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

描述（由申请人提供）：该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。核心假设是Bb和其他螺旋体含有一种独特的交联鞭毛钩蛋白FlgE，这是最佳运动所必需的。下面的两个目的是研究交联的识别和形成。了解FlgE交联的合成和结构，并可能导致抑制这种交联的新药的开发。这些药物有可能用于治疗莱姆病和其他螺旋体疾病。具体目标1。根据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。为了验证这一假设，将从生长细胞中纯化钩-基底复合物，并在大肠杆菌中合成重组Bb FlgE （rFlgE）并按照描述进行纯化。钩基体复合物和rFlgE将用胰蛋白酶等试剂消化，产物将用质谱法分离和分析。肽模式的差异将表明哪些肽是交联的。在交联中涉及的特定氨基酸和键的性质将使用肽图谱和串联质谱法确定。具体目标2。我们假设，改变Bb flgE中特定残基的位点定向突变导致挂钩蛋白flgE无法交联。携带这种突变的细胞也被认为具有改变的运动性。为了验证这些假设，将使用两种方法来构建flgE突变体。首先，在含有克隆Bb flgE的质粒flgE/pBSV2中构建特异性突变。该突变体已被证明是flgE非运动插入突变体SC-E1的补充。将诱变的质粒电穿孔到SC-E1中，并进行如下所述的分析。其次，将使用最近开发的反选择技术构建染色体编码的flgE中的特定突变。通过这两种方法产生的突变体将测试FlgE的合成、FlgE交联、鞭毛钩的产生和长度以及运动性。所采用的方法包括用于FlgE合成和交联的western blotting，用于鞭毛钩合成和长度测定的透射电子显微镜，以及用于评估运动改变的群体平板试验和计算机细胞跟踪；调查人员在使用所有这些技术方面都有丰富的经验。所获得的结果应该允许鉴定参与FlgE交联的氨基酸以及这种交联对Bb运动的影响。

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.