Mechanisms of phagosome formation, maturation and acidification: Implications for intracellular infection

Tuberculosis is the leading infectious cause of death worldwide, and is characterized by the intracellular survival Mycobacterium tuberculosis (MTB) within host phagocytes. Internalized pathogens become sequestered into vacuoles called phagosomes, which normally mature rapidly to become acidic, microbicidal organelles. MTB thwarts phagosomal maturation and therefore survives within the phagosome. To understand and treat infection with MTB, it is essential to understand the mechanisms governing phagocytosis, phagosomal acidification, and phagosomal maturation.; Two mechanisms essential for the process of Fc receptor-mediated phagocytosis are described. In the first, it was determined that the cytoskeletal rearrangements and intracellular signalling cascade that occur in response to Fc g -receptor ligation require the small GTPase Rho. Inhibition of Rho by microinjection of macrophages with C3 exotoxin altered cell morphology, and impaired the ability of Fc g receptors to cluster within the membrane, thereby inhibiting the signalling in response to particle adherence. Secondly, we determined that contrary to current dogma, phagocytosis was accompanied by an increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery profoundly inhibited phagocytosis, indicating that exocytosis of endomembranes is essential for particle internalization.; To determine the mechanisms governing phagosomal acidification, we investigated whether proton transporters active at the cell surface were present and functional in phagosomal membranes. We found that both the sodium-proton exchanger and the vacuolar type H+-ATPase were expressed and functional on phagosomes, although the V-ATPase predominated in acidifying phagosomes containing inert and mycobacterial particles.; Finally, two protein family members with potential roles in phagosomal maturation were studied. First, we identified that three isoforms of syntaxins, which are general regulators of vesicular transport, were expressed in macrophages and recruited to the phagosome. Second, we examined the effects of the Natural Resistance Associated Macrophage Protein (Nramp), which confers resistance to murine mycobacterial infection. We found that expression of Nramp in phagosomes containing live mycobacteria resulted in increased phagosomal acidification compared to Nramp-deficient macrophages. This effect was associated with increased phagosome-lysosome fusion and increased delivery of V-ATPases to the mycobacterial phagosome, demonstrating that Nramp plays a central role in phagosomal maturation.