Mechanisms of phagosomal development and the maturation arrest of Mycobacterium tuberculosis phagosomes

Mycobacterium tuberculosis continues to be a potent pathogen infecting a third of the world's population. Crucial to its ability to infect the host is its capacity to parasitize and multiply within macrophages. Phagosomes containing the tubercle bacilli do not fuse with lysosomes, defining one of the central paradigms of M. tuberculosis pathogenesis. The focus of my research was to elucidate the mechanisms governing phagosome maturation. The arrest of mycobacterial phagosome (MPC) maturation was previously associated with an abnormal pattern of endosomal Rabs, characterized by the accumulation of Rab5 and exclusion of Rab7. Here, the maturation block was further resolved to an event between the acquisition of the Rab5 effectors hVPS34, a PI-3-kinase, and EEA1. EEA1 mediates vesicle tethering and membrane fusion. Unlike latex bead phagosomes (LBC), which recruited EEA1, MPC excluded EEA1, a phenomenon directly linked to phagosome maturation arrest.;In subsequent studies, evidence was found to suggest that EEA1-dependent phagosome maturation occurs via interactions with Syntaxin 6 on vesicles from the TGN. Unlike LBC, MPC excluded Syntaxin 6. It was also found that EEA1 is required for Syntaxin 6 and Cathepsin D acquisition by LBC. Since EEA1 and Syntaxin 6 physically interact, this suggests that interactions between MPC and TGN are disrupted, which resolves the mechanism that mediates the exclusion of Cathepsin D and M6PR from these phagosomes. Additional studies uncovered the degradation of Cellubrevin on MPC. In contrast to LBC, MPC possessed a degraded Cellubrevin product. Since Cellubrevin interacts with Syntaxin 6 in TGN-to-endosome trafficking, this implicates an additional strategy used by this organism to segregate its phagosome from the default maturation pathway.;Mycobacterial alterations of membrane trafficking are not limited to the acquisition of late endocytic markers. It was also discovered that the recycling of Syntaxin 4 is attenuated on MPC. Since Syntaxin 4 functions in endosomes-to-plasma membrane trafficking, these results suggest that the characteristic retention of plasma membrane markers by MPC may be linked to the inhibition of Syntaxin 4 function.;Taken together, this thesis further resolves the fundamental processes of phagosome maturation by uncovering alterations caused by M. tuberculosis during the arrest of this pathway.