The long term objective of this proposal is to determine the molecular mechanisms of periodontal disease pathogenesis by Bacteroides forsythus. Although B. forsythus has been implicated as an important periodontal pathogen, especially in the early stages of disease, very little is known about its virulence factors. B. forsythus possesses two enzyme activities which are known to be virulence factors in other pathogens; a """"""""trypsin-like"""""""" protease and a sialidase. New data is presented characterizing the protease as a 79 kDa cell envelope-associated arginine-X cleaving serine protease. The protease has been partially purified and peptide fragments for sequencing have been generated. Additional new data shows that B. forsythus has a gene homologous to a probe derived from the sialidase gene of Bacteroides fragilis. The studies proposed will address the hypothesis that the trypsin-like serine protease and sialidase activities of B. forsythus are virulence factors, possibly targeted at host cell-surface glycoproteins. The experimental design will be to first isolate clones of the two genes by screening a B. forsythus genomic library (Specific Aims 1 and 3). The cloned genes will be sequences and expressed in E. coli. The substrates specificities of the enzymes will be tested with a range of natural proteins and glycoproteins to determine their potential host targets (Specific Aims 2 and 4). Inactivated mutants of the two genes will be constructed in vitro and introduced into B. forsythus recipients using IncP plasmid-based suicide vectors to produce protease-negative and sialidase-negative single, isogenic mutants by allelic exchanges (Specific Aim 5). The mutants will permit the testing in animal models of the hypothesis that the protease and sialidase are important virulence factors of B. forsythus. The genetic methods developed will be important in future studies of other B. forsythus virulence factors, and their role in pathogenesis. The results of this study will contribute to improvements in oral health by increasing our knowledge and understanding of the mechanisms of periodontal disease pathogenesis. Understanding these mechanisms will be vital for developing improved preventative and therapeutic treatments for periodontal disease.
