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

实现细菌信号的高通量分析和单细胞成像

• 批准号: 10522177
• 负责人: Ming Chen Hammond
• 金额: $ 35.48万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522177
• 关键词: Affect; Animal Model; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Behavior; Binding; Bioinformatics; Biological Assay; Biology; Biosensor; Cell Culture Techniques; Cell physiology; Cells; Charge; Chemicals; Cholera; Clinical; Combating Antibiotic Resistant Bacteria; Complex; Development; Diet; Drug Compounding; Drug Design; Dyes; Electron Microscopy; Environment; Environmental Risk Factor; Enzymes; Flow Cytometry; Fluorescent Dyes; Food Poisoning; Foundations; Funding; Gene Expression; Genetic; Goals; Gram-Negative Bacteria; Growth; Health; Health Status; Human; Image; Image Cytometry; Inflammatory Bowel Diseases; Light; Link; Mammalian Cell; Measures; Messenger RNA; Methods; Microbial Biofilms; Microbiology; Modeling; Molecular Genetics; Molecular Structure; Monitor; Noise; Nutrient; Organism; Outcome; Pathway interactions; Periodicity; Permeability; Phase; Physiological; Process; Production; Property; RNA; Regulation; Regulator Genes; Regulatory Pathway; Research; Signal Transduction; Signaling Molecule; Speed; Structure; Structure-Activity Relationship; Surface; Technology; Time; Toxin; Typhoid Fever; Urinary tract infection; base; cell motility; cellular imaging; enzyme activity; genetic analysis; gut colonization; gut health; gut microbiota; high throughput analysis; high throughput screening; improved; in situ imaging; insight; molecular imaging; next generation; novel; pathogen; pathogenic bacteria; prebiotics; programs; public health relevance; ratiometric; real time monitoring; screening; small molecule; tool; uptake

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.

项目摘要 启用细菌信号的高通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，包括一个 用于环状DI-GMP。该信号控制细菌是否附着在表面上，形成粘性生物膜并分泌 毒素。我们的主要目标之一是确定改变的营养，其他化学物质和环境投入 不同细菌中的环状DI-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。但是，发现主要输入 仍然具有挑战性，因为每个细菌都有许多环状DI-GMP信号传导酶，该信号为 瞬时产生，高电荷且丰度低，筛选方法仍然是关键的瓶颈。 因此，该建议将开发下一代荧光生物传感器，以增强高通量，定量 直接在细胞中筛选酶活性（AIM 2）。然后，这些生物传感器将用于发现主要 与环状DI-GMP相关的广泛的小分子结合结构域的输入和其他信号 酶（目标3）。此外，要了解调节环状DI-GMP的环境因素，这 提案将开发一种新型的生物传感器，以在生物膜中进行环状DI-GMP的原位成像（AIM 3）。在 长期，该项目旨在告知个性化饮食以治疗炎症性肠病并促进 肠道健康。 对于该项目的续约，原始范围也已扩展以研究小型的渗透性 分子进入细菌细胞。渗透率过程包括被动渗透，主动吸收和有效 外排机制，对于细菌生长，信号传导和抗生素耐药性至关重要。该提议将 开发高通量测定法，该测定能够实时监测细胞中的小分子渗透性（AIM 1）。该测定将用于了解影响分子结构和遗传因素 荧光染料和细胞内临床抗生素的积累。从长远来看，这个新目标将改善 使用这些染料和抗生素治疗的化学生物学工具。