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.