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.

项目摘要 细菌在自由速度的生活方式与现有的无柄群落之间交替，称为生物膜。这 生物膜生命周期包括三个阶段：创始人的细胞附着，生物膜成熟和分散。全球 病原体弧菌霍乱在感染过程中形成生物膜，而生物膜分散对于疾病传播至关重要。 虽然定义了促进V.霍乱生物膜形成的组件，但几乎一无所知。 霍乱生物膜分散。我开发了一种实时显微镜方法，可以检查整个 生物膜生命周期（包括分散）在V.霍乱中。使用这种成像技术和高含量遗传 筛选，我已经确定并开始表征V.霍乱生物膜分散所需的组件； 信号转导蛋白，基质拆卸酶和促进生物膜出口的运动函数。现在， 我的总体目标是定义在时空分布在时空分布的信号传导机制 单细胞分辨率。关于信号转导组件，具有最极端生物膜的突变体 我屏幕上的分散失效表型在完全未表征的两组式调节中有缺陷 系统。该电路由我命名为DBFS（用于生物膜传感器的分散）的传感器组成，响应 我命名为DBFR的调节剂（用于散布生物膜调节剂）和一个小的分泌蛋白 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，必须存在第二个未知的传感器激酶并磷酸化DBFR。我提出了一个模型 其中两个受不同刺激调节的传感器在DBFR上收敛以控制V.霍乱生物膜分散。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：（目标1） 确定DBFR如何整合来自两个传感器的信息以控制生物膜扩散； （目标2）定义如何 小蛋白质VC1637控制生物膜扩散； （AIM 3）确定生物膜分散是如何发生在单个 细胞水平。拟议的研究将揭示如何通过定义霍乱弧菌的分散。 分子级信号传导事件，影响各个细胞，从而导致人口范围从生物膜中退出。 此外，这项工作可以揭示可以操纵以激活生物膜扩散的目标 开发减少霍乱感染持续时间的治疗方法。我的K99培训将完成 在我的导师教授邦妮·巴斯勒（Bonnie Bassler）的指导下 充满活力的智力环境。我已经获得了几位合作者的支持，这些合作者是主题专家 对我来说是全新的，例如蛋白质组学和生物物理理论。此外，我计划进一步发展 通过参加微生物学会议，蛋白质组学课程，生物物理学和实验室的参加 管理，并通过参加科学写作研讨会。到R00阶段的开始， 我将获得并结合我现有的专业知识，使我能够实现自己的职业目标 独立的学术研究员解决生物学的基本问题。