ABSTRACT Diffusible signal factors (DSFs), long-chain fatty acids with a characteristic cis-2 unsaturation, are produced and used by several genera of gram-negative bacteria as quorum-sensing signals. We have found that the DSF cis-2 hexadecenoic acid (c2-HDA) is extremely potent in inhibiting expression of Salmonella functions necessary for colonization of the intestine and have found this compound to be present in the murine large intestine. As no mammalian source of fatty acids harboring a 2-cis unsaturation has been described, these findings strongly suggest that constituents of the gut microbiota produce and excrete DSFs that inhibit Salmonella virulence. We hypothesize that Salmonella uses the signals of these bacteria to balance its virulence functions, essential but also costly to the fitness and survival of the invading bacteria, with colonization and proliferation of the Salmonella population. Gut microbial metabolites may therefore serve multiple coordinated purposes in pathogens, balancing virulence functions with those required for proliferation within a host and thus affecting pathogen survival in the gut by multiple means. Here we propose to: Aim 1: Use complementary approaches to identify bacteria of the human gut microbiome that produce inhibitory DSFs and characterize their products; Aim 2: Identify the constellation of functions regulated in Salmonella by DSFs and identify mechanisms of this control, and; Aim 3: Using established murine models of Salmonella infection, characterize the biological function and translational relevance of c2-HDA to understand its mechanism of action and to support the eventual development of novel therapeutics, such as live biotherapeutic products, for the control of human salmonellosis.