PROJECT SUMMARY ABSTRACT Infections by Salmonella present a constant threat to human health in our country and throughout the world. Yet, our progress toward controlling salmonellosis has been largely fruitless; antibiotics are rarely warranted, and, when used, frequently fail due to resistant strains. To control this important foodborne pathogen, it is essential to understand the means by which it colonizes and induces disease. Chemical signals of the intestine, including those produced by both the animal host and the microbiota, can repress Salmonella virulence by reducing its ability to invade the intestinal epithelium. We propose that this signaling defines the fine balance between virulence and growth of the pathogen. We have found that a novel class of chemicals produced by species of the Gammaproteobacteria, termed diffusible signal factors (DSFs), potently represses invasion. DSFs are quorum-sensing molecules that we have found to exist in the large intestine of mice in sufficient concentration to inhibit Salmonella invasion. They therefore represent both a novel instance of inter- species signaling and a means by which Salmonella disease and carriage is modulated by its biological environment. The long-term goal of this work is to identify practical means to inhibit Salmonella invasion in humans and thus to reduce clinical and sub-clinical salmonellosis. Our objectives are to understand how invasion-inhibiting compounds function, and to investigate their efficacy in preventing disease. Our central hypothesis is that the resident microbiota of the large intestine produce chemical signals that repress Salmonella invasion, and that these signals thus dictate the balance between virulence and growth. We aim to test the specific hypotheses that: 1) Intestinal chemical signals (including both DSFs and other microbiota- derived compounds) modulate Salmonella virulence by controlling the proportion of the pathogen population capable of invasion to dictate disease and carriage; 2) Signaling molecules of varying structures bind within a single binding pocket of AraC-type invasion regulators, but utilizing different binding moieties, thus dictating activity and competition among these signals, and; 3) Signals repressive for invasion can be produced in animals using recombinant bacteria to reduce both clinical signs of salmonellosis and intestinal colonization by this pathogen. The work described here is significant and innovative as it has potential to identify a novel means of pathogen control that does not rely upon antibiotics but instead targets attributes essential to colonization and virulence.