Actinobacillus actinomycetemcomitans (Aa) has been implicated as the causative agent of Localized Aggressive Periodontitis (LAP), a severe form of periodontal disease that can lead to premature tooth loss in adolescents. Attachment of Aa to oral tissue is a pre-requisite for infection and has been shown to be mediated by the autotransporter protein, Aae. Preliminary data have shown that binding of Aa is specific for buccal epithelial cells (BECs) derived from humans and Old World monkeys, while binding to BECs derived from dog, rat, sheep, and New World monkey does not occur. To date, the aae gene responsible for specificity of Aa binding has not been fully characterized and its associated BEC receptor has not been identified. The goal of this application is to fully characterize the Aa/BEC adhesin-receptor interaction. The first Specific Aim of this application is to map the domain of the Aa adhesin (Aae) that confers adhesion specificity and accounts for binding to oral epithelium. To this end we will examine naturally occurring and genetically engineered variants of the aae autotransporter gene, and perform deletion analysis, random mutagenesis and site-directed mutagenesis of the gene.
The second Aim of this application is to identify the gene(s) responsible for the BEC receptor that interacts with the Aae adhesin. Biochemical and molecular approaches will use the adhesin-binding domain characterized in the first Specific Aim to identify its complementary receptor on human BECs. cDNA expression libraries will be constructed and used to transfect human epithelial cell lines (MKP A549) that are deficient in binding. Pools of A549 transformants will be probed with radiolabeled Aae adhesin to select clones that bind the adhesin so that receptor positive clones can be isolated and the gene (s) responsible for Aae receptor expression can be identified. The longterm goal of this application is to use this information to identify targets for therapeutic agents that can interfere with attachment of Aa to oral tissues and thus prevent colonization leading to LAP. ? ?
