Regulation and localization of flp pili: an under-investigated system

项目总结 菌毛是细菌以多种方式使用的细菌表面结构。它们可以用来 运动性、遗传交换和表面附着，通常是病原体的关键毒力因素。一个 在许多细菌病原体中都发现了一种特殊类型的菌毛，包括创伤弧菌和 Aggregatibacter放线菌伴生菌，被人类微生物组的一些成员用来 在肠道中建立永久性。尽管蝶泳很重要，但人们对它的研究还不够深入。 其他菌毛系统的深度，并有许多未探索的方面和好奇心。在这份提案中，我们概述了 利用一种为研究提供许多技术优势的模型细菌探索Flp菌毛的实验 菌毛：新月形杆菌。Flp系统的一个共同特征是菌毛蛋白基因受到调控。 与其余的菌毛基因分开。在Crescentus中，转录激活子CtrA与四个 Pilin启动子中的位点。初步数据表明，一个位点诱导表达，而其余的 位点从其上游位置抑制和/或延迟转录，这一现象以前没有在 细菌。通过突变不同的CtrA结合位点和 测量表达时间和大小的变化。的直接相互作用和潜在的协作性 CTRA在菌毛蛋白启动子上的结合将通过生化分析进行评估。最后，对桩基的规范 亚基将通过突变而改变，以及对菌毛蛋白亚基的差异调节的影响及其如何 有助于菌毛的合成以及生理后果，如噬菌体感染和表面 将检查附件(目标1)。耳垂的另一个共同特征是它通常是两极分化的。 本地化。定位取决于TadZ蛋白，但该蛋白如何定位是一个谜。蛋白 交互作用技术将被用来识别和表征导致TADZ的因素，从而确定FLP Pilus，变得两极本土化。此外，这种生物体中Flp菌毛的整个蛋白质补体 将通过使用一种新的纯化策略对完整的菌毛进行纯化来确定，这种方法具有 以前从未实现过(目标2)。这些研究将提供对FLP中保留的特征的洞察 菌毛系统，从而提供了对许多人类致病和共生的重要结构的洞察 有机体。

PROJECT SUMMARY Pili are bacterial surface structures that are used by bacteria in many ways. They can be used for motility, genetic exchange, and surface attachment, and are often critical virulence factors for pathogens. A particular type of pilus, the flp pilus, is found in many bacterial pathogens, including Vibrio vulnificus and Aggregatibacter actinomycetemcomitans, and is used by some members of the human microbiome to establish permanence in the intestinal tract. Despite its importance, the flp pilus is not nearly studied to the depth of other pilus systems, and has many unexplored facets and curiosities. In this proposal we outline experiments to explore the flp pilus using a model bacterium that provides many technical benefits for studying pili: Caulobacter crescentus. A common characteristic of the flp system is that the pilin gene is regulated separately from the rest of the pilus genes. In C. crescentus, the transcriptional activator CtrA binds to four sites in the pilin promoter. Preliminary data indicates that one site induces expression, while the remaining sites inhibit and/or delay transcription from their upstream positions, a phenomenon not previously reported in bacteria. Regulation of the pilin promoter will be explored by mutating different CtrA-binding sites and measuring changes in expression timing and magnitude. The direct interaction and potential cooperativity of CtrA binding at the pilin promoter will be assessed using biochemical assays. Lastly, regulation of the pilin subunit will be altered by mutagenesis, and the effect of differential regulation of the pilin subunit and how it contributes to pilus synthesis as well as physiological consequences such as phage infection and surface attachment will be examined (Aim 1). Another common characteristic of the flp pilus is that it is often polarly localized. Localization depends on the TadZ protein, but how that protein is localized is a mystery. Protein interaction techniques will be used to identify and characterize the factors that cause TadZ, and thus the flp pilus, to become polarly localized. Additionally, the entire protein complement of the flp pilus in this organism will be determined by purification of whole intact pili using a novel purification strategy, something that has never before been accomplished (Aim 2). These studies will provide insight into features conserved among flp pili systems, and thus provide insight into a structure important for many human pathogenic and commensal organisms.