Mechanisms of adherence of P.gingivalis to matrix proteins via fimbrial cryptie receptor

牙龈卟啉单胞菌通过菌毛隐秘受体粘附基质蛋白的机制

• 批准号: 08457480
• 负责人: HAMADA Shigeyuki
• 金额: $ 4.67万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457480/
• 关键词: Porphyromonas gingivalis; fimbriae; protease; fibronectin; Arginine; Peptide

Porphyromonas gingivalis 381, a suspected periodontopathogen, possesses fimbriae on its cell surface. The organism is known to produce proteases which can degrade the host cell surface matrix proteins. In this study, we investigated the effect of protease on the binding of the purified P.gingivalis fimbriae to cultures fibroblasts or matrix proteins. A protease that can hydrolyze benzoyl-L-arginine p-nitro-anilide was obtained from P.gingivalis 381 cells by sonication in phosphate-buffered 0.2% Triton X-100 and was purified by column chromatography. The protease degrade various host proteins, including collagen and fibronectin, and cleave the C-terminus of the arginine residue in peptides. However, P.gingivalis fimbriae were not degraded by this protease activity. When cultured fibroblasts were partially treated with the protease, the binding of the purified P.gingivalis fimbriae to the fibroblast monolayr was increased significantly. Similarly, binding of the fimbriae to the collagen … More or fibronectin immobilized on the microtiter wells was also enhanced. Addition of these host matrix proteins efficiently inhibited the binding of fimbriae to the fibroblast monolayr. The binding assay of fimbriae using dipeptidyl ligand affinity column chromatography demonstrated a clear interaction between fimbriae and the arginine residue. We then analyzed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the BIAcore system. 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, suggesting than 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 layr in the oral cavity through fimbriae-Arg interaction (a novel host-parasite relation ship). Less

牙龈卟啉单胞菌381是一种可疑的牙周病病原菌，其细胞表面具有菌毛。已知该生物体产生可降解宿主细胞表面基质蛋白的蛋白酶。在这项研究中，我们研究了蛋白酶对纯化的牙龈卟啉单胞菌菌毛与培养成纤维细胞或基质蛋白结合的影响。用0.2%Triton X-100磷酸盐缓冲液超声处理牙龈卟啉单胞菌381细胞，得到一种能水解苯甲酰-L-精氨酸对硝基苯胺的蛋白酶，并经柱层析纯化。蛋白酶降解各种宿主蛋白质，包括胶原蛋白和纤连蛋白，并切割肽中精氨酸残基的C-末端。然而，牙龈卟啉单胞菌菌毛不被这种蛋白酶活性降解。当用蛋白酶部分处理培养的成纤维细胞时，纯化的牙龈卟啉单胞菌菌毛与成纤维细胞单层的结合显著增加。类似地，菌毛与胶原蛋白的结合 ...更多信息 或固定在微量滴定威尔斯孔上的纤连蛋白也得到增强。这些宿主基质蛋白的加入有效地抑制了菌毛与成纤维细胞单层的结合。使用二肽基配体亲和柱色谱法的菌毛的结合测定证明了菌毛和精氨酸残基之间的明确相互作用。然后，我们通过使用BIAcore系统分析了菌毛和固定化纤连蛋白（通过纯化的蛋白酶完整或部分降解的纤连蛋白）的相互作用。BIAcore图谱表明菌毛和蛋白酶降解的纤连蛋白之间的相互作用增强。我们还表明，通过BIAcore分析的菌毛的降解的纤连蛋白的特异性结合。酶联生物素-亲和素法检测生物素化菌毛与固定化纤维连接蛋白的结合。纯化的蛋白酶增强了菌毛与固定化纤连蛋白的结合。加入L-Arg或C-末端含有Arg残基的寡肽可抑制这种增强作用，表明牙龈卟啉单胞菌菌毛可能具有与C-末端的Arg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的精氨酸特异性蛋白酶可以暴露基质蛋白分子中的一个隐蔽位，即宿主基质蛋白的C-末端Arg残基，从而使生物体可以通过菌毛-Arg相互作用（一种新的宿主-寄生虫关系）粘附到口腔中的表面层。少

项目成果

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Amano, A., Fujiwara, T., Nagata, H., Kuboniwa, M., Sharma, A., Sojar, H.T., Genco, R.J., Hamada, S.and Shizukuishi, S.: "Porphyromonas gingivalis fimbriae mediate coaggregation with Streptococcus oralis through specific domains" Journal of Dental Research

Amano, A.、Fujiwara, T.、Nagata, H.、Kuboniwa, M.、Sharma, A.、Sojar, H.T.、Genco, R.J.、Hamada, S. 和 Shizukuishi, S.：“牙龈卟啉单胞菌菌毛介导与

Kontani,M.et al.: "Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease" Molecular Microbiology. 24 6. 1179-1187 (1997)

Kontani，M.等人：“牙龈卟啉单胞菌通过其自身精氨酸特异性蛋白酶暴露的菌毛隐性受体与基质蛋白的粘附”分子微生物学。

Kontani, M.et al.: "Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease" Molecular Microbiology. 24・6. 1179-1187 (1997)

Kontani，M.等：“牙龈卟啉单胞菌通过其自身的精氨酸特异性蛋白酶暴露的菌毛隐性受体与基质蛋白的粘附”《分子微生物学》24·6（1997）。