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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要实现细菌信号的高通量分析和单细胞成像环状二核苷酸（CDN）是一类新兴的信号分子，位于细菌和细菌的交叉点上。主持人互动。在细菌细胞内，CDN 充当化学信号，控制不同的细胞程序定植（环二-GMP）、应激反应（环二-AMP）和表面接触（环AMP-GMP）。此外，这三种细菌 CDN 和一种新发现的哺乳动物 CDN（称为 cGAMP）被发现可以刺激哺乳动物细胞中通过称为 STING（刺激物的刺激物）的蛋白质受体的先天免疫信号传导途径干扰素基因）。因此，了解 CDN 水平如何受到环境和宿主输入的调节将有助于增进我们对细菌与宿主相互作用的了解，包括细菌病原体和宿主免疫反应。然而，获得这些关键机制见解的主要障碍是很难观察这些化学信号在不同尺度和系统中的水平变化。因此，该提案的广泛目标是开发能够实现高通量的发光和荧光生物传感器对从多个细胞到单个细胞（目标 1）、从培养物到宿主内（目标 2）以及从个体物种到群落（目标 3）。我们之前确定了一种新型的基因编码生物传感器，基于RNA的荧光（RBF）生物传感器，具有足够的灵敏度和选择性来跟踪和定量低丰度细胞内代谢物，包括 CDN。以我们早期发明的开启技术为基础用于环二 GMP 和环二 AMP 的 RBF 生物传感器，我们将开发设计策略来制造比率 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状态（目标 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氏菌，这是哺乳动物细胞内的李斯特氏菌病。我们将与肯塔基大学的史蒂文森教授合作研究伯氏疏螺旋体蜱虫体内的伯氏疏螺旋体是莱姆病的病原体。使 CDN 信号传导的研究成为可能细菌和模型微生物群落中，我们将采用广泛宿主载体系统进行基因组整合 RBF 生物传感器基因（目标 3）。此外，为了能够研究先天免疫信号 cGAMP，我们将进行高通量选择以制造新型 RBF 生物传感器（目标 4）。最后我们将开发生物发光共振能量转移 (BRET) 生物传感器，可用于定量环二 GMP 粗裂解物，并具有未来用于整体动物成像的潜力（目标 1）。与沃特斯教授合作密歇根州立大学，我们将使用这些新型 BRET 生物传感器来分析霍乱弧菌的反应，人类肠道胆汁酸是霍乱的病原体。