Members of the MAC complex have recently core to the fore as one of the principle microbial infections in individuals immunocompromised by Acquired Immunodeficiency Syndrome caused by the HIV virus. MAC, and other pathogenic mycobacteria species, have evolved to survive within the macrophage, a cell more usually associated with the eradication of invading microbes. This interaction enables MAC to avoid complement an humoral responses, and poses problems for effective chemotherapy. Recent work in the laboratory has extended greatly our appreciation of the nature of the intracellular compartment inhabited by these bacteria. The vacuoles have a high pH and are extremely dynamic, intersecting readily with the sorting/recycling endosomal system of the host cell. However, despite the increase in our appreciation of the compartment several key questions still remain, the most notable being how MAC exerts its influence over the phagosome and their host cell. This project contains detailed approaches designed to address the following specific question. (1) How do mycobacterial species prevent phagosome maturation? (2) How do activated macrophages overcome this suppression and kill the infecting microbe. (3) How do MAC modulate their host cell and bystander macrophages to suppress localized cellular immune responses and so avoid macrophage activation? The approaches outlined represent novel procedures that maximize exploitation of the newly emergent genome databases and mutagenesis protocols. We have applied our cell biological expertise to development of new genetic screens to isolate and characterize mutants deficient in different aspects of intra-macrophage survival. These studies will proceed in close interaction with our continued cell biological analyses of trafficking pathways into and out of the MAC vacuole, and transfer between infected macrophages and bystander cells. Finally, mutants shown to be defective in factors key to intra-macrophage survival and persistence will be examined in the murine model for their potential as attenuate strains suitable for use as vaccines against MAC and M. tuberculosis. The combination of biochemical, cell biological, and gene approaches described in this project is supported by strong collaborations and interactions with the other members of the program, and the results should enhance our understanding of intracellular survival by MAC, and other pathogenic mycobacteria.