PROJECT SUMMARY Antibiotic-resistant bacterial infections are becoming increasingly more prevalent, and Staphylococcus aureus infections in particular have high rates of antibiotic resistance, prompting research into alternative treatment strategies. S. aureus vaccines have been developed, but all have failed in clinical trials to date, likely due to the ability of S. aureus to blunt the immune system and block protective immune responses. S. aureus produces many virulence factors that promote disease, including an arsenal of toxins, which directly target and kill host immune cells. Several S. aureus toxins specifically bind to human cells, and are not active in the mouse, rendering it difficult to dissect the specific role of toxins in the disease process. Few models exist to study interactions between S. aureus communities, the toxins they produce, and their human cell targets. S. aureus causes a wide range of disease manifestations in the human host, from skin and soft tissue infections to bacteremia and systemic spread to deep tissues. When S. aureus enters the bloodstream, bacteria are trapped in the liver, and then spread to the kidney to form large lesions called abscesses. Abscesses contain a central core of tightly clustered bacteria, with concentric layers of necrotic and live neutrophils, and an outer layer of macrophages. Because it is difficult to penetrate abscesses with antibiotics, they can persist follow drug treatment, and may represent one of the reservoirs responsible for recurrent infections. Better understanding of the interactions between bacteria within these structures, and the interactions with surrounding host cells, will be critical in developing future therapeutics to more efficiently eliminate these structures. This proposal will utilize fluorescent transcriptional reporters to determine the spatiotemporal expression patterns of S. aureus toxin expression within kidney abscesses, and will also develop an in vitro system to study host-pathogen interactions within abscesses. We hypothesize that direct interactions with neutrophils, and diffusible antimicrobials from macrophages, promotes expression of virulence factors specifically at the periphery of abscesses. We will utilize fluorescent reporter strains and immunofluorescence microscopy to determine whether toxin expression patterns change over the course of kidney abscess formation in our mouse model, and determine if this is dictated by the presence of neutrophils and macrophages. We will also develop an in vitro model of abscess formation using 3D agarose droplets to encapsulate bacteria and adhere host cells to the droplet surface. In this model, we will visualize the dynamics of virulence factor expression in the presence and absence of mouse and human primary phagocytes using live imaging fluorescence microscopy. Establishing these robust systems to study S. aureus spatial patterning and host-pathogen interactions will enable us to uncover key S. aureus vul...