The goal of this study is to investigate the specific molecular interactions which determine adhesion in the oral cavity. The ability of a microorganism to adhere to a host surface is an essential prerequisite for successful colonization and infection. Although it is recognized that the surface properties of bacteria may ultimately determine the outcome of host-parasite interactions, the precise nature of the bacterial surface components mediating these interactions remains largely obscure. In some cases, bacterial surface appendages such as fimbriae or fibrillae have been identified. In other instances, bacteria have been shown to contain binding proteins or adhesins on their cell surface, and a lectin-like mechanism of attachment has been proposed. Our general research aims continue to be the identification and characterization of microbial surface proteins involved in bacterial attachment. We have prepared a large number of well-characterized glycopeptides as structural analogues of salivary glycoproteins. These reagents will be valuable tools in many aspects of this project (Specific Aims 1-5). The present proposal expands our earlier studies with particular emphasis on the purification and characterization of the bacterial lectins recently identified. Specifically our aims are to: (1) Screen oral bacteria for lectin-like properties. We will continue to systematically screen many oral bacterial strains to identify binding sites for sialic acid, galactose, N-acetylglucosamine, and fucose using glycopeptide analogues containing the appropriate terminal sugar. Whole cell chromatography and direct binding assays will be employed as techniques to accomplish this aim. (2) Identify the oligosaccharide determinants of salivary glycoproteins involved in specific interactions with oral bacteria. The identified determinants will be used as probes for lectin purification and characterization. (3) Characterize microbial lectins on the basis of size by SDS-PAGE. We will use 125I- photoactivatable glycopeptide reagents (125I-PAGP) and protein blotting techniques for this purpose. (4) Purify microbial lectins involved in interactions with salivary glycoproteins. We will initially focus on completing our studies with the sialic acid lectins of S. sanguis and S. mitis, then purify the galactose binding lectin from F. nucleatum. Finally, we will begin purification of newly identified lectins. (5) Prepare polyclonal and monoclonal antibodies to lectin preparations and purified lectin(s). These antibodies will be used as immunological reagents to purify and characterize identified lectins. (6) Characterize purified lectins. We will investigate the following parameters: size, subunits, compositional analysis, structural analysis and specificity. The purification and characterization of these bacterial surface proteins provide ultimate proof of their role in attachment and a basis for a more complete understanding of host-parasite relationships.
