ISOLATION AND TESTING OF P GINGIVALIS VIRULENCE GENES

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The long range goal of this revised application is to isolate new virulence genes from Porphyromonas gingivaIis and to characterize them along with previously isolated genes. The results are expected to improve our general understanding of the pathogenic mechanisms employed by this organism in periodontal disease causation. The application has three specific aims. The first is entitled "Construction and Use of a Reporter Gene to Isolate and Characterize P. gingivalis Virulence Genes". A promoterless reporter gene encoding tetracycline resistance will be randomly inserted into the P. gingivalis chromosome using homology provided by cloned chromosomal fragments. The resulting library will be enriched and screened for clones which express the reporter gene function during infection in the murine abscess model but which do not express it during laboratory cultivation. Such clones win likely be labeled with the reporter gene in genes that are important to the survival and virulence of P. gingivalis during the infectious process. In Specific Aim 2, entitled "Cloning and Genetic Characterization of Reporter Gene-Labeled Virulence Genes", the labeled genes will be recovered by marker rescue techniques. Partial sequencing of the 5 prime ends of the genes will enable us to determine those likely to be of greatest importance with regard to P. gingivalis pathogenicity. The sequencing data will also provide us with the necessary information to synthesize probes which can be used to clone the entire P. gingivalis gene into Escherichia coli. Isogeneic mutants of selected virulence genes will be constructed using insertional inactivation methods. The pathogenic potential of these mutants will be compared to their parent using the murine abscess model and, in certain cases, the gnotobiotic rat model. Specific Aim 3 of the application is entitled "Genetic and Biochemical Characterization of P. gingivaIis Surface Antigens and Virulence Factors". The virulence genes isolated in Specific Aim 2 win be genetically and biochemically characterized. In addition, we will perform the same characterizations on previously cloned P. gingivalis genes encoding hemagglutinin and two other unidentified putative surface antigens. Their role as putative virulence factors will be tested by installing them in the reporter gene construct developed in Specific Aim l and examining their expression during infection in the murine abscess model.