PROJECT SUMMARY Staphylococcus aureus is a major human pathogen that causes significant morbidity and mortality in both hospital- and community-acquired infections. The appearance of multidrug-resistant strains has compounded this problem, galvanizing efforts aimed at identifying new therapeutic targets. Staphylococcal infections are characterized by the formation of tissue abscesses which represent the primary site of interaction between a bacterial microcolony and the innate immune response of the host. We have discovered that the staphylococcal abscess exhibits remarkable molecular heterogeneity, challenging efforts focused on designing therapeutics or vaccines for the treatment and prevention of S. aureus infections. In this proposal, we will address this challenge through the development of a cutting-edge multi-modal imaging platform that will define the molecular inventory of the S. aureus abscess and reveal the contribution of host antimicrobials to the molecular composition of this infectious interface. This proposal combines our expertise in immunology, infection biology, mass spectrometry, small animal imaging, machine learning, and computer vision to develop an integrated multimodal visualization method for studying microbial communities and tissue abscesses. Our unique approach will reveal the proteins, lipids, small molecules, and transcripts that define the staphylococcal abscess. Through genetic inactivation of critical innate immune effectors, we will uncover how the host response to infection drives alterations in these lesions. These experiments will test the hypotheses that exposure to environmental stresses encountered within the host have a powerful effect on molecular abundance and distribution within the staphylococcal microcolony, and that the molecular environment of the abscess is highly variable and changes according to the stage of abscess development. Taken together, our next-generation imaging capability will (i) map molecular heterogeneity within microbial communities in response to environmental stressors, (ii) define molecular heterogeneity in tissue abscesses, and (iii) determine molecular profiles and discover spatio-molecular host and microbial factors that define the lifecycle of the staphylococcal abscess. These studies will uncover new targets for therapeutic intervention against this important human pathogen, and the techniques developed as a result of this proposal will be broadly applicable to all physiologically relevant processes, profoundly impacting biomedical research.