Role of cytolethal distending toxin in periodontitis

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DESCRIPTION (provided by applicant): As one of the most common bacterial diseases of humans, periodontal disease afflicts as much as 50% of the adult population and results in destruction of the connective tissues and bone that anchor the teeth. Aggregatibacter (Actinobacillus) actinomycetemcomitans (Aa) is an oral bacterium that is associated with various forms of human periodontal diseases. Interestingly, the factors produced by Aa that are involved in virulence appear to target host immune cells rather than the tissues that support the teeth. We have identified a toxin in Aa that is related to the E. coli cytolethal distending toxin (Cdt), but differs by targeting lymphocytes rather than epithelial cells. Our hypothesis is that the Aa Cdt causes immunosuppression, thus allowing Aa to successfully colonize the human gingiva in high numbers. However, the specific role that Cdt plays in the pathogenesis of human periodontal disease remains to be determined. Our preliminary data and data from the closely related H. ducreyi Cdt on macrophages and dendritic cells suggest that the toxin induces cell death in non-proliferating immune cells. These data suggest that cell cycle progression is not required for cytotoxicity and that the mechanism of Cdt action may be more complicated than previously thought. The goal of this study is to understand if Cdt contributes to periodontal disease by targeting non- proliferating immune cells, and to determine if the toxin represents a potential therapeutic target for intervention against human periodontal diseases. In our first Aim, we will determine if the mechanism of Cdt intoxication is similar for non-proliferating immune cells and epithelial cells. This will be accomplished by measuring lethality, binding, internalization, and DMA damage to non-proliferating macrophages, dendritic cells, and lymphocytes compared to proliferating lymphocytes and epithelial cells. In our second Aim, we will generate a mutant of Aa that is unable to express Cdt in order to assess the virulence of this organism using a mouse model of periodontitis. Mice will be infected orally with either normal or Cdt-defective Aa organisms and the degree of alveolar bone loss supporting the teeth, number of bacteria, and viability of infiltrating immune cells will be measured. The successful demonstration of an in vivo role for this toxin in periodontal disease will confirm a role for Cdt in Aa infections. Understanding the biological role of Cdt and its mechanism of action will allow us to investigate it as a new potential target for therapeutic intervention of periodontal disease, thus providing an alternative to the use of antibiotics. Furthermore, compounds that inhibit immunosuppression caused by Cdt may also have utility to increase immune function in other immunosuppressive diseases.