The novel cross-linking of the flagellar hook protein of Borrelia burgdorferi

描述（由申请人提供）：本项目的长期目标是更好地了解伯氏疏螺旋体（Bb）的运动性，因为它与发病机制有关。中心假设是，Bb和其他螺旋体物种包含一个独特的交联的鞭毛钩蛋白FlgE是必不可少的最佳运动。下面的两个目标是研究交叉连接的识别和形成。了解FlgE交联合成和结构，可能会导致抑制这种交联的新药的开发。这些药物可能用于治疗莱姆病和其他螺旋体疾病。具体目标1.基于已发表的和初步的数据，我们假设Bb FlgE鞭毛钩蛋白是共价交联的。为了检验这一假设，将从生长的细胞中纯化钩-基体复合物，并在大肠杆菌中合成重组Bb FlgE（rFlgE）并如所述纯化。用胰蛋白酶等试剂消化钩状基体复合物和rFlgE，并通过质谱法分离和分析产物。肽模式的差异将指示哪些肽是交联的。将使用肽图谱和串联质谱法确定交联中涉及的特定氨基酸和键的性质。具体目标2。我们推测，定点突变，改变特定的残基在Bb flgE的钩蛋白FlgE无法交联的结果。携带这种突变的细胞也被假设具有改变的运动性。为了检验这些假设，将使用两种方法来构建flgE突变体。首先，将构建含有克隆的Bb flgE的质粒flgE/pBSV 2中的特定突变。该突变体已被证明可补充flgE非运动插入突变体SC-E1。将诱变的质粒电穿孔到SC-E1中，并如下所述进行分析。第二，将使用最近开发的反选择技术构建染色体编码的flgE中的特异性突变。将测试通过两种方法产生的突变体的FlgE合成、FlgE交联、鞭毛钩产生和长度以及运动性。所采用的方法包括FlgE合成和交联的蛋白质印迹法，鞭毛钩合成和长度测定的透射电子显微镜，以及群板测定和计算机化细胞跟踪来评估改变的运动性;研究人员使用所有这些技术都有丰富的经验。所获得的结果应允许参与FlgE交联的氨基酸的鉴定和这种交联对Bb运动的影响。 公共卫生相关性：细菌的移动能力对它们中的许多人致病很重要;许多细菌通过旋转鞭毛游泳，其中一个组成部分是灵活的“钩”。螺旋体在世界范围内引起许多疾病，它们似乎表现出独特的钩蛋白交联。通过了解这种交联，可以开发新的药物来靶向这种交联并对抗螺旋体引起的疾病。

DESCRIPTION (provided by applicant): The long-term goal of this project is to better understand Borrelia burgdorferi (Bb) motility as it relates to pathogenesis. The central hypothesis is that Bb and other spirochete species contain a unique cross-linking of the flagellar hook protein FlgE that is essential for optimal motility. The two aims below investigate the identification and formation of the cross-links. Understanding FlgE cross-link synthesis and structure and can potentially lead to the development of novel drugs that inhibit this cross-linking. These drugs could potentially be used to treat Lyme borrelosis and other spirochetal diseases. Specific aim 1. Based on published and preliminary data, we hypothesize that the Bb FlgE flagellar hook proteins are covalently cross-linked to one another. To test this hypothesis, hook- basal-body complexes will be purified from growing cells, and recombinant Bb FlgE (rFlgE) will be synthesized in Escherichia coli and purified as described. The hook basal body complexes and rFlgE will be digested with agents such as trypsin, and the products will be separated and analyzed by mass spectrometry. Differences in the peptide patterns will indicate which peptides are cross-linked. The specific amino acids and nature of the bond involved in the cross-linking will be determined using peptide mapping and tandem mass spectrometry. Specific aim 2. We hypothesize that site-directed mutations that alter specific residues in Bb flgE result in the inability of the hook protein FlgE to be cross-linked. Cells bearing such mutations are also hypothesized to have altered motility. To test these hypotheses, two approaches will be used to construct flgE mutants. First, specific mutations in the plasmid flgE/pBSV2 containing cloned Bb flgE will be constructed. This mutant has been shown to complement the flgE non-motile insertion mutant SC-E1. The mutagenized plasmids will be electroporated into SC-E1 and analyzed as described below Second, specific mutations in the chromosomally encoded flgE will be constructed using a recently developed counter-selection technique. Mutants generated by both methods will be tested for synthesis of FlgE, FlgE cross-linking, flagellar hook production and length, and motilty. The methods employed include western blotting for FlgE synthesis and cross-linking, transmission electron microscopy for flagellar hook synthesis and length determination, and swarm plate assays and computerized cell tracking to assess altered motility; the investigators have extensive experience using all these techniques. The results obtained should allow for the identification of the amino acids involved in FlgE cross-linking and the effects of this cross-linking on Bb motility. PUBLIC HEALTH RELEVANCE: The ability of bacteria to move is important for many of them to cause disease; many swim by rotating their flagella, one component of which are the flexible 'hooks'. Spirochetes cause many diseases world-wide, and they appear to exhibit a unique cross-linking of hook proteins. By understanding this cross-linking, novel drugs can be developed to target this cross-linking and combat spirochete-caused diseases.