Epitope structure of fibronectin that recognized by bacterial adhesins

纤维连接蛋白（Fn）是一种主要的细胞外基质蛋白，并且似乎在感染的第一阶段介导各种微生物对宿主组织的粘附。金黄色颗粒菌是一种常见于健康人口腔中的营养变异链球菌，常从感染性心内膜炎患者中分离到。我们以前曾报道过这种细菌的Fn结合能力与心内膜炎动物模型的感染性之间存在联系。我们在此报告说，Fn具有一个新的结合位点的各种细菌，包括这种生物体，该地区可能是更相关的微生物的发病机制比N-末端区域已被广泛研究，从这个角度来看。制备了抗人Fn的单克隆抗体（mAb），其中一个克隆具有抑制G.选择adiacens。使用通过Sequ获得的Fn的多肽片段进行蛋白质印迹分析 ...更多信息 用嗜热菌蛋白酶和赖氨酰内肽酶进行的限制性消化表明，抑制性mAb的表位位于整个Fn多肽的中心部分（105 kDa细胞结合片段），该片段缺乏N-和C-末端部分，前者包含许多细菌物种的已知结合区域。金黄色葡萄球菌和化脓性链球菌也能够结合到固定在测定板上的中心105 kDa细胞结合片段，除了已知的结合到N-末端部分，而G.其中，一株大肠埃希菌和一株白僵菌仅分布于中部。S.金黄色葡萄球菌和化脓性链球菌与N-末端Fn片段的结合被测定培养基中过量的游离Fn抑制。与此相反，粘附的四个物种的中央105 kDa的片段不抑制可溶性Fn，但由mAb。考虑到哺乳动物体液中高浓度的可溶性Fn，Fn的中心部分可能比N-末端区域在体内细菌粘附中具有更重要的作用。少

Fibronectin (Fn) is a major extracellular matrix protein and appears to mediate adherence of various microorganisms to host tissues at the first stage of infections. Granulicatella adiacens, a dominant nutritionally variant streptococci (NVS) harboring in the healthy human oral cavities, is often isolated from patients with infective endocarditis as the causative agent. We have previously reported a link between the Fn-binding ability of this specie and the infectivity in an animal model of endocarditis. We report herein that Fn possesses a novel binding site for various species of bacteria including this organism, and the region may be more relevant to microbial pathogenesis than the N-terminal region which has been extensively investigated from this point of view. Monoclonal antibodies (mAbs) were generated against human Fn, and one clone with an ability to inhibit the Fn-binding of G. adiacens was selected. Western blotting analyses using polypeptide fragments of Fn obtained by sequ … More ential limited digestions with thermolysin and lysyl endopeptidase showed that the epitope for the inhibiting mAb resided in the central part of the whole Fn polypeptide (105 kDa cell-binding fragment) which lacked both the N- and C-terminal parts, the former contains the known binding regions for many bacterial species. Staphylococcus aureus and Streptococcus pyogenes were also capable of binding to the central 105 kDa cell-binding fragment immobilized onto assay plates, in addition to the known binding to the N-terminal part while G. adiacens and an Echerichia coli were only to the central part. The adherence of S. aureus and S. pyogenes to N-terminal Fn fragment was inhibited by excess amounts of free Fn in the assay medium. In contrast, adherence of the four species to the central 105 kDa fragment was not inhibited by soluble Fn, but by the mAb. Considering the high concentrations of soluble Fn in the mammalian body fluids, the central part of Fn may have more important roles than the N-terminal region in the bacterial adherence in vivo. Less