Tuberculosis (TB) is a highly heterogeneous human disease that develops in some, but not all, individuals who inhale 1-3 infectious bacilli of Mycobacterium tuberculosis (Mtb). TB disease can range from pulmonary disease that is mild and self-resolving, to severe, or can disseminate to extrapulmonary sites. Understanding the complexity of TB pathogenesis requires a holistic approach that integrates human and animal model studies. Although there is growing evidence that Mtb strain-dependent factors drive different infection outcomes, the in vivo mechanisms that govern these outcomes are poorly understood. In this project, we start with clinical Mtb strains associated with different pathogenic outcomes (e.g., high transmission or disseminated disease) or mutant strains harboring modifications in gene candidates, identified in Core A and Project 1, to identify polymorphisms in Mtb strains associated with distinct human clinical outcomes. Mice infected with these clinical or mutant Mtb strains will be used to investigate how they govern three distinct stages of pathogenesis: 1) establishing infection at the site of aerosol inhalation, 2) dissemination to distant sites, and 3) interactions with host immunity in distinct lung macrophage populations. These studies will leverage our group’s recent advances in mouse TB models, including identifying the earliest cellular events after aerosol Mtb infection, tracking Mtb dissemination using a physiologic, ultra low-dose (ULD) infection model in which mice are infected with 1-3 CFUs of aerosolized Mtb, and assessing paired host and Mtb transcriptomes in distinct pulmonary macrophage types. Multiparameter confocal microscopy and advanced quantitative image analysis will be used to determine how these Mtb strain- dependent factors shape immune cell interactions at infection sites. The overall goal of the proposed experiments is to gain insight into molecular and cellular mechanisms of pathogenesis at distinct stages of Mtb infection to inform development of novel host- and pathogen-directed interventions.