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: 9368567
• 负责人: Ming Chen Hammond
• 金额: $ 32.36万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 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 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9368567
• 关键词: Affinity; Animals; Bacteria; Bacterial Genome; Behavior; Bile Acids; Biological Assay; Biosensor; Borrelia burgdorferi; Cell Culture Techniques; Cell physiology; Cells; Cellular biology; Chemicals; Cholera; Collaborations; Combating Antibiotic Resistant Bacteria; Communities; Cyclic AMP; Decision Making; Development; Diet; Dinucleoside Phosphates; Energy Transfer; Escherichia coli; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Future; Gene Expression; Genes; Genomics; Goals; Grant; Health Status; Human; Image; Immune response; Immune signaling; Individual; Interferons; Intestines; Kentucky; Knowledge; Ligands; Light; Link; Listeria monocytogenes; Listeriosis; Lyme Disease; Mammalian Cell; Maps; Michigan; Microbial Biofilms; Microscope; Modeling; Molecular; Natural Immunity; Organism; Outcome; Pathway interactions; Periodicity; Production; Proteins; RNA; Race; Reader; Reagent; Regulation; Reporting; Research; Second Messenger Systems; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Surface; Symbiosis; System; Technology; Ticks; Toxin; Transfer RNA; United States National Institutes of Health; Vibrio cholerae; Water; Work; animal imaging; aptamer; arm; base; biological adaptation to stress; cell motility; cellular imaging; complex biological systems; design; gut microbiota; high throughput analysis; high throughput screening; imaging modality; imaging platform; in vivo; innovation; insight; invention; microbial community; nanomolar; novel; pathogen; prebiotics; programs; public health relevance; ratiometric; receptor; response; small molecule; spatiotemporal; tool; vector; whole animal imaging

PROJECT SUMMARY Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals Cyclic dinucleotides (CDNs) are an emerging class of signaling molecules at the intersection of bacterial and host interactions. Within bacterial cells, CDNs act as chemical signals that control distinct cellular programs for colonization (cyclic di-GMP), stress response (cyclic di-AMP), and surface contact (cyclic AMP-GMP). Furthermore, these three bacterial CDNs and a newfound mammalian CDN called cGAMP are found to stimulate an innate immune signaling pathway in mammalian cells through a protein receptor called STING (Stimulator of Interferon Genes). Thus, understanding how CDN levels are regulated by environmental and host inputs would advance our knowledge of bacterial-host interactions, on both the side of bacterial pathogens and the host immune response. However, the major roadblock to obtaining these critical mechanistic insights has been the difficulty in observing changes in the levels of these chemical signals across scales and systems. Thus, the broad goals of this proposal are to develop luminescent and fluorescent biosensors that enable high-throughput analysis and imaging of CDNs from many to single cells (Aim 1), from cultures to within hosts (Aim 2), and from individual species to communities (Aim 3). We previously established that a new type of genetically-encoded biosensors, RNA-based fluorescent (RBF) biosensors, have sufficient sensitivity and selectivity to track and quantitate low abundance, intracellular metabolites including CDNs. Building on our earlier invention of turn-on RBF biosensors for cyclic di-GMP and cyclic di-AMP, we will develop design strategies to make ratiometric RBF biosensors for these CDNs that can report on the signaling status of bacterial pathogens within hosts (Aim 2). In collaboration with Prof. Portnoy at UC Berkeley, we will study Listeria monocytogenes, the causative agent of listeriosis, within mammalian cells. In collaboration with Prof. Stevenson at U Kentucky, we will study Borrelia burgdorferi, the causative agent of Lyme disease, in the tick. To enable the study of CDN signaling in diverse bacteria and in model microbial communities, we will employ a broad-host vector system for genomic integration of RBF biosensor genes (Aim 3). Furthermore, to enable the study of the innate immune signal cGAMP, we will perform high-throughput selections to make novel RBF biosensors (Aim 4). Finally, we will develop bioluminescent resonance energy transfer (BRET) biosensors that can be applied to quantitate cyclic di-GMP in crude lysates and have future potential for whole animal imaging (Aim 1). In collaboration with Prof. Waters at Michigan State, we will use these novel BRET biosensors to analyze the response of Vibrio cholerae, the causative agent of cholera, to human intestinal bile acids.

项目摘要 启用细菌信号的高通量分析和单细胞成像 环状二核苷酸（CDN）是细菌和 主机互动。在细菌细胞中，CDN充当化学信号，控制不同的细胞程序 定植（环状DI-GMP），应力反应（环状DI-AMP）和表面接触（环状AMP-GMP）。 此外，发现这三个细菌CDN和新发现的哺乳动物CDN被发现刺激 哺乳动物细胞中的先天免疫信号通路通过称为刺激的蛋白质受体（刺激剂 干扰素基因）。因此，了解如何通过环境和主机输入调节CDN水平 在细菌病原体和宿主的一侧都提高了我们对细菌宿主相互作用的了解 免疫反应。但是，获得这些关键机理见解的主要障碍是 难以观察跨尺度和系统的这些化学信号水平的变化。因此， 该提案的广泛目标是开发发光和荧光生物传感器，使高通量能够 从许多细胞到单个细胞（AIM 1）的CDN分析和成像，从培养到宿主内部（AIM 2），从 各种物种到社区（目标3）。我们以前确定了一种新型的遗传编码 生物传感器，基于RNA的荧光（RBF）生物传感器，对跟踪和选择性具有足够的灵敏度和选择性 定量低丰度，包括CDN在内的细胞内代谢产物。建立我们较早的上交发明 RBF生物传感器用于环状DI-GMP和循环DI-AMP，我们将制定设计策略以制造比率RBF 这些CDN的生物传感器可以报告宿主内细菌病原体的信号传导状态（AIM 2）。在 与加州大学伯克利分校的Portnoy教授合作，我们将研究单核细胞增生李斯特菌， 李斯特氏病，在哺乳动物细胞内。与肯塔基州史蒂文森教授合作，我们将学习Borrelia Burgdorferi是莱姆病的病因，在壁虱中。为了研究不同的CDN信号传导 细菌和模型微生物群落中，我们将采用基因组整合的广泛主持媒介系统 RBF生物传感器基因（AIM 3）。此外，为了实现先天免疫信号CGAMP的研究，我们将 执行高通量选择以制造新型的RBF生物传感器（AIM 4）。最后，我们将发展 生物发光谐振能传递（BRET）生物传感器可用于定量环状二-GMP 粗裂解物，并具有将来的整个动物成像的潜力（AIM 1）。与Waters教授合作 密歇根州立大学，我们将使用这些新颖的BRET生物传感器来分析Vibrio Cholerae的响应， 霍乱的致病药物，对人肠胆汁酸。