Biomolecular analysis on iron acquisition mechanism and pathogenicity of periodontopathic bacteria

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Iron is an essential nutrient for periodontopathic bacteria as well as it is for most living organisms. Our previous study showed that Porphyromonas gignivalis utilized hemoglobin as an effective iron source and that its hemoglobin receptor was identical to the catalytic domain of lysine-specific cysteine proteinase (KGP). In this study, we first investigated the effects of hemoglobin on the growth of various periodontopathic bacteria. Actinobacillus actinomycetemcomitance, Prevotella intermedia and Prevotella nigrescence effectively utilized hemoglobin as an iron source. 65 kDa outer membrane protein of A. acdnomycetemoomitans, 60 kDa of P.intermedia and 41, 56 and 59 kDa of P.nigrescens were identified as hemoglobin-binding proteins. Next, the active domain of KGP showing hemoglobin-binding activity was investigated with KGP fragments, which were prepared based on the DNA sequence. It was found that amino-terminal portion in catalytic domain of P.gingivalis KGP showed strong -hemoglobin-binding activity by dot blot assay. KGP is one of major proteases of P.gingivais and it is believed to play a critical role in the pathogenesis of P.gingivalis. Therefore, we constructed DNA vaccine targeted for P.gingivalis KGP and evaluated the effect of the DNA vaccine as an immunogen against P.gingivalis challenge in a murine model. The DNA vaccine was injected into the quadriceps muscles weekly for a total of four inoculations. Following immunization, specific IgG antibodies in serum were clearly induced. Immunization of the DNA vaccine conferred a significant amount of protection against P gingivalis lethal challenge compared with that in control animals : These results suggest that KGP acts as a hemoglobin-binding protein and can also be useful as an immunogen to induce a protective response to P.gingivalis infection