This way out: Spatiotemporal regulation of Vibrio cholerae biofilm dispersal

出路：霍乱弧菌生物膜扩散的时空调控

• 批准号: 10188774
• 负责人: Andrew A. Bridges
• 金额: $ 10.92万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188774
• 关键词: Alleles; Bacteria; Binding; Biochemistry; Biology; Biophysics; Cell Fractionation; Cell-Matrix Junction; Cells; Communities; Educational workshop; Environment; Enzymes; Epitopes; Event; Exhibits; Failure; Gene Expression; Genes; Genetic Screening; Goals; Growth; Heterogeneity; Imaging Techniques; Individual; Infection; Knowledge; Lead; Life Style; Light; Liquid substance; Luciferases; Mass Spectrum Analysis; Measures; Mentors; Microbial Biofilms; Microbiology; Microscopy; Modeling; Molecular; Monitor; Names; Operon; Peptide Hydrolases; Phase; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Population; Process; Production; Proteins; Proteomics; Regulation; Regulon; Reporter; Research; Research Personnel; Resolution; Role; Severity of illness; Signal Transduction; Stimulus; Swimming; System; Time; Training; Universities; Vibrio cholerae; Vibrio cholerae infection; Western Blotting; Work; Writing; bacterial genetics; career; cell motility; comparative; disease transmission; extracellular; genetic analysis; mutant; new technology; pathogen; professor; promoter; response; screening; sensor; spatiotemporal; symposium; theories; therapy development; tool; transcriptome sequencing

PROJECT SUMMARY Bacteria alternate between a free-swimming lifestyle and existing in sessile communities known as biofilms. The biofilm lifecycle consists of three stages: founder cell attachment, biofilm maturation, and dispersal. The global pathogen Vibrio cholerae forms biofilms during infection and biofilm dispersal is critical for disease transmission. While the components facilitating V. cholerae biofilm formation are defined, almost nothing is known about V. cholerae biofilm dispersal. I developed a real-time microscopy approach that permits examination of the entire biofilm lifecycle, including dispersal, in V. cholerae. Using this imaging technique and high-content genetic screening, I have identified and begun characterizing components required for V. cholerae biofilm dispersal; signal transduction proteins, matrix disassembling enzymes, and motility functions that promote biofilm exit. Now, my overarching goal is to define the signaling mechanisms that coordinate biofilm dispersal in space and time at single-cell resolution. Regarding signal transduction components, the mutant with the most extreme biofilm dispersal-failure phenotype from my screen is defective in a wholly uncharacterized two-component regulatory system. This circuit is composed of a sensor that I named DbfS (for Dispersal of Biofilm Sensor), a response regulator that I named DbfR (for Dispersal of Biofilm Regulator), and a small secreted protein of no known function, VC1637, that is encoded in the dbfS-dbfR operon and controls DbfS activity. In addition, my genetic analyses show that a second, unknown, sensor kinase must exist and phosphorylate DbfR. I propose a model in which two sensors, regulated by different stimuli, converge on DbfR to control V. cholerae biofilm dispersal. I will use the tools of microscopy, bacterial genetics, proteomics, biochemistry, and biophysics theory to: (Aim 1) determine how DbfR integrates information from two sensors to control biofilm dispersal; (Aim 2) define how the small protein, VC1637, controls biofilm dispersal; (Aim 3) determine how biofilm dispersal occurs at the single- cell level. The proposed research will reveal how dispersal is coordinated in V. cholerae by defining the molecular-level signaling events, impinging on individual cells, that lead to population-wide exit from biofilms. Moreover, this work could reveal targets that can be manipulated to activate biofilm dispersal, possibly guiding development of treatments that reduce the duration of V. cholerae infection. My K99 training will be completed under the guidance of my mentor Professor Bonnie Bassler at Princeton University where I am immersed in a vibrant intellectual environment. I have enlisted the support of several collaborators who are experts in topics that are wholly new to me, such as proteomics and biophysical theory. In addition, I plan to further my growth through participation in microbiology conferences, attendance of courses in proteomics, biophysics, and lab management, and by partaking in scientific writing workshops. By the start of the R00 phase, the knowledge that I will have gained, combined with my existing expertise, will enable me to achieve my career goal of being an independent academic researcher tackling fundamental problems in biology.

项目概要 细菌在自由游动的生活方式和存在于被称为生物膜的固着群落中交替存在。这 生物膜生命周期由三个阶段组成：创始细胞附着、生物膜成熟和分散。全球 病原体霍乱弧菌在感染过程中形成生物膜，生物膜的扩散对于疾病传播至关重要。 虽然促进霍乱弧菌生物膜形成的成分已被确定，但对霍乱弧菌几乎一无所知。 霍乱生物膜扩散。我开发了一种实时显微镜方法，可以检查整个 霍乱弧菌的生物膜生命周期，包括传播。利用这种成像技术和高内涵遗传 通过筛选，我已经确定并开始表征霍乱弧菌生物膜传播所需的成分； 信号转导蛋白、基质分解酶和促进生物膜退出的运动功能。现在， 我的首要目标是定义协调生物膜在空间和时间上扩散的信号机制 单细胞分辨率。关于信号转导成分，生物膜最极端的突变体 我的屏幕中的分散失败表型在完全未表征的双组分调节中存在缺陷 系统。该电路由我命名为 DbfS（生物膜分散传感器）的传感器组成，响应 我将其命名为 DbfR（生物膜调节器的分散）的调节器，以及一种未知的小分泌蛋白 函数 VC1637，在 dbfS-dbfR 操纵子中编码并控制 DbfS 活动。另外，我的遗传 分析表明，必须存在第二种未知的传感器激酶并磷酸化 DbfR。我提出一个模型 其中两个受不同刺激调节的传感器汇聚在 DbfR 上以控制霍乱弧菌生物膜的扩散。我 将使用显微镜、细菌遗传学、蛋白质组学、生物化学和生物物理学理论的工具来：（目标 1） 确定 DbfR 如何整合来自两个传感器的信息来控制生物膜扩散； （目标 2）定义如何 小蛋白质 VC1637 控制生物膜分散； （目标 3）确定生物膜分散是如何在单点发生的 细胞水平。拟议的研究将通过定义霍乱弧菌的传播是如何协调的 分子水平的信号事件，影响单个细胞，导致整个群体退出生物膜。 此外，这项工作可以揭示可操纵以激活生物膜分散的目标，可能指导 开发缩短霍乱弧菌感染持续时间的治疗方法。我的K99培训即将完成 在我的导师普林斯顿大学 Bonnie Bassler 教授的指导下，我沉浸在 充满活力的智力环境。我已经获得了几位合作者的支持，他们都是该主题的专家 这对我来说是全新的，例如蛋白质组学和生物物理理论。此外，我计划进一步成长 通过参加微生物学会议、参加蛋白质组学、生物物理学和实验室课程 管理，并参加科学写作研讨会。在 R00 阶段开始时，我们知道 结合我现有的专业知识，我将获得的知识将使我能够实现成为一名 解决生物学基本问题的独立学术研究员。