Deviant Expression of Rab5 on Phagosomes Containing the Intracellular PathogensMycobacterium tuberculosisandLegionella pneumophilaIs Associated with Altered Phagosomal Fate

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Abstract
The intracellular human pathogensLegionella pneumophilaandMycobacterium tuberculosisreside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. TheL. pneumophilaphagosome exists completely outside the endolysosomal pathway, and theM. tuberculosisphagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development ofL. pneumophilaandM. tuberculosisphagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-typeL. pneumophilanever acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutantL. pneumophila(which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast,M. tuberculosisphagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads,M. tuberculosis, and avirulentL. pneumophilabut not wild-typeL. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-typeL. pneumophilaphagosomes, functional Rab5 persists onM. tuberculosisphagosomes. The absence of Rab5 on theL. pneumophilaphagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on theM. tuberculosisphagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.