Tuberculosis, caused by Mycobacterium tuberculosis, is one of the greatest infectious causes of disease and mortality worldwide. The problem has been exacerbated in recent years by the emergence of drug resistant strains and the concurrent HIV epidemic. There is no reliable vaccine against tuberculosis and there is increasing evidence that natural infection and disease cannot prevent subsequent reinfection. Thus host immunity is only partially effective in preventing and eliminating infection. To enhance our understanding of the basic interplay between pathogenic mycobacteria and host immunity, we will use a closely related pathogenic species Mycobacterium marinum. M. marinum causes a systemic tuberculosis-like disease in ectotherms, and a peripheral granulomatous disease in humans called fish tank granuloma. M. marinum is a powerful model for the study of tuberculosis owing to its genetic and pathogenic similarities to M. tuberculosis, its relatively rapid growth, and the existence of multiple natural hosts that serve as animal models. Adult zebrafish develop caseating granulomatous tuberculosis similar to active human tuberculosis. Mm also produces granulomatous disease in zebrafish larvae, which allow live monitoring of host-pathogen interactions due to their optical transparency. Using the embryo model in conjunction with a variety of fluorescence-based tools we developed in M. marinum, we have been able to monitor and modulate the essential stages of infection, dissemination and granuloma formation in real-time. Here, we will test a series of M. marinum mutants in the zebrafish to explore the molecular mechanisms by which mycobacteria resist and even usurp host immunity to persist. We will develop additional techniques to assess the subcellular localization of intracellular M. marinum during infection, and use a pH sensitive GFP reporter to determine correlate phagosomal acidification to intrabacterial acidification. Finally, we will continue our studies of the RD1 virulence locus. Specifically, we will pursue the identity of, and the molecular mechanism by which, RD1-secreted determinants induce the production of host matrix metalloproteinase-9 by epithelial cells, thus facilitating granuloma formation and bacterial expansion. The information obtained from our proposed experiments will further our fundamental knowledge about tuberculosis.