PROJECT SUMMARY Despite high levels of antibiotic exposure, some individual bacterial cells survive multiple courses of antibiotic treatment due to antibiotic tolerance, which is a transient phenotypic change associated with reduced metabolism or slowed growth. It remains unclear what drives the formation of tolerant cells within host tissues, and whether the antimicrobials generated by the host immune system may contribute to the formation of antibiotic tolerant bacterial cells. To investigate the impact of host-derived stresses, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), on antibiotic tolerance, we are probing the growth of the human pathogen, Yersinia pseudotuberculosis, in a mouse model. Within host tissues, this microbe replicates to form clonal clusters of extracellular bacteria that directly interface with a layer of neutrophils that are, in turn, enveloped by a layer of macrophages. The bacterial subpopulation at the outer edge of the microcolony responds to neutrophil contact by upregulating expression of the anti-phagocytic, type III secretion system (T3SS). These host cell-associated bacteria are part of a larger peripheral layer, which responds to and detoxifies diffusible nitric oxide (NO) originating from distal macrophages. This system clearly allows us to distinguish the spatial location of individual bacterial cells, and to determine if host cell interactions, or interactions with the antimicrobials they produce, are contributing to the formation of antibiotic tolerant bacterial populations. We hypothesize that antibiotic tolerant bacteria emerge within host tissues due to innate immune cell-derived ROS and RNS. We have recently developed a novel reporter system to detect antibiotic (doxycycline) exposure within individual bacterial cells in host tissues, and will use this tool to: 1) define the roles of immune cell-derived ROS and RNS in promoting of antibiotic tolerance, 2) determine the pathways utilized by doxycycline-tolerant subpopulations within host tissues. In addition, we have also developed an innovative agarose droplet-based system to model growth within host tissues, which will enable kinetic analyses, and provide a more thorough understanding of how bacterial communities interact with immune cells in vivo. The extent of bacterial heterogeneity within host tissues has been largely unexplored, making this a novel, understudied aspect of the disease process, and the focus of research in my lab. Identifying and understanding the cues that promote the formation of tolerant bacterial subpopulations has important implications for the design of novel therapeutics, which need to target all subpopulations to effectively eliminate bacteria within host tissues.