The goal of this revised application is to isolate and characterize new virulence genes from Porphyromonas gingivalis. The methodology (first described in Salmonella) is based on a novel approach termed IVET (in vivo expression technology) that facilitates for the positive selection and subsequent screening of bacterial genes that are induced in host tissues. The knowledge gained will broaden our understanding of the virulence mechanism(s) conferring pathogenicity on P. gingivalis. It will also contribute to the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease.
Specific Aim one will be to test the IVET vector constructed for P. gingivalis. A transcriptional fusion of a known P. gingivalis promoter with the promoterless chloramphenicol acetyltransferase (cat) and luciferase (luxAB) genes will be inserted in the P. gingivalis V2308 chromosome using homology provided by the promoter fragment. cat and luxAB expression will be tested in P. gingivalis, then in a mouse model. Luciferase activity will offer a sensitive real-time, non-invasive, non- radioactive reporter for measuring promoter activation.
Specific Aim two will identify and characterize, host-induced gene(s) isolated from P . gingivalis V2308. The promoterless cat-luxAB reporter genes will be randomly inserted in the chromosome of P. gingivalis using homology provided by cloned chromosomal fragments. Using a murine model, the library will be enriched and screened for strains that show promoter activation during the infection (chloramphenicol resistance) but not on laboratory medium (absence of luciferase activity). These strains would likely carry operon fusions that are important in host survival and virulence of P. gingivalis. The operon fusions will be recovered by marker rescue techniques. Partial sequencing of the region 5' to the reporter genes and knowledge of their genomic map location will facilitate the identification of those genes that maybe of greatest significance in P. gingivalis virulence. These genes will be clones in E. coli and further characterized. From the host-induced genes isolated in the study, we anticipate the discovery of new virulence genes which will launch an independent extramurally funded research program.
