Project Summary/Abstract (Overall) This application is a competitive renewal of a program project grant entitled, “The intersection of innate and adaptive immunity to intracellular pathogens.” A major goal of this P01 is to identify and characterize innate immune pathways that are triggered, avoided, or manipulated by intracellular pathogens and to determine the role of these pathways during infection and immunity. More specifically, in this P01 we focus on the microbiology and immunobiology of three diverse facultative intracellular bacterial pathogens; Listeria monocytogenes, Legionella pneumophila, and importantly, M. tuberculosis. An overall theme of this application is that the innate immune system detects key metabolites and virulence activities of intracellular bacterial pathogens, including cytosolic invasion or access via dedicated bacterial secretion systems. For example, all three pathogens under investigation activate STING, either by direct secretion of a cyclic-di- nucleotide (CDN) or by activation of host cell cGAS in response to bacterial DNA, to activate STING, culminating in critical host responses including activation of a type I IFN and autophagy. In Project 1, Portnoy extends his studies on the roles of c-di-AMP and STING by examining how c-di-AMP secretion affects L. monocytogenes pathogenesis and dissemination to the placenta and brain during an oral model of infection. In Project 2, Cox asks how M. tuberculosis uses its ESX-1 type VII secretion system to perforate phagosomal membranes, activate STING and induce production of type I IFN, which promotes infection, but also activates ubiquitin-mediated autophagy responses, which serves to limit infection. However, while type I IFNs are generally accepted to play a crucial role in orchestrating anti-viral immunity, the roles of type I IFNs in the responses to bacteria are complex and often exacerbate infection by bacterial pathogens. In Project 3, Vance presents preliminary data suggesting that type I IFN-dependent induction of the interleukin-1 receptor antagonist (IL-1Ra) is an important mechanism by which type I IFNs exacerbate bacterial infections. We also explore the idea that CDNs are just one example of immunostimulatory bacterial metabolites by analyzing two other bacterial metabolites that lead to the activation of what are classified as innate or invariant T-cells, including MAIT cells and Vg9Vd2 T-cells. We will ascertain the relevance of these metabolites during L. monocytogenes and M. tuberculosis infection (Projects 1, 2 and 4). In Project 4, Stanley propose to follow up on her preliminary data showing the CDN-based protein vaccines result in a robust and durable tuberculosis vaccines especially when introduced intranasally that is associated with induction of a protective Th17 response. Stanley proposes to evaluate a number of vaccine strategies using CDNs in combination with modified BCG and L. monocytogenes-based vectors.