Shared Resource Core B – Abstract This integrated P01 program relies on the premise that the biophysics of respiratory droplets impacts M. tuberculosis (Mtb) payload, physiology and culturability, and thus influences its ability to transmit and infect a new host. To study the aerobiology of infection and identify the bacterial genes required for effective transmission, we will develop a tractable model of simulated Mtb transmission and apply it to compare infectivity of wild type versus mutant Mtb strains, from cavity caseum to terminal lung airways. The mouse is the most widespread animal model of aerosol infection used to investigate TB disease, bacterial genetic requirements and response to therapy. There is extensive experimental evidence supporting efficient lung ‘seeding’ by aerosolized Mtb droplets. Our group has access to two large animal BSL3 facilities and has a long-standing expertise in developing and optimizing new animal models to address challenging questions that require translational tools. We have also developed tools to generate cavity caseum in a modified rabbit model of active TB, and variations of caseum surrogate that rely on foamy macrophage lysate and mucosalivary secretion mimics as the starting point. These matrices will be optimized and used (i) in in vitro screens of Mtb mutants to identify and validate candidate genes required for survival of Mtb during the transitions between host-like and environmental conditions (with Projects 1 and 2: identification of genes and adaptive metabolic responses that Mtb requires to survive transmission stresses), (ii) as the starting material to aerosolize Mtb bacilli in experiments of controlled transmission to mice, to qualitatively and quantitively mimic overall features of human respiratory secretion dynamics in the bioaerosol generator, and (iii) to test the hypothesis that some drugs may impair transmission despite accumulating in caseum and cavities at concentrations that are sub-bactericidal. With support and guidance from the Aerobiology Project 4, we will combine and leverage in vitro, ex vivo and in vivo tools to develop and optimize a mouse model of simulated transmission by controlled aerosol infection. The model will be applied to confirm the contribution of Mtb genes and pathways – identified in vitro – in the successful multi-step transition from one host to another. Mutants with demonstrated loss of fitness for transmission in mice will progress to testing between-animal transmission in guinea pigs (Project 3: Mechanisms of cough in M. tuberculosis transmission). Thus Core B activities serve the objectives of each project and will be closely guided by Aerobiology Project 4.