Protein-protein covalent bonding and Treponema motility

描述(申请人提供)：齿密螺旋体(TD)是牙周疾病的主要参与者，牙周疾病会导致牙齿脱落，并与包括心血管疾病在内的全身疾病有关。TD与梅毒螺旋体梅毒相似，梅毒螺旋体是世界范围内发病率和死亡率的主要原因。TD和梅毒螺旋体都对特定的抗生素产生了抗药性。本研究的长期目标是开发治疗螺旋体疾病的新药，TD作为模型系统。TD和其他螺旋体是高度侵袭性的细菌，因为它们独特的运动方式。螺旋体周质鞭毛(PFS)是运动和致病所必需的。钩状结构是所有细菌PFS的重要组成部分。该钩子由多个FlgE蛋白组成。中心假设是TD和其他螺旋体的FlgE蛋白是共价交联的，以加强钩子的最佳运动性和毒力。其他有外部鞭毛的细菌缺乏交联链。了解FlgE交联物的结构和合成可能导致开发抑制交联物的新药来治疗牙周病、梅毒和其他螺旋体疾病。下面的三个目标解决了这一假设。具体目的1.我们假设TD FlgE鞭毛挂钩蛋白是共价交联的。为了验证这一假设，我们将使用从TD中分离的钩蛋白和在体外形成的交联型FlgE蛋白来确定交联物的精确化学结构。这种方法利用了质谱学，以及如何在其他系统中破译交联。具体目的2.我们假设TD FlgE的定点突变将导致钩蛋白在体外和体内都不能交联。该方法包括体外诱变rflgE，并将选择的突变引入TD细胞并分析其表型。携带这种突变的细胞被假设已经改变了运动性。具体目的3.我们假设交联型FlgE在TD致病中是必不可少的。为了验证这一假设，我们将分析交联性缺陷的突变株，并使用两种已建立的毒力分析方法与野生型进行比较。所获得的结果应该使我们能够确定FlgE交联物在TD发病机制中的作用。

DESCRIPTION (provided by applicant): Treponema denticola (Td) is strongly implicated as a major participant in periodontal diseases, which cause tooth loss and are implicated in systemic diseases including cardiovascular disease. Td is similar to the syphilis spirochete T. pallidum, which is a major cause of morbidity and mortality world-wide. Both Td and T. pallidum have developed resistance to specific antibiotics. The long term goal of this research is to develop new drugs to treat spirochetal diseases, with Td serving as the model system. Td and other spirochetes are highly invasive bacteria due to their unique mode of motility. Spirochete periplasmic flagella (PFs) are essential for motility and virulence. The hook structure is an essential component of all bacterial PFs. The hook consists of multiple FlgE proteins. The central hypothesis is that the FlgE proteins of Td and other spirochetes is covalently cross-linked to strengthen the hook for optimal motility and virulence. Other bacteria with external flagella lack cross-links. Understanding the structure and synthesis of FlgE cross-links could lead to development of novel drugs that inhibit cross-linking to treat periodontal disease, syphili and other spirochetal diseases. The three aims below address this hypothesis. Specific aim 1. We hypothesize that the Td FlgE flagellar hook proteins are covalently cross- linked. To test this hypothesis, we will determine the precise chemical structure of the cross-link using hook proteins isolated from Td and cross-linked FlgE proteins formed in vitro. The approach utilizes mass spectrometry and how cross-linking is deciphered in other systems. Specific aim 2. We hypothesize that site-directed mutations in Td FlgE will result in the inability of the hook protei to be cross-linked both in vitro and in vivo. The approach incorporates in vitro mutagenesis of rflgE, and introducing select mutations into Td cells and analyzing their phenotypes. Cells bearing such mutations are hypothesized to have altered motility. Specific aim 3. We hypothesize that cross-linking FlgE is essential for Td to cause disease. To test this hypothesis, we will analyze mutants defective in cross-linking and make comparisons to the wild-type using two established virulence assays. The results obtained should allow us to determine the role of FlgE cross-linking in Td pathogenesis.