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

基本信息

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
项目类别：
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

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

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项目摘要

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 pili是细菌在许多方面使用的细菌表面结构。它们可用于运动，遗传交换和表面附着，通常是病原体的关键毒力因子。一个在许多细菌病原体中发现了特定类型的菌毛，flp pilus，包括颤音和聚集的放线菌cotemcetemcomitans，并被人类微生物组的某些成员使用在肠道中建立永久性。尽管它很重要，但FLP pilus并未接近研究其他Pilus系统的深度，并具有许多未开发的方面和好奇心。在此提案中，我们概述了使用模型细菌探索FLP Pilus的实验，该模型细菌为研究提供了许多技术优势 pili：caulobacter crescentus。 FLP系统的一个共同特征是调节PILIN基因与其他菌毛基因分开。在C. crescentus中，转录激活剂CTRA与四个 PILIN启动子中的站点。初步数据表明一个站点诱导表达，而其余的站点抑制和/或延迟从其上游位置转录，这种现象先前未在细菌。通过突变不同的CTRA结合位点，将探索PILIN启动子的调节测量表达时序和幅度的变化。直接互动和潜在的合作 PILIN启动子的CTRA结合将使用生化测定法进行评估。最后，对皮林的规定亚基将因诱变而改变，以及pilin亚基的差异调节及其的影响促进菌毛合成以及生理后果，例如噬菌体感染和表面将检查附件（AIM 1）。 flp pilus的另一个常见特征是它通常是极地的本地化。定位取决于tadz蛋白，但是该蛋白如何定位是一个谜。蛋白质相互作用技术将用于识别和表征引起tadz的因素，从而识别和表征FLP Pilus，将成为两极分化的局部化。此外，整个蛋白质补充了该生物的Flp pilus 将通过使用新颖的纯化策略纯化整个完整的pili来确定从来没有完成过（AIM 2）。这些研究将洞悉FLP中保守的特征 pili系统，因此可以洞悉对许多人类病原体和共生重要的结构有机体。