title: Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate.
abstract: The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome 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 of L. pneumophila and M. tuberculosis phagosomes, 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-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes 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 avirulent L. pneumophila but not wild-type L. 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-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, Center for Health Sciences, Los Angeles, California 90095, USA. dclemens@mednet.ucla.edu
authors.forename: D L
authors.initials: DL
authors.lastname: Clemens
authors.forename: B Y
authors.initials: BY
authors.lastname: Lee
authors.forename: M A
authors.initials: MA
authors.lastname: Horwitz
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006361:Heating; D006801:Humans; D007424:Intracellular Fluid; D016952:Legionella pneumophila / Q000276:immunology*; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010588:Phagosomes / Q000276:immunology* / Q000382:microbiology; D011817:Rabbits; D011990:Receptors, Transferrin / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D011990:Receptors, Transferrin; D020696:rab5 GTP-Binding Proteins