Tuberculosis constitutes a global health threat of growing proportions. Development of improved strategies for prevention and treatment of tuberculosis will be facilitated by an improved understanding of the cell biology of Mycobacterium tuberculosis. We have established that, whereas phagosomes containing dead M. tuberculosis proceed to phagolysosomes, live M. tuberculosis arrests the maturation of its phagosome at an early endosomal stage. However, the molecular basis underlying this arrested maturation has not been determined. We propose to examine the interaction of the M. tuberculosis phagosome with the host cell by immunoelectron microscopy and by biochemical and proteomic analysis of isolated phagosomes obtained at sequential times after incubation of human macrophages with live or dead M. tuberculosis. We shall determine the host cell protein composition of purified M. tuberculosis phagosomes by metabolic radiolabeling, 1- and 2-D gel electrophoresis, tryptic digestion, and mass spectrometry based proteomics. We shall explore the role of proteins identified by this approach by expressing these molecules as epitope tagged constructs in an adenoviral expression system and determining their localization using immunofluorescence and immunoelectron microscopy. When possible, we shall express dominant positive or negative mutants of these proteins and examine the effect of this on the phenotype of the M. tuberculosis phagosome. As overexpression of various host proteins alters the maturational state of the phagosome, we shall employ destabilized and inducible bacterial GFP expression as direct probes to assess bacterial metabolic activity within phagosomes of various phenotypes. In a similar fashion, we shall also explore the role of known components of the host cell membrane trafficking machinery, including Rab- and Rho- GTPases, and their downstream effectors. Because the lipid composition of the phagosome has an important impact on its capacity to participate in intracellular signaling, interaction with Rab and Rho effector proteins, and interaction with the host cell cytoskeleton, we shall also determine the lipid composition of the M. tuberculosis phagosome by metabolic radiolabeling, chromatographic analysis, and mass spectrometry. These experiments will advance our understanding of the cell biology and pathogenic mechanisms of M. tuberculosis and facilitate the development of new strategies to combat tuberculosis. ? ?
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