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 环状二核苷酸（CDN）是一类新兴的信号分子，其位于细菌和 主机交互。在细菌细胞内，CDN作为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，发现这三种细菌CDN和一种新发现的哺乳动物CDN（称为cGAMP）可以刺激 哺乳动物细胞中的先天免疫信号传导途径，通过称为STING的蛋白质受体（刺激因子） 干扰素基因）。因此，了解CDN水平如何受环境和主机输入的调节， 促进我们对细菌-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，获得这些关键的机械见解的主要障碍是 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 从许多细胞到单细胞的CDN分析和成像（Aim 1），从培养物到宿主内的CDN分析和成像（Aim 2）， 3、个体物种到群落（Aim 3）。我们之前已经确定了一种新的基因编码的 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 定量低丰度的细胞内代谢物，包括CDN。基于我们早期发明的 对于环二GMP和环二AMP的RBF生物传感器，我们将开发设计策略以使比率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菌， 哺乳动物细胞内的线粒体病。我们将与肯塔基州的史蒂文森教授合作，研究疏螺旋体 莱姆病的病原体--伯氏螺旋体。为了能够在不同的环境中研究CDN信号， 在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 的RBF生物传感器基因（目的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，其可以应用于定量环二GMP， 粗裂解物，并具有未来的潜力，为整个动物成像（目标1）。与沃茨教授合作， 密歇根州立大学，我们将使用这些新的BRET生物传感器来分析霍乱弧菌的反应， 霍乱的病原体，对人体肠道胆汁酸。

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.