Elucidation of molecular mechanism on periodontal bacterial adherence, invasion and signal transduction to human cell

在这项研究中，我们证明了人甘油醛-3-磷酸脱氢酶（GAPDH）和牙龈卟啉单胞菌fimhrise之间的相互作用是特异性和高亲和力，通过生物分子相互作用分析系统（BIAcore）。用各种蛋白酶如赖氨酰内肽酶消化人GAPDH，并通过HPLC收集片段。通过BIAcore测量每个GAPDH片段与牙龈卟啉单胞菌菌毛的结合活性，并且用质谱仪和氨基酸测序仪分析显示最强结合活性的片段。由于发现牙龈卟啉单胞菌菌毛的结合结构域存在于人GAPDH的165 - 185位氨基酸残基，因此我们合成了人GAPDH的165 - 185位氨基酸残基的多肽，并利用共聚焦显微镜检测了该多肽对牙龈卟啉单胞菌粘附和侵袭人牙龈成纤维细胞的抑制作用。该合成肽以剂量依赖性方式抑制牙龈卟啉单胞菌对人成纤维细胞的粘附和侵袭。接下来，我们通过与上述相同的方法鉴定了口腔链球菌GAPDH对牙龈卟啉单胞菌菌毛的结合结构域，并发现口腔链球菌GPADH的菌毛结合结构域与人GAPDH的菌毛结合结构域存在于相同的区域。因此，我们研究了牙龈卟啉单胞菌菌毛结合区的合成肽对牙龈卟啉单胞菌与链球菌形成生物膜的抑制作用。口腔该肽还抑制牙龈卟啉单胞菌S。结果表明，合成的牙龈卟啉单胞菌菌毛结合区多肽抑制牙龈卟啉单胞菌生物膜的形成和牙龈卟啉单胞菌对人成纤维细胞的粘附和侵袭，提示该多肽可能是一种有效的预防牙周炎的药物。

In this study, we demonstrated that interaction between human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Porphyromonas gingivalis fimhrise was specific and high affinity via a biomolecolar interaction analysis system (BIAcore). Human GAPDH was digested with various proteinasas such as lysylendopeptidase and fragments were collected by HPLC. Binding activity of each GAPDH fragment to P. gingivalis fimbriae was measured via BIAcore, and the fragment which demonstrated the strongest binding activity was analyzed with mass spectrometer and amino acid sequencer. Since it was found that the binding domain for P. gingivalis fimbriae existed in amino acid residues 165-185 of human GAPDH, we synthesized the peptide corresponding to amino acid residues 165-185 of human GAPDH, and examined the inhibitory effect of the peptide on P. gingivalis adherence and invasion to human gingival fibroblast with confocal microscpe. The synthetic peptide inhibited P. gingivalis adherence and invasion to human fibroblast in a dose-dependent manner. Next, we identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae by the same method as we described above, and found that the fimbrial binding domain of S oralis GPADH existed in the same region as that of human GAPDH. Therefore, we investigated the inhibitory effect of the synthetic peptide corresponding to P. gingivalis fimbriae-binding region on biofilim formation of P. gingivalis with S. oralis. The peptide also inhibited P. gingivalis S. oralis biofilm formation in a dose-dependent manner.In conclusion, the synthetic peptide corresponding to P. gingivalis fimbriae-binding region inhibited P. gingivalis biofilm formation and P. gingivalis adherence and invasion to human fibroblast, indicating that the peptide might be a potent preventive against periodontitis.