Elucidation of molecular mechanism of binding between periodontopathic bacteria and oral streptococci, and development of agents which inhibit forming of dental biofilm

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In this study, firstly, we examined glyceraldehydes-3-phosphate dehydrogenase (GAPDH) activities on cell surfaces of various oral streptococci which colonize on the tooth surface in the early stage, and their coaggregation activities with Porphyromonas gingivalis. The GAPDH activity on the cell surface of oral streptococci was correlated strongly with the coaggregation activity, therefore, we purified cell surface GAPDHs from five strains of oral streptococci which showed strong cell surface GAPDH and coaggregation activities, and analyzed their interactions with P. gingivalis fimbriae via a biomolecular interaction analysis system (BIAcore). The GAPDHs demonstrated high affinity with P. gingivalis fimbriae and their DNA sequences possessed high degree of homology with one another (more than 95%). Next, we constructed and purified Streptococcus oralis ATCC 9811 recombinant GAPDH (rGAPDH). rGAPDH was digested with various proteinases and applied to reverse-phase HPLC. Peaks were collected and P. gingivalis fimbriae-binding ability of each peak was measured by BIAcore. Amino acid sequence of the fraction which exhibited high binding affinity was determined with mass spectrometer and amino acid analyzer. The binding domain of rGAPDH for P.gingivalis fimbriae existed in amino acid residues 166-183. The synthetic peptide corresponding to the domain inhibited the coaggregation between P.gingivalis and oral streptococci.Moreover, we developed a new method for screening of heterotypic biofilms with a plasmid integration library of Streptococcus gordonii and investigated genes of S.gordonii required to support a heterotypic biofilm community with P.gingivalis. We found S.gordonii governed the development of heterotypic oral biofilms through multiple genetic pathways.