Fibronectin-binding Protein of Treponema Denticola in Periodontal Disease

描述（由申请人提供）：牙周炎是世界上第二常见的疾病，由包括密螺旋体在内的微生物引起。纤维连接蛋白（FN）是一种多功能黏附糖蛋白，分布于血浆、细胞外基质和细胞表面。细菌fn结合蛋白在细菌粘附和侵袭宿主组织中起重要作用。先前的研究发现，齿状霉与FN结合，并被抗FN抗体阻断。然而，关于fn结合蛋白及其在齿状霉发病机制中的作用的知识非常有限。研究人员通过基因组计划鉴定出一种新的纤维连接蛋白/纤维蛋白原结合蛋白基因fbp（基因ID: 2739776），该基因与原核生物fn结合蛋白的n端片段同源。(FbpA pfam05833)。根据CDD数据库，FbpA是一个同源的fn结合蛋白基因家族，有来自不同细菌的超过117个成员。fbp在齿状弓形虫发病机制中的作用以及fbp与FN之间的相互作用尚未被研究。在初步研究中，我们在大肠杆菌中表达了重组Fbp，并鉴定出rFbp具有FN结合特性。rFbp能以浓度依赖的方式阻断树突天牛与FN的结合，最大阻断程度为42%。我们还制作了抗rfbp抗体，通过Western blot证实了Fbp在齿状体中表达。我们认为fbp是一个重要的毒力因子。研究假设Fbp通过介导与宿主FN的相互作用而参与了齿状小蠊的毒力。通过比较fbp突变株和野生型菌株的表型，分析fbp的毒力作用，并通过研究fbp蛋白与FN的相互作用来表征fbp的功能。具体来说，在Aim 1中，我们计划在齿齿田鼠中制造fbp的等基因突变体。我们将在小鼠模型中比较fbp突变体与野生型齿状舌鼠在FN结合、细胞附着和毒力方面的差异。此外，我们将通过基因互补拯救fbp突变体，以确认fbp的功能。在Aim 2中，我们将筛选一个随机的肽库来确定Fbp与FN的结合位点，随后通过Fbp的位点特异性突变来验证结合位点。此外，我们还将设计和测试合成的Fbp肽抑制树突菌与FN结合的能力。我们期望找到一种肽，它可以防止牙齿田鼠粘附到宿主组织，这可能是一种抗感染因子。综上所述，这是首个齿状霉黏附素突变体，也是首个系统鉴定Fbp蛋白结合位点的方法。我们认为该项目新颖独特，不仅有助于我们了解fbp在齿状霉发病机制中的作用，也有助于我们了解其他携带fbp基因的细菌的发病机制。长期目标是深入了解牙周菌对牙周组织的附着和侵袭机制，并表征FbpA蛋白家族的生物学功能，为预防细菌感染提供新的策略。

DESCRIPTION (provided by applicant): Periodontitis is the second most common disease worldwide and is induced by microorganisms including Treponema denticola. Fibronectin (FN) is a multifunctional adhesive glycoprotein that is distributed in plasma, the extracellular matrix, and on the surface of cells. Bacterial FN-binding proteins play important roles in bacterial adhesion and invasion of host tissues. Previous research has found that T. denticola bound FN which was blocked by anti-FN antibodies. However, the knowledge about FN-binding proteins and their roles in the pathogenesis of T. denticola is very limited. The genome project of T. denticola has identified a new putative fibronectin/fibrinogen-binding protein gene, fbp (Gene ID: 2739776), which is homologous to an N-terminal segment of the prokaryotic FN-binding protein. (FbpA, pfam05833). FbpA is a family of homologous Fn-binding protein gene with over 117 members from different bacteria according to the CDD database. The role of fbp in pathogenesis of T. denticola and the interactions between Fbp and FN has not been investigated. In preliminary studies, we expressed recombinant Fbp in E. coli, and identified that rFbp has FN binding properties. The binding of T. denticola to FN was blocked by rFbp in a concentration dependent manner to a maximum level of 42%. We also make anti-rFbp antibodies to confirm that Fbp is expressed in T. denticola by Western blot. We propose that fbp is an important virulence factor. The research hypothesis is that Fbp contributes to the virulence of T. denticola by mediating interaction with host FN. The purpose of this proposal is to analyze the virulence roles of fbp by comparing the phenotypes of fbp mutant and wildtype strains, and to characterize the functions of Fbp by studying the interactions between Fbp protein and FN. Specifically, in Aim 1 we plan to make isogenic mutants of fbp in T. denticola. We will compare fbp mutant with wildtype T. denticola in FN binding, cell attachment, and virulence in a mouse model. In addition, we will rescue fbp mutants by genetic complementation to confirm the function of fbp. In Aim 2, we will screen a random peptide library to identify the binding site of Fbp for FN and subsequently verify the binding site by site-specific mutagenesis on fbp. In addition we will also design and test the capacity of synthetic Fbp peptide to inhibit the binding of T. denticola to FN. We expect to find a peptide which prevents the adherence of T. denticola to host tissue which may serve as an anti-infection factor. In summary, this is first adhesin mutant of T. denticola and first systemic approach to identity the binding site of Fbp protein. We believe that the proposed project is novel and unique and will help us to understand not only the role of fbp in the pathogenesis of T. denticola, but also in other bacteria with fbp genes. The Long term goal is to understand in depth the mechanism of attachment and invasion of T. denticola to periodontal tissues and to characterize the biological function of the FbpA protein family in order to provide new strategies to prevent bacterial infections.