MOLECULAR GENETIC STUDIES OF PORPHYROMONAS GINGIVALIS PROTEASES

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Porphyromonas gingivalis has been implicated as a contributing etiologic agent of adult periodontitis. It is epidemiologically associated with adult periodontitis in specific patient populations and it can induce periodontitis in an experimental animal model. There is growing evidence that several putative virulence factors (e.g., capsule, adhesion, membrane vesicles, and hydrolytic enzymes) may contribute to its pathogenicity. In general, proteases of P. gingivalis are likely to function as virulence factors which destroy soft and hard connective tissue as well as key plasma proteins that would otherwise mediate protective host functions. In preliminary work, we have successfully cloned in E. coli a P. gingivalis gene that specifies an enzyme which hydrolyzes the C3 complement protein. Our immediate objective is to characterize this determinant genetically and to specifically test the role of this protease in the virulence of P. gingivalis infection. Specific aims during the proposed funding period include: 1) Characterization of the cloned P. gingivalis C3 protease gene. This will include: subcloning and characterization of the gene in E. coli, the determination of the complete nucleotide sequence, a systematic study of its expression in P. gingivalis, and comparison of this gene to other protease genes cloned from P. gingivalis; 2) A comparative examination of clinical isolates of P. gingivalis using the C3 protease gene as a probe to evaluate gene polymorphisms in strains isolated from stable and progressive disease sites of periodontitis patients; 3) The employment of allelic exchange mutagenesis to construct mutants carrying the defective C3 protease gene. These mutants will be characterized genetically, biochemically and immunologically. They will be evaluated in an in vivo rodent model; and, 4) Generation of a genomic map of all the protease genes that have been cloned from P. gingivalis in order to establish baseline information on genetic organization and to help in setting the stage for studies aimed at exploring the potential regulation of virulence genes in P. gingivalis. Our long term objective is to gain a comprehensive understanding of the precise role of proteases in the pathogenicity of P. gingivalis and from the knowledge, develop strategies to aid in the control and prevention of periodontitis.