The resident microbiota lining gut and respiratory mucosae provide critical defense against invading pathogens and overgrowth of resident pathobionts, a mechanism termed colonization resistance (CR). This mechanism incorporates both direct pathways of competitive exclusion of pathogens and indirect pathways that enhance host immunity. Although CR is best understood in the context of enteric infection, gut bacteria are also found in healthy lungs where they impact pulmonary cytokine levels and alveolar macrophagefunction, thus influencing the risk of pneumonia. Therefore, pulmonary CR may involve both interbacterial antagonism and indirect immunomodulation. There is a critical need to understand how bovine enteropathogens impact CR in the gut and lung.My long-term goal is to understand the relationships among Salmonella spp., the microbiota and host immunity in relation to animal health and food safety. Salmonella enterica threaten both bovine and human health. Salmonella Dublin (SeD) is a cattle-adapted serotype that causes systemic infections and predominantly manifests as pneumonia in calves, in contrast to the generalist serotypes that primarily cause localized enteritis. Salmonella has evolved several mechanisms to overcome CR. Salmonella exploits the inflammatory response to promote gut colonization. In addition, type VI secretion systems (T6SS) inject toxic effector proteins to kill bacteria and eukaryotic cells. Despite years of study, the mechanisms by which SeD overcome CR are poorly described. My preliminary data suggest that gut associated lung bacteria (GALB) may impact pulmonary pathogen colonization in calves. I hypothesize that GALB induce pulmonary immunity to resist SeD infection and SeD uses T6SS to overcome CR in the gut and lung. I will test my hypothesis in the following objectives:Objective 1: Establish the role of GALB in pulmonary immune function and resistance against SeD infection. (A) Evaluate the relationship among immune cell populations, cytokine levels and the lung microbiota in bronchoalveolar fluid of healthy calves. (B) Assess the effects of GALB priming on SeD control in vitro. Hypothesis: GALB stimulate low-grade inflammation that enhances alveolar macrophageimmune function, promoting pulmonary resistance against SeD.Objective 2: Determine the role of SeD T6SSs in subversion of direct CR. (A) Establish the requirement for T6SS in SeD colonization of the calf gut. (B) Determine the conditions in which T6SS are expressed and establish the role of T6SS in interbacterial competition between SeD and GALB in vitro. Hypothesis: The 2 SeD T6SSs (SPI-6 and SPI-19) allow SeD to overcome CR by disrupting the microbiota.