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

牙龈卟啉单胞菌381是一种疑似牙周病原体，其细胞表面有菌毛。已知该生物体能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假单胞菌菌毛与培养的成纤维细胞或基质蛋白结合的影响。以0.2%磷酸缓冲液Triton X-100为底物，经超声波处理，从牙龈假单胞菌381细胞中获得一种能水解苯甲酰-L-精氨酸对硝基-苯胺的酶，并用柱层析对其进行纯化。该酶降解多种宿主蛋白，包括胶原和纤维连接蛋白，并裂解多肽中精氨酸残基的C末端。然而，牙龈假单胞菌菌毛并未被该酶活性降解。当该酶部分作用于培养的成纤维细胞时，纯化的牙龈假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…的结合固定在微滴定孔上的纤维连接蛋白的数量也有所增加。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。二肽配体亲和柱层析对菌毛的结合分析表明菌毛与精氨酸残基之间存在明显的相互作用。然后，我们利用Biacore系统分析了菌毛和固定化纤维连接蛋白(被纯化的酶完整或部分降解的纤维连接蛋白)之间的相互作用。Biaccore图谱显示菌毛和可降解酶的纤维连接蛋白之间的相互作用增强。通过Biaccore分析，我们还发现菌毛与降解的纤维连接蛋白具有特异性结合。采用生物素亲和素法检测固定化纤维连接蛋白与生物素化菌毛的结合力。纯化的蛋白水解酶增强了固定化纤维连接蛋白与菌毛的结合。这种增强作用被L-精氨酸或C-末端含有精氨酸残基的寡肽所抑制，这表明牙龈假单胞菌菌毛可能具有与C-末端的精氨酸残基紧密结合的能力。综上所述，这些研究表明，牙龈假单胞菌精氨酸特异的蛋白酶可以暴露基质蛋白分子中的一个隐表位，即宿主基质蛋白的C端Arg残基，从而使有机体能够通过菌毛-Arg相互作用(一种新的宿主-寄生虫关系)附着在口腔的表层。较少

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