This way out: Spatiotemporal regulation of Vibrio cholerae biofilm dispersal

项目摘要 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之间交替。的 生物膜生命周期包括三个阶段：创始细胞附着、生物膜成熟和扩散。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是至关重要的。 虽然促进霍乱弧菌生物膜形成的成分已经确定，但对霍乱弧菌几乎一无所知。 胆固醇生物膜扩散。我开发了一种实时显微镜方法， 生物膜生命周期，包括扩散，在霍乱弧菌。利用这种成像技术和高含量的遗传 筛选，我已经确定并开始表征霍乱弧菌生物膜扩散所需的组分; 信号转导蛋白、基质分解酶和促进生物膜排出的运动功能。现在我想， 我的首要目标是确定在空间和时间上协调生物膜扩散的信号机制， 单细胞分辨率。关于信号转导成分，具有最极端生物膜的突变体 从我的屏幕分散失败表型是在一个完全不典型的双组分调节缺陷 系统该电路由一个我命名为DbfS（生物膜分散传感器）的传感器组成， 我将其命名为DbfR（生物膜调节器的分散），以及一种未知的小分泌蛋白质。 在dbfS-dbfR操纵子中编码并控制DbfS活性的功能VC 1637。此外，我的基因 分析表明，必须存在第二种未知的传感激酶并使DbfR磷酸化。我提出一个模型 其中两个受不同刺激调节的传感器会聚在DbfR上以控制霍乱弧菌生物膜扩散。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：（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;（目的2）定义 小蛋白，VC 1637，控制生物膜分散;（目的3）确定生物膜分散如何发生在单- 细胞水平。拟议的研究将揭示如何通过定义 分子水平的信号事件，冲击单个细胞，导致整个种群从生物膜中退出。 此外，这项工作可以揭示可以操纵以激活生物膜分散的靶点， 开发减少霍乱弧菌感染持续时间的治疗方法。我的K99培训将完成 在我的导师普林斯顿大学的邦妮·巴斯勒教授的指导下，我沉浸在一个 充满活力的智力环境。我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，我还计划进一步发展 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R 00阶段开始时， 结合我现有的专业知识，将使我能够实现我的职业目标， 研究生物学基本问题的独立学术研究员。

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.