We propose to study the role of Actinobacillus actinomycetemcomitans as an etiologic agent in the development and progression of periodontal disease. This microorganism synthesizes a leukotoxin (LT) which may act as a unique virulence factor in the pathogenicity of the microorganism. The LT has been shown to kill polymorphonuclear leukocytes and monocytes from humans and some nonhuman primates (i.e. Macaca fascicularis) but not other mammalian species. Therefore, we propose to examine the critical nature of this virulence factor in experimental periodontal disease in M. fascicularis. The project will incorporate three major areas of studies on the LT: 1) Molecular biological identification of the LT gene, including: purification and sequencing of the protein, analysis of the gene (Tox) in wild type Tox+ and Tox- A. actinomycetemcomitans, and transformation experiments to produce isogenic recombinant Tox+ and Tox A. actinomycetemcomitans; 2) Immunological (antibody) approaches to investigating the structure/function of LT using production of mouse polyclonal and monoclonal antibodies and nonhuman primate antibodies to LT for examination of: binding domains on LT, avidity of antibody, diversity of antibody, and functional inhibition of LT; and, 3) Immunological approaches to defining the critical importance of LT in the virulence (periodontal disease in M. fascicularis) of A. actinomycetemcomitans by: elicitation of elevated anti-LT antibody in nonhuman primates, immunotoxin targeting and elimination of specific anti-LT B lymphocytes. These studies have been designed to incorporate the most recent biological technologies in order to initiate the next phase in periodontal research; definition of the critical virulence mechanism of periodontopathogens. The results from these investigations will address a specific hypothesis concerning the importance of the LT in the virulence of A. actinomycetemocomitans. In addition, the studies will provide the basis for defining the protective role of antibodies in periodontal disease, and thereby promote experiments to utilize this protective principle for a specific directed attack on the pathogenic microorganisms.
