Delineation of unique flagellar proteins in spirochetes

螺旋体是一组研究很少的医学上重要的细菌。螺旋体的能动性是由 周质鞭毛，其在周质空间内驻留和旋转。这些细菌的能动性对于 迄今为止研究的所有致病性螺旋体的毒力，包括伯氏疏螺旋体。虽然运动性发挥这样的作用， 虽然周质鞭毛在细胞中起着重要作用，但我们对周质鞭毛的组装、运作或组织知之甚少。 螺旋体虽然周质鞭毛的许多组成部分在细胞中具有高度保守的对应物， 外部鞭毛来自模式生物肠道沙门氏菌和大肠杆菌，一些独特的成分 周质鞭毛的形态特征明显区别于外鞭毛。最重要的是，我们最近的研究 提供了第一个证据，即新的螺旋体特异性成分，称为周质环， 对于鞭毛的组装和定向、这些细菌的独特形态和运动性是必不可少的。 然而，很少有人知道的基因编码的周质衣领或他们的功能，在任何 螺旋体基于我们的初步数据，我们假设周质领由多个 新的螺旋体特异性蛋白质，并且这些蛋白质中的每一种在鞭毛组装中发挥不同的作用， 螺旋体独特的形态和运动。为了解决这一假设，我们提出了两个具体目标。 目的1是鉴定组成大领复合物的蛋白质，确定它们的天然细胞结构 并在B中起作用。burgdorferi。此外，为了扩展B的相关性。Burgdorferi周质领研究， 我们建议确定这些新的鞭毛蛋白或它们的功能是否在其他螺旋体中是保守的。 目标1有望通过利用生物信息学、遗传学、各种生化测定和低温冷冻来实现。 电子断层摄影术我们希望能够识别编码项圈复合物结构的蛋白质，它们的功能， 螺旋体的位置、结构和组装。目标2提出了确定顺序装配 细胞被膜中的周质领蛋白，并了解新领蛋白的相互作用 与其他突出的鞭毛蛋白及其对周质所需扭矩增加的影响 鞭毛在粘性或复杂的介质中旋转，如哺乳动物组织。我们计划实现这一目标 目的是使用各种突变，生化，和冷冻电子断层扫描。我们希望了解 周质鞭毛组装在螺旋体中，它们的总体影响在B中。burgdorferi。知识 从这个项目中获得的是理解新的生物合成，组装和功能的基础 鞭毛蛋白不仅存在于B.但在其他医学上重要的疾病中， 无法培养的螺旋体，如梅毒螺旋体。这些研究也可以导致在结构上的应用- 基于药物设计，以破坏电机组装，因此阻止莱姆病的传播以及其他 螺旋体传播的疾病

Spirochetes are a group of poorly studied medically significant bacteria. The motility of spirochetes is driven by periplasmic flagella, which reside and rotate within the periplasmic space. These bacterial motility is crucial for virulence by all pathogenic spirochetes studied to-date including Borrelia burgdorferi. While motility plays such a vital role, we know very little about the periplasmic flagellar assembly, operation or organization in any spirochete. Although many components of the periplasmic flagellum have highly conserved counterparts in the external flagella from the model organisms Salmonella enterica and Escherichia coli, some unique components of the periplasmic flagella clearly distinguish them from external flagella. Most importantly, our recent studies have provided the first evidence that the novel spirochete-specific component known as the periplasmic collar is essential for flagellar assembly and orientation, the distinctive morphology and motility of these bacteria. However, very little is known about the genes encoding the periplasmic collar or their function in any spirochete. Based on our preliminary data, we hypothesize that the periplasmic collar is comprised of multiple novel spirochete-specific proteins, and that each of these proteins plays a distinct role in flagellar assembly, spirochetes distinctive morphology and motility. To address this hypothesis, we propose two specific aims. Aim 1 is to identify the proteins that make-up the large collar complex, determine their native cellular structure and function in B. burgdorferi. Moreover, to extend the relevance of B. burgdorferi periplasmic collar studies, we propose to determine if these novel flagellar proteins or their function is conserved in other spirochetes. Aim 1 is expected to be accomplished by using bioinformatics, genetics, various biochemical assays and cryo- electron tomography. We expect to identify the proteins encoding the collar complex structure, their function, location, structure, and assembly in the spirochetes. Aim 2 is proposed to determine the sequential assembly of the periplasmic collar proteins in the cell envelope and to understand the interactions of novel collar proteins with other prominent flagellar proteins and their impacts on increased torque required for the periplasmic flagella to rotate in viscous or complex medium such as the mammalian tissues. We plan to accomplish this aim using various mutational, biochemical, and cryo-electron tomography. We expect to understand how the periplasmic flagella are assembled in the spirochete and their overall impacts in B. burgdorferi. The knowledge gained from this project is fundamental to understand the biosynthesis, assembly, and function of the novel flagellar proteins not only in B. burgdorferi and Leptospira but also in other medically significant yet uncultivable spirochetes such as Treponema pallidum. These studies can also lead to applications in structure- based drug design to disrupt motor assembly, therefore blocking the spread of Lyme as well as other spirochete-borne diseases.