PROJECT SUMMARY Polymicrobial communities are ubiquitous in the human body and their behaviors are critical drivers of both health and disease. Bacteria are social organisms; thus, the behavior of the group is driven not only by the composition of the community, but also by interactions between the constituents and their surrounding environment. A key principle shared among all communities, is that space matters. Groups organize to maximize acquisition of goods, minimize exposure to toxic compounds, and optimize communication. Our recent data reveal bacteria can communicate with members of distant species and respond by changing how they spatially structure their communities. My laboratory seeks to understand how bacteria communicate between species by observing them in their native environment, tracking their movements, and listening to and decoding their languages. We utilize Pseudomonas aeruginosa, the most notoriously problematic opportunistic pathogen in multiple types of polymicrobial infections, including chronic wounds and lung infections in cystic fibrosis (CF) patients. We recently reported that P. aeruginosa establishes infections with other important pathogens in the lungs of patients with CF for decades, remaining unresponsive to intense antibiotic therapies and causing lung decline and early death. In this proposal, we begin by visualizing the spatial landscape of polymicrobial communities in situ, in transplanted lungs from CF patients and animal models of polymicrobial infection. By combining next-generation tissue clearing and fluorescent in situ hybridization, we are able to visualize the spatial positioning of species on a range of spatial scales, in relation to each other and host structures. This will yield an unprecedented view of microbes during infection and provide a platform for visualizing communities in other organs rich in polymicrobial communities, such as the gastrointestinal tract. Next, we will systematically decode the interspecies signaling language. We designed high-throughput screens to identify genes necessary for signaling and the repertoire of signaling molecules. Using live-imaging techniques pioneered by my lab, we visualize and track the movement of bacterial cells, gene expression, and dynamics of motility appendages, upon modulation of the identified pathways. Initial studies reveal P. aeruginosa responds by activating multiple signaling systems, which tune the direction of movement and activate virulence systems. Collectively, these studies will construct a comprehensive picture of interspecies communication and enlighten how interactions exacerbate disease.