Amylase, the most abundant enzyme in saliva, binds to several species of oral streptococci. Amylase-binding streptococci (ABS) are present in significant numbers in developing human dental plaque. Little is understood of the cariogenicity of ABS such as S. gordonii, despite their early colonization of teeth, substantial acidogenicity, and characteristic ability to bind to the major enzyme in saliva (amylase). It is possible that amylase in oral salivary pellicles serves as an adhesion receptor for ABS. Adherent streptococci may bind amylase from saliva and hydrolyze dietary starch into substrates that can be metabolized by them. Thus, amylase-binding may be crucial to the long- term colonization of these bacteria in the mouth. Once established, ABS may contribute to dental caries. Our recent data suggest that these organisms are cariogenic in a well-characterized rat model, especially in the presence of dietary sucrose. The long term goals of our research are therefore to define the structural basis for streptococcal amylase-binding and to determine its role in oral bacterial colonization and cariogenicity.
Two Specific Aims are proposed. We will: 1) construct stable mutations introduced into the amylase-binding protein A (abpA) gene, and assess the functional consequences of this mutation in in vitro models of microbial adhesion as well as in a well-characterized rat model (as described below). Further efforts will purify recombinant AbpA and assess the functionality of this molecule; 2) assess the ecological importance of amylase binding to well characterized laboratory strains and fresh isolates of S. gordonii by comparing the colonization and persistence abilities of amylase binding-negative (ab-) mutants and their genetically complemented revertants with their wild-type progenitors (ab+) in the well-characterized specific-pathogen free Osborne Mendel (SPFOM) model. These studies will be done under the influence of starch-, sucrose - and in some cases glucose-containing diets, while providing infectious challenges before and after the teeth erupt. With these studies we seek to understand the molecular basis for, and the ecological significance of, the interaction of amylase with early colonizing bacteria of the mouth. Such studies are crucial to better understand oral biofilm formation, which causes dental caries and periodontitis.
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