Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals

项目摘要 实现细菌信号的高吞吐量分析和单细胞成像 我们的研究旨在了解细菌如何感知化学信号来调节不同的行为。我们 已经发明了不同类型的生物传感器来快速测量细菌中的关键信号分子，其中包括一种 环二GMP这种信号控制细菌是否附着在表面，形成粘性生物膜，并分泌 毒素我们的主要目标之一是确定营养物质、其他化学物质和环境投入的变化 不同细菌中的环二GMP水平。我们最近展示了一种成功的方法， 基于结构的生物信息学分析和实验筛选。然而，主要输入的发现 仍然具有挑战性，因为每种细菌都含有许多环状二GMP信号传导酶， 瞬时产生、高电荷和低丰度，并且筛选方法仍然是关键瓶颈。 因此，该提案将开发下一代荧光生物传感器，以提高高通量，定量 直接在细胞中筛选酶活性（目的2）。这些生物传感器将用于发现主要的 与环二GMP和其他信号传导相关的广泛小分子结合结构域的输入 酶（目的3）。此外，为了了解调节环二GMP的环境因素，本文 该提案将开发一种新型的生物传感器，用于对生物膜中的环二GMP进行原位成像（目的3）。在 从长远来看，该项目旨在为个性化饮食提供信息，以治疗炎症性肠病，并促进 肠道健康 对于本次更新的项目，原有的研究范围也有所扩大，渗透性小 分子进入细菌细胞。渗透过程包括被动渗透、主动吸收和主动吸收。 外排机制，并且对细菌生长、信号传导和抗生素抗性至关重要。这项建议会 开发一种能够实时监测细胞中小分子渗透性的高通量测定法（Aim 1）。该检测将被应用于了解分子结构和遗传因素，影响 荧光染料和临床抗生素在细胞内的积累。从长远来看，这一新目标将提高 使用这些染料和抗生素治疗的化学生物学工具。

PROJECT SUMMARY Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals Our research aims to understand how bacteria perceive chemical signals to regulate different behaviors. We have invented different types of biosensors to rapidly measure key signaling molecules in bacteria, including one for cyclic di-GMP. This signal controls whether bacteria attach to surfaces, form sticky biofilms, and secrete toxins. One of our major goals is to identify nutrients, other chemicals, and environmental inputs that change cyclic di-GMP levels in different bacteria. We recently demonstrated a successful approach that combines structure-based bioinformatics analysis and experimental screening. However, the discovery of primary inputs remains challenging because each bacterium harbors many cyclic di-GMP signaling enzymes, the signal is transiently produced, highly charged, and low in abundance, and the screening method remains a key bottleneck. Thus, this proposal will develop next-generation fluorescent biosensors to enhance high-throughput, quantitative screening of enzyme activity directly in cells (Aim 2). These biosensors then will be applied to discover primary inputs for a widespread small molecule binding domain associated with cyclic di-GMP and other signaling enzymes (Aim 3). In addition, towards understanding environmental factors that regulate cyclic di-GMP, this proposal will develop a new type of biosensor to perform in situ imaging of cyclic di-GMP in biofilms (Aim 3). In the long term, this project aims to inform personalized diets to treat inflammatory bowel diseases and promote gut health. For this renewal of the project, the original scope also has been expanded to study the permeability of small molecules into bacterial cells. The permeability process includes passive permeation, active uptake, and active efflux mechanisms, and is critical to bacterial growth, signaling, and antibiotic resistance. This proposal will develop a high-throughput assay that enables real-time monitoring of small molecule permeability in cells (Aim 1). The assay will be applied to understand both the molecular structures and genetic factors that affect accumulation of fluorescent dyes and of clinical antibiotics inside cells. In the long term, this new aim will improve chemical biology tools that use these dyes and antibiotics treatments.