CELL ADHERENCE OF DENTAL PLAQUE FORMING STREPTOCOCCI

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The studies described in this project are designed to examine events occurring during the early stages of plaque formation by Streptococcus mutans (Streptococcus sobrinus), particularly the events mediating sucrose-dependent cell adherence and colonization. Enzymes and other proteins involved in the formation of glucans and the specific adherence of S. mutans cells to these glucans are being purified and characterized. Emphasis has been placed upon a few proteins considered to be of key importance in early plaque formation: 1. A primer-independent glucosyltransferase (GTF-Si) may contribute the priming dextran needed by other glucosyltransferases (GTF) for the synthesis of the plaque matrix. GTF-Si as well as the primer-dependent GTF will be purified to homogeneity by immunoaffinity chromatography and their individual roles in the early stages of glucan synthesis determined in recombination experiments utilizing radiolabeled substrates. 2. Streptococcus mutans releases both dextranase and a reversible inhibitor of dextranase to the extracellular milieu. The inhibitor and dextranase may mediate the levels of primer dextran present in early colonies of S. mutans, thus regulating the nature of the extracellular produced products by primer-dependent GTF. Kinetic studies of the mechanism of inhibitor action on dextranase function will be conducted using reversed phase HPLC to monitor the effects of inhibition on hydrolysis of low MW substrates by dextran and HPLC size exclusion chromatography to analyze the kinetics of inhibitor-enzyme complex formation and reversal. 3. A dextran- binding enzyme produced by S. mutans forms branches on linear dextrans in the absence of sucrose, thus converting them to more- efficient primers for the GTF enzymes, rendering them resistant to dextranase action and contributing to the complexity of the water-insoluble glucans. Kinetic studies of this enzyme will be conducted using reversed phase HPLC capable of detecting early reaction products. Studies of glucan binding by the GTF enzymes will be directed to a comparison of binding characteristics with those known for the major glucan binding protein (GBP#1). Panels of monoclonal antibodies to the GTF, dextranase, inhibitor, branching enzyme and GBP#1 will be utilized to isolated those proteins, in studies of extracellular and cell- associated protein complements, and for comparisons of the structures of those proteins.