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

本研究首先检测了早期在牙齿表面定植的各种口腔链球菌细胞表面的甘油醛-3-磷酸脱氢酶（GAPDH）活性，以及它们与牙龈卟啉单胞菌的共聚集活性。口腔链球菌细胞表面GAPDH活性与共聚集活性密切相关，因此，我们从5株具有较强细胞表面GAPDH和共聚集活性的口腔链球菌中纯化细胞表面GAPDH，并通过生物分子相互作用分析系统（BIAcore）分析它们与牙龈卟啉菌菌毛的相互作用。GAPDHs与牙龈假单胞菌菌毛具有较高的亲和性，DNA序列具有较高的同源性（95%以上）。接下来，我们构建并纯化了口腔链球菌ATCC 9811重组GAPDH （rGAPDH）。rGAPDH经多种蛋白酶消化后，应用反相高效液相色谱法测定。采集各峰，用BIAcore软件测定各峰牙龈卟啉菌毛的结合能力。利用质谱联用仪和氨基酸分析仪测定了具有高结合亲和力的部分的氨基酸序列。rGAPDH的结合域存在于牙龈假单胞菌菌毛的氨基酸残基166 ~ 183。该结构域对应的合成肽抑制牙龈假单胞菌与口腔链球菌的共聚集。此外，我们开发了一种利用戈登链球菌质粒整合文库筛选异型生物膜的新方法，并研究了支持与牙龈卟啉单胞菌异型生物膜群落所需的戈登链球菌基因。我们发现S.gordonii通过多种遗传途径控制着异型口腔生物膜的发育。

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.