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

在本研究中，我们首先检测了早期定植于牙面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。因此，我们从5株细胞表面GAPDH和共聚集活性较强的口腔链球菌中分离纯化了细胞表面GAPDH，并利用生物分子相互作用分析系统(Biacore)分析了它们与牙龈假单胞菌菌毛的相互作用。GAPDHs与牙龈假单胞菌菌毛具有很高的亲和力，它们的DNA序列具有高度的同源性(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)。用不同的酶消化重组人谷氨酸脱氢酶，并应用于反相高效液相色谱。收集各峰，用Biacore法测定各峰的菌毛结合能力。用质谱仪和氨基酸分析仪测定了具有高结合亲和力的组份的氨基酸序列。牙龈假单胞菌菌毛的rGAPDH结合区存在于166-183个氨基酸残基。此外，我们建立了一种新的方法，利用戈登链球菌质粒整合文库筛选异型生物被膜，并研究了支持与牙龈假单胞菌形成异型生物被膜群落所需的基因。我们发现，戈登葡萄球菌通过多条遗传途径控制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.