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

flp pili 的调控和本地化：一个尚待研究的系统

• 批准号: 10046802
• 负责人: Patrick David Curtis
• 金额: $ 41.46万
• 依托单位: UNIVERSITY OF MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046802
• 关键词: Actinobacillus actinomycetemcomitans; Adhesions; Affect; Affinity; Animal Model; Architecture; Bacteria; Bacterial Pili; Bacteriophages; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Caulobacter crescentus; Cell Cycle; Cells; Characteristics; Complement; Consensus; Curiosities; Data; Ensure; Fimbriae Proteins; Gel; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Human; Human Microbiome; Infection; Intestines; Knowledge; Measures; Methods; Microbial Biofilms; Modeling; Mutagenesis; Mutate; Mutation; Organism; Pathogenesis; Pathogenicity; Physiological; Pilum; Positioning Attribute; Process; Protein Secretion; Proteins; Proteomics; PubMed; Regulation; Reporter; Reporting; Research; Rest; Role; Scheme; Signal Transduction; Site; Structural Genes; Structure; Surface; System; Techniques; Testing; Time; Transcription Coactivator; Vibrio vulnificus; Virulence Factors; Work; appendage; cell motility; daughter cell; experimental study; extracellular; fitness; genetic regulatory protein; insight; interest; member; novel; pathogen; pathogenic bacteria; pilus genes; promoter; protein structure; protein-histidine kinase; recruit; response; structured data; yeast two hybrid system

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.

项目摘要 皮利是细菌以多种方式使用的细菌表面结构。它们可用于 运动性、遗传交换和表面附着，并且通常是病原体的关键毒力因子。一 在许多细菌病原体中发现了一种特殊类型的菌毛，flp菌毛，包括创伤弧菌和 放线菌共生聚集杆菌，并且被人类微生物组的一些成员用于 在肠道中建立持久性。尽管flp菌毛很重要， 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 使用为研究提供许多技术益处的模式细菌探索flp菌毛的实验 皮利：新月柄杆菌。flp系统的一个共同特点是菌毛蛋白基因受到调控 与其他菌毛基因分开。In C. crescentus，转录激活因子CtrA结合四个 菌毛蛋白启动子中的位点。初步数据表明，一个位点诱导表达，而其余的 位点抑制和/或延迟从其上游位置的转录，这是以前没有报道过的现象， 细菌将通过突变不同的CtrA结合位点来探索菌毛蛋白启动子的调节， 测量表达时间和幅度的变化。的直接相互作用和潜在的协同性， 将使用生物化学测定法评估菌毛蛋白启动子处的CtrA结合。最后，对桩柱的监管 亚基将被突变改变，以及菌毛蛋白亚基的差异调节的效果以及它如何被改变。 有助于菌毛合成以及生理后果，如噬菌体感染和表面 将检查附件（目标1）。flp菌毛的另一个共同特点是它经常是极性的 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于识别和表征导致TadZ的因素，从而导致flp 菌毛，成为极地本地化。此外，在这种生物体中， 将通过使用新的纯化策略纯化完整的皮利来确定， 从未实现过（目标2）。这些研究将提供深入了解flp中保守的特征 皮利系统，从而提供了对许多人类病原体和真菌的重要结构的深入了解。 有机体