Phosphatidylinositol phosphates and early Rab family V members controlling <italic>Mycobacterium tuberculosis</italic> phagosome maturation arrest.

Abstract

Interference with the progression of phagolysosome biogenesis allows <italic> Mycobacterium tuberculosis</italic> to survive inside the macrophages. The molecular mechanisms underlying this process are yet to be fully elucidated. Using four-dimensional confocal microscopy, the roles of phosphatidylinositol 3-phosphate (PI3P) and Rab22a in phagosomal maintenance and phagosome maturation arrest have been identified. Modulation of these two molecules on mycobacterial phagosome brings about phagosome maturation arrest. PI3P is a phosphoinositide that is crucial for phagosome maturation. All types of phagosomes examined were positive for PI3P immediately after phagocytosis. Phagosomes harboring live mycobacteria experienced a loss after the initial PI3P burst and remained PI3P negative. In contrast, both model phagosomes containing latex beads, and heat killed mycobacteria do not experience a loss and remained positive for PI3P during the later stages of maturation, although the PI3P levels were dissimilar in amount and in production dynamics. The lack of PI3P on phagosomes containing live mycobacteria was due to: (i) the inhibition of the calcium/calmodulin/calmodulin kinase II cascade that recruits the Type III phosphatidylinositol 3-kinase hVPS34 to the phagosomes, and (ii) the secretion of a heat-sensitive mycobacterial PI3P phosphatase, SapM, that hydrolyzes PI3P to PI. Taken together, these results suggest that mycobacteria have developed two independent means to remove PI3P from the phagosome resulting in maturation arrest. Rab22a is a less studied member of the group V Rab family of early endocytic GTP-binding proteins. Time lapse confocal microscopy revealed that phagosomes harboring live mycobacteria accumulated Rab22a in contrast to model latex bead phagosomes. Rab22a is also differentially retained for extended periods of time when compared to the transient recruitment of other members of the group V family of Rabs, Rab5 and Rab21. Recruitment of Rab22a to model phagosomes harboring dead mycobacteria increased the presence of recycling endosomal markers, reduced phagosomal acidification and diminished the activity of degradative hydrolases. Our findings assign a function in mycobacterial phagosome maturation arrest to Rab22a; <italic> M. tuberculosis</italic> specifically and actively recruits this GTPase to orchestrate maintenance of the mycobacterial phagosome maturation arrest at an early endocytic stage.