GENETIC AND BIOCHEMICAL STUDY ABOUT FUNCTION AND SECRETION MECHANISM OF EXTRACELLULAR PROTEINASES OF PERIODONTOPATHOGENIC MICROORGANISMS

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1. We constructed RGP-deficient mutants of Porphyromonas gingivalis via disruption of the RGP gene by use of suicide plasmid systems. In the course of construction, we found that two different RGP genes existed on the chromosome of P.gingivalis. The RGP-null (rgpA rgpB double) mutant showed deficiency in the ability to adhere to erythrocyte and the inhibition of bactericidal action of PMN,indicating that RGP is one of the major virulence factors of P.gingivalis.2. We investigated the role of RGP in the formation of P.gingivalis fimbriae by use of the rgp mutants. The RGP-null mutant possessed very few finbriae. Immunoblot analysis using antifimbrilin suggests that RGP may be involved in processing of the fimbrilin.3. We cloned and sequenced the second RGP gene. The gene (rgpB) encoded 736 amino acids. Comparison between rgpA and rgpB revealed that rgpB did not possess most of the hemagglutinin domain, and suggested that gene conversion might have occurred between their proteinase domain regions. The results provide one of the hypotherical scenarios of generation of the two rgp genes that they have been generated through duplication of an ancestor rgp gene, insertion of the hemagglutinin domain region into one copy of the resulting two rgp genes (or deletion of the region from one rgp), and homologous recombination between the proteinase domain regions of the two rgp genes.