Kontani M, Kimura S, Nakagawa I, Hamada S
Department of Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka, Suita-Osaka, Japan.
Mol Microbiol. 1997 Jun;24(6):1179-87. doi: 10.1046/j.1365-2958.1997.4321788.x.
Porphyromonas gingivalis, a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin-avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of L-Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae-Arg interaction (a novel host-parasite relationship).
牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，已知其与牙周炎的发病机制有关。牙龈卟啉单胞菌菌毛是从细胞表面伸出的蛋白质附属物，可能有助于该菌黏附于宿主细胞表面。我们之前曾提出，牙龈卟啉单胞菌产生的精氨酸特异性蛋白酶可增强纯化菌毛与成纤维细胞或基质蛋白的黏附。在本研究中，我们更详细地揭示了该蛋白酶增强菌毛结合的机制。从牙龈卟啉单胞菌381细胞中获得并纯化了精氨酸特异性蛋白酶和菌毛。然后，我们使用基于表面等离子体共振原理的光学生物传感器实时生物分子相互作用分析（BIAcore）系统，分析了菌毛与固定化纤连蛋白（完整的或经纯化蛋白酶部分降解的纤连蛋白）之间的相互作用。BIAcore分析图谱显示菌毛与蛋白酶降解的纤连蛋白之间的相互作用增强。我们还通过BIAcore分析表明菌毛与降解的纤连蛋白存在特异性结合。通过酶联生物素-抗生物素蛋白测定法检测了生物素化菌毛与固定化纤连蛋白的结合。纯化的蛋白酶增强了菌毛与固定化纤连蛋白的结合。在菌毛结合反应中加入L-精氨酸或C末端含有精氨酸残基的寡肽可抑制这种增强作用，这表明牙龈卟啉单胞菌菌毛可能具有与C末端精氨酸残基紧密结合的能力。综上所述，这些研究表明牙龈卟啉单胞菌精氨酸特异性蛋白酶可在基质蛋白分子中暴露一个隐蔽表位，即宿主基质蛋白的C末端精氨酸残基，从而使该菌能够通过菌毛-精氨酸相互作用（一种新型宿主-寄生虫关系）黏附于口腔表层。
