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

项目总结 实现细菌信号的高通量分析和单细胞成像 环二核苷酸(CDN)是一类新兴的信号分子，位于细菌和 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(循环di-GMP)、应激反应(循环di-AMP)和表面接触(循环AMP-GMP)。 此外，这三种细菌CDN和一种新发现的哺乳动物CDN cGAMP被发现能够刺激 哺乳动物细胞中一种通过称为刺激物(Stimator)的蛋白质受体的先天性免疫信号通路 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 从细菌病原体和宿主两个方面促进我们对细菌与宿主相互作用的了解 免疫反应。然而，获得这些关键的机械论见解的主要障碍是 很难观察到这些化学信号在不同尺度和系统中的水平变化。因此， 这项计划的主要目标是开发发光和荧光生物传感器，使高通量 从多个细胞到单个细胞(目标1)、从培养物到宿主内(目标2)和从 从个体物种到群落(目标3)。我们之前证实了一种新型的基因编码的 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 量化低丰度的细胞内代谢物，包括CDN。建立在我们早期发明的开机基础上 环状双环戊二烯和环状双环戊二烯的径向基函数生物传感器，我们将开发设计策略来制造比率径向基函数。 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……里面 与加州大学伯克利分校的波特诺教授合作，我们将研究单核细胞增多性李斯特菌，这是 哺乳动物细胞内的李斯特菌病。与肯塔基大学的史蒂文森教授合作，我们将研究疏螺旋体 莱姆病的病原体伯氏杆菌。使CDN信号在不同领域的研究成为可能 细菌和模型微生物群落中，我们将使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，我们将发展 生物荧光共振能量转移(BRET)生物传感器应用于环二核苷酸的定量研究 粗制的裂解物，未来有可能用于全动物成像(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， 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.