PROJECT SUMMARY A critically understudied part of Listeria monocytogenes (Lm) pathogenesis is colonization of the mammalian gallbladder. The gallbladder is the primary bacterial reservoir for several enteric pathogens, including Lm, Salmonella, and Campylobacter, and serves as the source of fecally secreted bacteria that has the potential to increase transmission of these foodborne pathogens. Here, we will dissect the nature of Lm growth in the gallbladder and identify the bacterial metabolic and stress response pathways critical for colonization of the murine gallbladder. To that end, we present preliminary data from our novel ex vivo model of Lm gallbladder colonization using organs from non-human primates (NHP). These results have set the stage for investigations into specific genes and pathways necessary for Lm replication in the gallbladder lumen. The objectives of this proposal will be accomplished with three Specific Aims: (1) Define the gallbladder as a replicative niche for Lm. To do this, we will determine the localization of Lm in the gallbladder and define the metabolites available in the gallbladder lumen. (2) Identify the Lm genes required for colonization of the gallbladder. Here, fluorescent reporter strains will be engineered to identify the Lm signaling pathways that are activated in the gallbladder during infection. Additionally, we will evaluate specific Lm genes and pathways for their roles in pathogenesis, with a focus on gallbladder colonization. (3) Determine the role of the immune response to Lm ∆rex infection. Our approach is technically innovative as it takes advantage of our novel model of NHP gallbladder colonization to globally assess bacterial genes important for replication in this unique niche. Results from these studies will be significant as they will uncover how bacterial pathogens replicate in the gallbladder and have the potential to uncover therapeutic targets to reduce colonization and spread of enteric pathogens.