Project Summary/Abstract (Project 2, Cox) Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a major cause of human morbidity and mortality, particularly in the developing world. The spread of antibiotic resistant strains of M. tuberculosis has increased the urgency to develop new vaccines with greater efficacy than Bacille Calmette- Guerin (BCG), which is ineffective to prevent pulmonary infection in adults, by far the most common form of TB. The ESX-1 secretion system of Mycobacterium tuberculosis is a key virulence determinant that is required for intracellular growth and for eliciting distinct innate immune responses, autophagy and type I interferon (IFN) during infection. BCG is extremely similar to M. tuberculosis but it lacks ESX-1 and thus cannot trigger these powerful innate immune pathways. The overarching goal of this project is to identify the M. tuberculosis factors that specifically activate these responses, and use this to create BCG strains that engage these pathways during vaccination. In Aim 1, we investigate ESX-1 secreted substrates that are required for perforating phagosomal membranes in collaboration with Project 1, and use a new system to express these factors in BCG to restore perforation but without the rest of the ESX-1 secretion system. In Aim 2, we explore two hypotheses for how M. tuberculosis limits targeting to autophagy, a powerful anti-bacterial host defense mechanism. In Aim 3, we will utilize our existing knowledge, as well as information from Aims 1 and 2, to engineer BCG strains to specifically test the role of autophagy and type I IFN in promoting immunity. Moreover, we will collaborate with Projects 1, 3, 4, and Core B, to compare and combine our recombinant BCG strategy with STING-targeted adjuvants (Project 4) and bacterial metabolites (Project 1) to identify synergies between these approaches, and to test their efficacy in novel models of vaccination (Projects 3 and 4). Thus, these studies will not only provide deeper mechanistic understanding of how autophagy and anti-viral signaling are activated, but may lead directly to the creation of new TB vaccines.