SUMMARY The larger goal of the project is to identify infection-dependent weaknesses of Gram-negative bacterial pathogens that could be exploited chemically. We focus on the human pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and its survival within mammalian cells and use cell culture infection models and small molecules as tools. We and others have observed that some small molecules that are not effective against Gram-negative bacteria in media prevent pathogen survival during infection of cells and animals. Gram-negative bacteria likely become vulnerable during infection to small molecules because a variety of host innate immune mechanisms permeabilize the bacterial cell envelope and/or occupy efflux pumps, enabling compounds to reach a periplasmic or cellular target. When laboratory media imitates host innate immunity by permeabilizing the outer membrane, these compounds inhibit bacterial growth. We have identified small molecules that under broth conditions that permeabilize the outer membrane or in protoplasts, dissipate bacterial inner membrane voltage without physically disrupting the lipid bilayer. Membrane voltage disruption could compromise bacteria by, for instance, interfering with energy production and/or activating stress- response signaling. The compounds do not disrupt mammalian membranes at concentrations that kill S. Typhimurium in macrophages and at least one of the compounds reduces tissue colonization in mice, demonstrating in vivo potential for this approach. The Specific Aims will interrogate five distinct molecule classes to determine whether targeting the inner membrane should be pursued as a novel approach to fighting Gram-negative bacterial infections.