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: 10455425
• 负责人: Andrew A. Bridges
• 金额: $ 2.89万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455425
• 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.

项目总结 细菌在自由游泳的生活方式和固定的生物膜群落之间交替存在。这个 生物膜的生命周期包括三个阶段：创始细胞附着、生物膜成熟和扩散。《环球报》 霍乱弧菌在感染过程中形成生物膜，生物膜扩散是疾病传播的关键。 虽然促进霍乱弧菌生物膜形成的成分已经定义，但对霍乱弧菌几乎一无所知。 霍乱菌生物被膜扩散。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，包括扩散。利用这种成像技术和高含量的基因 通过筛选，我已经鉴定并开始鉴定霍乱弧菌生物被膜扩散所需的成分； 促进生物膜退出的信号转导蛋白、基质分解酶和运动功能。现在, 我的首要目标是定义在空间和时间上协调生物膜扩散的信号机制 单元格分辨率。关于信号转导成分，具有最极端生物膜的突变体 我屏幕上的分散-失败表型在一个完全没有特征的双组分调控中是有缺陷的 系统。这个电路由一个传感器组成，我称之为dBFS(用于生物膜传感器的扩散)，一个响应器 我命名为DbfR(用于生物膜的扩散调节)的调节器，以及一个未知的小分泌蛋白 函数VC1637，它编码在dBFS-DBFR操作子中，并控制dBFS活动。另外，我的基因 分析表明，必须存在第二个未知的传感器激酶，并使DbfR磷酸化。我提出了一个模型 其中，受不同刺激调节的两个传感器会聚在DbfR上，以控制霍乱弧菌生物膜的扩散。我 将利用显微镜、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)确定如何 小蛋白VC1637控制生物膜的扩散；(目标3)确定生物膜如何在单个- 细胞水平。这项拟议的研究将通过定义霍乱弧菌中的 分子水平的信号事件，影响单个细胞，导致整个种群退出生物膜。 此外，这项工作可能会揭示可以被操纵以激活生物膜扩散的靶标，这可能会指导 开发缩短霍乱弧菌感染持续时间的治疗方法。我的K99训练就会完成 在普林斯顿大学我的导师邦妮·巴斯勒教授的指导下，我沉浸在 充满活力的智力环境。我已经得到了几位合作者的支持，他们都是主题方面的专家 对我来说是全新的，比如蛋白质组学和生物物理学理论。此外，我计划进一步发展我的事业 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，了解到 我将获得，结合我现有的专业知识，将使我能够实现我的职业目标，成为一名 研究生物学基本问题的独立学术研究人员。