Fibronectin-binding Protein of Treponema Denticola in Periodontal Disease

描述(申请人提供)：牙周炎是全世界第二常见的疾病，由包括齿密螺旋体在内的微生物引起。纤维连接蛋白(FN)是一种多功能的粘附性糖蛋白，分布于血浆、细胞外基质和细胞表面。细菌Fn结合蛋白在细菌对宿主组织的黏附和侵袭中起着重要作用。先前的研究发现，齿状毛滴虫与Fn结合，而Fn被抗Fn抗体所阻断。然而，关于FN结合蛋白及其在齿状毛滴虫发病机制中的作用的了解非常有限。齿纹夜蛾基因组计划已经发现了一个新的可能的纤维连接蛋白/纤维蛋白原结合蛋白基因fBP(基因ID：2739776)，它与原核生物纤维连接蛋白结合蛋白的N端片段同源。(FbpA，ppam05833)。根据CDD数据库，FbpA是一个同源的Fn结合蛋白基因家族，有117多个成员来自不同的细菌。FBP在齿状毛滴虫发病机制中的作用以及FBP和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，并鉴定了rFBP具有FN结合特性。RFBP以浓度依赖的方式阻断齿状毛滴虫与Fn的结合，最高可达42%。我们还制备了抗rFBP的抗体，通过Western印迹证实FBP在齿纹夜蛾中得到了表达。我们认为FBP是一个重要的毒力因子。研究假设是FBP通过介导与寄主Fn的相互作用而促进了齿纹夜蛾的毒力。本研究的目的是通过比较FBP突变株和野生型菌株的表型来分析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。具体地说，在目标1中，我们计划在T.denticola中制造FBP的等基因突变体。我们将在小鼠模型中比较FBP突变体和野生型齿状毛滴虫在Fn结合、细胞附着和毒力方面的差异。此外，我们还将通过遗传互补的方法拯救FBP突变体，以确认FBP的功能。在目标2中，我们将筛选一个随机的多肽文库来确定FBP与FN的结合部位，并随后通过FBP上的定点突变来验证结合部位。此外，我们还将设计并测试合成的FBP多肽抑制齿状毛滴虫与Fn结合的能力。我们希望找到一种能够阻止齿状毛滴虫附着在宿主组织上的多肽，这可能是一种抗感染因子。综上所述，这是第一个齿纹夜蛾粘附素突变体，也是第一个系统鉴定FBP蛋白结合部位的方法。我们相信，该项目是新颖和独特的，将有助于我们不仅了解FBP在齿状毛滴虫发病机制中的作用，而且也有助于了解其他携带FBP基因的细菌的作用。其长期目标是深入了解齿状毛滴虫对牙周组织的附着和侵袭机制，并对FbpA蛋白家族的生物学功能进行表征，从而为预防细菌感染提供新的策略。 公共卫生相关性：牙周炎是全球第二大常见病，与齿密螺旋体细菌感染有关。本研究将研究最近发现的齿状毛滴虫纤维连接蛋白结合蛋白基因(FBP)的致病性，作为预防细菌感染和牙周病的新策略的基础。

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. PUBLIC HEALTH RELEVANCE: Periodontitis is the second most common disease worldwide and is associated with infection by the bacteria Treponema denticola. This study will investigate pathogenicity of a recently identified fibronectin binding protein gene (fbp) of T. denticola as a basis for new strategies to prevent bacterial infection and periodontal disease.