PROJECT SUMMARY How microbial pathogens evade host cellular defenses remains unclear. A full molecular understanding of host-pathogen interactions will aid in the development of new approaches to treat infection. There is a particular need to identify new classes of chemicals that target Gram-negative human pathogens. We therefore developed an in-cell, high content microscopy-based screening platform (called SAFIRE) that identifies compounds which prevent the accumulation of GFP- expressing Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages. We are now at the stage of establishing compound mechanism of action, including defining why compounds enable the macrophage to survive while the bacterium is killed. The compound we propose to study, JD1, is not sufficiently developed to be a lead compound for drug development but is instead at the stage of basic biological discovery. These efforts may reveal new facets of host-pathogen biology, new targets for Gram-negative bacterial pathogens, and possibly identify a chemical that could be improved for use as an antimicrobial. 0