We will examine factors influencing bacterial adherence to cementum and enamel in vitro. Our experimental strategy will focus on two steps that are crucial to understanding this complicated process. The first step is the selective adsorption of salivary and plasma components to cementum and enamel, a process that forms the pellicle substrate to which bacteria adhere. The second step is the adherence of bacteria to the pellicle. the proposed experimental strategy is as follows: First, we will characterize the mechanisms by which prominent pellicle glycoproteins purified from saliva and plasma adhere to human enamel and cementum by: a) using various enzymatic and chemical deglycosylation methods to isolate oligosaccharides and protein cores from purified proteins; b) using external radiolabeling techniques to study the composition of enamel and cementum pellicles formed from these fragments to determine whether adherence is mediated by the carbohydrate portions of purified glycoproteins, the protein portion, or both; and c) using liquid secondary ion mass spectrometry (LSIMS) to determine the sequence of the protein and/or carbohydrate portions of a pellicle glycoprotein that promote its adherence. Second, we will profile the bacterial binding characteristics of salivary and plasma proteins and glycoproteins by: a) identifying proteins that function as bacterial receptors by incubating nitrocellulose blots of electrophoretically separated proteins and glycoproteins with radiolabeled bacteria; b) characterizing the structural specificity of the bacteria-receptor interactions by including competitive inhibitors (e.g. oligosaccharides or peptides) in the incubation medium; and c) using LSIMS to determine the structure of the recognition sequence that specifies bacterial binding to the protein receptor. Third, we will profile the bacterial binding characteristics of salivary and plasma lipids by: a) identifying which components can function as bacterial receptors by incubating thin layer plates of chromatographically separated lipids with radiolabeled bacteria; b) characterizing the structural specificity of the bacteria-receptor interactions by including competitive inhibitors in the incubation medium and by screening on lipid standards of known structure; and c) using LSIMS to determine the structure of the recognition sequence that specifies bacterial binding to the lipid receptor. Fourth, we will use an in vitro adherence model in which radiolabeled bacteria are incubated with coated human enamel and cementum slabs to assess the biological significance of these studies by: a) determining which bacteria bind to pellicles formed from purified pellicle proteins as described in Aim 1; and b) determining whether the bacterial receptor activities identified using methods described in Aims 2 and 3 are retained when the same receptor molecules are coated onto cementum and enamel slabs. The results of these studies should yield important information concerning the structural basis for the bi-functional role of major pellicle components, which both bind to tooth surfaces and serve as bacterial receptors.
