This way out: Spatiotemporal regulation of Vibrio cholerae biofilm dispersal

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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.