we will determine the subcellular Iocalization of proteins in this locus and identify putative protein-protein interactions in vivo and in vitro. We previously demonstrated that Fapl glycosylation is involved in maturation of the biofilm. Using a combination of molecular and genetic analyses, we will dissect the function of these glycosylation genes in biofilm formation. Elucidation of the function of Fapl glycosylation and definition of the Fapl polypeptide structure/function relationships will contribute to the field of bacterial colonization in the oral environment and to the pathogenic mechanism of other important pathogens as their genomes indicate that they also possess Fapl-like molecules, and the flanking proteins.
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