Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, causes one of the most important diseases in the world. New strategies for prevention and treatment of tuberculosis require more understanding of the intracellular compartment that it occupies within the host cell. In the previous funding period the PI has made considerable progress in characterization of the M. tuberculosis phagosome. Of considerable importance, the virulent M. tuberculosis strain Erdman was shown to arrest the maturation of the phagosome at a stage that retains endosomal markers and the capacity to acquire exogenously added transferrin. The understanding of the basic biology of vesicular docking and fusion events has advanced with the recognition of the importance of rab-GTPases, RhoD, and SNAP-SNARE-NSF molecules in regulation of membrane trafficking within the cell. This proposal seeks to determine whether M. tuberculosis arrests the maturation of its phagosome by disrupting the distribution or function of a select group of ras-GTPases (rab 4,5,7,9, and 11 and RhoD) and SNARE proteins (syntaxins 2-4, cellubrevin, and VAMP-2). The presence or absence of these proteins on the M. tuberculosis phagosome will be determined using cryosection immunogold technique. Because these proteins are often present at low levels, this technique will be enhanced by using transfected cells overexpressing in an inducible manner the proteins of interest and their mutant forms, and assessing their effects on the maturation and intracellular growth of M. tuberculosis. Using permeabilized cells, it will be determined whether M. tuberculosis alters the delivery or the recycling of rab-GTPases on its phagosomes. These studies will help to elucidate the virulence mechanisms that enable M. tuberculosis to survive and multiply within its host cells and should facilitate the development of new strategies for prevention and treatment of tuberculosis.
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