Fibronectin-binding Protein of Treponema Denticola in Periodontal Disease

描述（由申请人提供）：牙周炎是全球第二大常见疾病，由包括齿垢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入宿主组织中起重要作用。以往的研究发现T.抗FN抗体可阻断其与FN的结合。然而，有关FN结合蛋白及其在T。齿垢非常有限。T. denticola已经鉴定了一个新的推定的纤连蛋白/纤维蛋白原结合蛋白基因，fbp（基因ID：2739776），其与原核FN结合蛋白的N-末端片段同源。(FbpA，pfam05833）。FbpA是一个同源的Fn结合蛋白基因家族，根据CDD数据库，其成员超过117个。fbp在T.尚未研究Fbp和FN之间的相互作用。在前期研究中，我们在大肠杆菌中表达了重组Fbp。coli，并鉴定了rFbp具有FN结合特性。T. rFbp以浓度依赖性方式阻断了Denticola对FN的作用，最高水平为42%。我们还制备了抗rFbp抗体以证实Fbp在T. Western blot检测结果。我们认为fbp是一个重要的毒力因子。 研究假设Fbp参与了T.介导与宿主FN的相互作用。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之间的相互作用来表征Fbp的功能。具体来说，在目标1中，我们计划在T.齿垢我们将比较fbp突变体和野生型T。在FN结合，细胞附着和小鼠模型中的毒力。此外，我们将通过基因互补的方法拯救fbp突变体，以证实fbp的功能。目的二：从随机肽库中筛选出Fbp与FN的结合位点，并通过定点突变验证其结合位点。此外，我们还将设计并测试合成的Fbp肽抑制T.牙菌斑到FN。我们希望能找到一种能抑制T.可以作为抗感染因子的宿主组织。 综上所述，这是T.首次系统性鉴定Fbp蛋白的结合位点。我们相信，该项目的提出是新颖和独特的，将有助于我们了解不仅在T。但也存在于其他具有FBP基因的细菌中。长期目标是深入了解T.目的：研究FbpA蛋白家族在牙周组织中的生物学功能，为预防细菌感染提供新的策略。 公共卫生相关性：牙周炎是世界上第二常见的疾病，与细菌齿垢密螺旋体感染有关。本研究旨在探讨新发现的T.牙周病是牙周病的一个重要组成部分。

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.