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

在这项研究中，我们通过生物分子相互作用分析系统（BIAcore）证明了人甘油醛-3-磷酸脱氢酶（GAPDH）与牙龈卟啉单胞菌（Porphyromonas gingivalis fihrise）的相互作用具有特异性和高亲和力。用赖氨酸多肽酶等多种蛋白对人GAPDH进行酶切，并采用高效液相色谱法收集片段。通过BIAcore检测GAPDH片段与牙龈假单胞菌菌毛的结合活性，并用质谱仪和氨基酸测序仪对结合活性最强的片段进行分析。由于在人GAPDH的165-185个氨基酸残基中发现牙龈假单胞菌菌毛的结合域，我们合成了与人GAPDH的165-185个氨基酸残基相对应的肽段，并用共聚焦显微镜检测了该肽段对牙龈假单胞菌对人牙龈成纤维细胞的粘附和侵袭的抑制作用。合成的肽以剂量依赖的方式抑制牙龈假单胞菌对人成纤维细胞的粘附和侵袭。接下来，我们用同样的方法鉴定了口腔链球菌GAPDH对牙龈卟啉菌毛的结合域，发现口腔链球菌GAPDH的结合域与人类GAPDH的结合域存在于同一区域。因此，我们研究了牙龈假单胞菌菌毛结合区对应的合成肽对口腔假单胞菌与牙龈假单胞菌形成生物膜的抑制作用。该肽还能以剂量依赖性的方式抑制口腔牙龈假单胞菌生物膜的形成。综上所述，合成的与牙龈假单胞菌菌毛结合区相对应的肽抑制了牙龈假单胞菌生物膜的形成，抑制了牙龈假单胞菌对人成纤维细胞的粘附和侵袭，提示该肽可能是一种有效的牙周炎预防药物。

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.