Studies on virulence regulation in Porphyromonas

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Porphyromonas gingivalis, a black-pigmented, gram- negative anaerobe, is widely implicated as an important etiological agent of periodontal disease. This bacterium expresses several potential virulence factors (e.g., capsule, LPS, fimbriae, membrane vesicles, and hydrolytic enzymes) that may contribute to its pathogenicity. Another virulence factor, the recA gene, confers resistance to the oxidative stress environment of the inflammatory periodontal pocket. The recA gene product is a key protein in DNA repair that protects P. gingivalis from DNA damage induced by bactericidal reactive oxygen derivatives generated in the periodontal pocket by neutrophils and transient air exposure. Our laboratory has identified two genes, vimA and bcp, that may be part of the recA transcription unit and may also function in virulence. Further, the vimA-mediated virulence modulation in P. gingivalis, may represent a novel posttranscriptional regulation of virulence factors in this organism. Because the BCP homologue may have peroxidase function, and gingipains are involved in heme accumulation which can inactivate H2O2, it might be considered an important strategy for the organism to coordinate its oxidative stress and proteolytic activities. This importance is further supported by observation that the recA locus promoter is active during infection of the murine host. Moreover, the promoter activity is affected by temperature, iron and calcium which are factors known to coordinately regulate the expression of other bacterial virulence genes. Our observations, taken together, may suggest an important role for the complex recA locus in the survival and virulence of P. gingivalis. It is our hypothesis that the bcp-recA-vimA transcriptional unit is important for virulence and protection against oxidative stress. Our overall objective is to elucidate the molecular mechanism(s) for the vimA-mediated virulence regulation and examine the relative importance of the bcp-recA-vimA operon in oxidative stress resistance in P. gingivalis. Specific aims for the proposed research are: 1) To characterize the bcp-recA-vimA transcriptional unit in P. gingivalis W83. This will include: a) mapping the transcription initiation site; b) verifying the promoter sequence upstream of the primary start site; c) evaluating the effect of the bcp gene on the function on the recA and vimA genes; 2) To examine the functional significance of the vimA mutation on protease activation in P. gingivalis W83; and 3) To evaluate the importance of the bcp-recA-vimA transcriptional unit in oxidative stress protection.