Despite our best efforts, infections of agricultural animals with Salmonella persist, posing threats to both animal health and food safety. Few, if any, measures have proven effective in reducing Salmonella carriage in production animals, the major source of this pathogen. Antibiotics are ineffective at curtailing infection and have served only to exacerbate the global crisis of antimicrobial resistance. Preventative measures, such as vaccines and probiotic approaches, have also been largely ineffective. The alternative then is to seek novel means to reduce Salmonella disease and carriage by preventing its colonization of livestock and poultry.Invasion of the intestinal epithelium by Salmonella, encoded by a type III secretion system, is essential to both virulence and carriage of this pathogen (1-4). We and others have shown that invasion can be induced or repressed by the chemical signals of the gut, emanating from both the microbiota and the host itself (5-8). Our recent studies demonstrate that a class of small molecules, collectively termed Diffusible Signal Factors (DSFs), inhibit three AraC-type transcriptional activators (HilD, HilC, and RtsA) that are essential to invasion, and thus profoundly reduce the expression of invasion genes. Importantly, as invasion is required for both disease and intestinal colonization by Salmonella, preventing invasion can reduce its carriage and spread. DSFs are cis-2 unsaturated fatty acids that function as quorum-sensing molecules in several gram-negative bacteria (9). A single gene from Cronobacter turicensis, termed rpfF and encoding an enoyl-CoA dehydratase, can be expressed in E. coli to produce cis-2 hexadecenoic acid (c2-HDA), a DSF that inhibits invasion when simply grown in co-culture with Salmonella. These findings, in combination with our insight into the molecular mechanisms of this control, provide a path to develop probiotic approaches to prevent Salmonella carriage. The long-range goal of our work is to create and implement means to control Salmonella in agricultural animals in lieu of antibiotics. The goal of this proposal is to practically and economically produce and deliver DSFs to animals as bioactive compounds that prevent Salmonella carriage and disease. We will do so by achieving these objectives:Objective 1. Determine the effects of recombinant DSF production on Salmonella invasion. We will identify the specific DSFs produced, determine their potency, and test their ability to prevent invasion when expressed by E. coli using chemical, biochemical, cell-culture and imaging methods.Objective 2. Efficiently produce recombinant DSF in E. coli. We will express DSF-producing enzymes constitutively and inducibly in a recognized probiotic strain of E. coli to identify those that effectively repress Salmonella invasion gene expression when grown in co-culture, while maintaining the biological fitness of the host strain.Objective 3. Use recombinant E. coli as a delivery system to reduce Salmonella colonization. We will employ DSF-producing E. coli in a relevant animal model to assess their ability to prevent intestinal carriage and fecal shedding of Salmonella. We will use chickens as the most appropriate agricultural animal to test the preventative potential of DSFs.