PROJECT SUMMARY/ABSTRACT This MIRA proposal outlines an integrated research program at the interface of chemistry and biology focused on cell-cell communication in bacteria, or ?quorum sensing? (QS). QS has a major impact on human health, with some of the most common pathogens utilizing this sensing mechanism to regulate virulence?i.e., the ability to initiate infection?once sufficient cells have amassed to overwhelm a host. Understanding the molecular mechanisms of QS, its role in mixed microbial communities, and its impact on both acute and chronic disease remain pressing and unaddressed challenges in the field. For example, our understanding of how QS signaling molecules interact with their target protein receptors to activate or inhibit QS pathways is limited to four species in Gram-negative bacteria. Further, with an increasing awareness of the importance of microbial communities (i.e., our ?microbiomes?) to human health, it is astonishing how little we know about the role of chemical signaling between these organisms in the maintenance (or disruption) of healthy microbial consortia. As bacteria use simple chemical signals to regulate QS, synthetic chemists and chemical biologists are well positioned to address these problems and other related challenges at the molecular level. With support from the NIH over the past decade, the PI has advanced the development of synthetic ligands that modulate QS signaling systems in Gram-negative bacteria and has shown that these ligands can strongly attenuate QS- controlled behaviors in many pathogens. This past work situates her ideally to lead this research project. The overall vision for this MIRA project is to build on the PI's 12-year foundation of results and leadership in this area and apply a chemical approach to expand the understanding of QS across multiple scales?from individual QS signal:receptor interactions to signaling in a single species to signaling within mixed bacterial populations to interactions of the community with a host. We will achieve this vision through the pursuit of three broad Goals: (1) the development of new small molecules capable of strongly modulating QS in Gram- negative bacteria with high potencies, stabilities, and defined modes of action; (2) the application of these molecules and new chemical strategies to delineate the biochemical mechanisms of QS; and (3) characterization of the roles of QS in mixed microbial environments relevant to human health. These three Goals will be pursued through an integration of chemical synthesis, chemical biology, bacteriology, biochemistry, structural biology, and genomics. Studies will be performed in the PI's laboratory at the UW? Madison and with a team of committed collaborators with expertise in QS and methods critical to this project. The overall outcome of this project will be a drastically increased and rigorously tested understanding of QS in bacteria and its role in biologically significant environments, and a suite of new and freely accessible research tools for the QS field. Our findings will shape the development of new methods to treat bacterial disease and will directly impact human health.