SUMMARY: Salmonella is the leading cause of bacterial foodborne poisoning with significant public health implications worldwide. Poultry products (eggs and poultry meat) are humans' main source of Salmonella infections. Human Salmonella serotypes do not cause disease in birds, which serve as the reservoir, but colonize their intestines. Salmonella can enter the human food chain during processing and humans can be exposed to infection through direct contact with infected poultry carcasses. Currently, Salmonella infections are controlled by antibiotics and/or vaccination; however, the emergence of antibiotic-resistant Salmonella strains is a growing source of concern. Additionally, live Salmonella vaccines are not effective at reducing food poisoning and they raise the risk of live bacteria entering the human food chain. Untreated Salmonella infections have been linked to a variety of chronic health issues such as colon and gallbladder cancers. According to the American Cancer Society, gallbladder and colon cancers have been associated with infections with two Salmonella serovars, S. Typhi and S. Enteritidis, respectively. Therefore, there is a critical need for antibiotic alternatives to control Salmonella infection in poultry and to mitigate the risk of Salmonella food poisoning, antimicrobial resistance, and cancer in humans. Quorum sensing autoinducer-2 (QS AI-2) plays a critical role in the pathogenicity, virulence, biofilm formation, motility, genetic competence, sporulation, and antibiotic production of several bacteria including Salmonella. Therefore, inhibition of QS AI-2 activity using QS inhibitors (QSI) is a potentially novel strategy for antibacterial development. Since QSIs do not interfere with the metabolic processes of a bacterial cell such as protein synthesis, DNA metabolism, and cell wall formation, which are the targets for the development of drug resistance, QSIs will not exert selection pressure on the bacteria during treatment, thus bacteria are less likely to develop resistance. We hypothesize that breaking the bacterial “conversation” by using QSIs makes pathogens less virulent and thus more susceptible to host immune responses. Through the screening of 1,900 compounds to identify the QS/AI-2 inhibitors against S. Typhimurium, we identified 10 compounds that inhibited the QS/AI-2 production of S. Typhimurium (95%; at 10M final concentration) without affecting bacterial growth. We compared the efficacy of these 10 compounds to the benchmark QSIs identified in previous studies and found that our compounds are more effective at lower concentrations. In this proposal, we will (Aim 1) identify and evaluate the efficacy of the selected QSIs in vitro, (Aim 2) evaluate the efficacy of the lead QSIs in infected 3D human intestinal pluripotent stem cell-derived organoids and in the reservoir animal host (chickens), and (Aim 3) identify the drug target(s) of the selected leads using drug-target binding st...