Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate - PubMed (original) (raw)

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

D L Clemens et al. Infect Immun. 2000 May.

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.

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Figures

FIG. 1

Growth of L. pneumophila and M. tuberculosis in THP-1, HeLa Tet-off, and HeLa-Rab5c cells. Monolayers of THP-1 macrophage-like cells, HeLa Tet-off cells, and HeLa-Rab5c cells expressing Rab5c were coincubated with L. pneumophila at a high MOI (2 × 108/ml) or a low MOI (2 × 107/ml) for 1 h (A), with L. pneumophila (2 × 108/ml) for 2 h (C), with M. tuberculosis (106/ml) for 2 h (B), or with M. tuberculosis (107/ml) for 2 h (D) at 37°C, washed, and incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and the number of CFU was determined by plating serial dilutions on CYE (A and C) or 7H11 (B and D) agar plates. The capacity of the bacteria to grow extracellularly in the culture medium was assessed by inoculating L. pneumophila (C) or M. tuberculosis (D) into wells containing only the culture medium or into parabiotic chambers in which the bacteria were separated from the HeLa cells by a 0.2-μm-pore-size filter. Although the bacteria are taken up much less efficiently by HeLa cells, they multiply, once inside, with a similar doubling time in HeLa cells and THP-1 cells (A and B). Overexpression of Rab5c does not alter the intracellular growth rate of L. pneumophila or M. tuberculosis in HeLa cells (C and D). The bacteria do not grow in the absence of cell monolayers or when separated from the monolayer in a parabiotic chamber (C and D). Data shown are the means ± the standard deviations of triplicate determinations.

FIG. 2

Distribution of staining for the human transferrin receptor in HeLa-Rab5c cells 2 days after infection with M. tuberculosis. HeLa-Rab5c cells stably transfected with the transferrin receptor gene were coincubated with M. tuberculosis and latex beads for 2 h, washed extensively, incubated at 37°C for 2 days, fixed, processed for cryoimmunoelectron microscopy, and stained by immunogold for the transferrin receptor. The number of transferrin immunogold particles per micrometer of phagosomal membrane, nuclear membrane, and plasma membrane was enumerated. Data are the percentages of the specified compartment whose membranes contain the indicated number of gold particles per micrometer. A total of 59 M. tuberculosis phagosomes and 48 latex bead phagosomes were evaluated.

FIG. 3

Quantitation of LAMP-1 immunogold staining in HeLa-Rab5c cells infected with L. pneumophila or M. tuberculosis. (A) HeLa-Rab5c cells were coincubated with wild-type or avirulent L. pneumophila for 15 min at 37°C and fixed immediately or coincubated at 37°C for 30 min, washed, and incubated for 6 or 8 h and then fixed. (B) HeLa-Rab5c cells expressing Rab5c were coincubated with latex beads and either live or heat-killed M. tuberculosis for 2 h and either fixed immediately or washed, incubated for 2 days at 37°C, and then fixed. After fixation, all cells were processed for cryoimmunoelectron microscopy and stained for LAMP-1. LAMP-1-bound immunogold particles were enumerated on phagosomal, nuclear, and plasma membranes. Data shown represent the mean and standard deviation of gold particle counts on at least 20 cells (each with at least one phagosome) on each of at least three electron microscopy grids. Wild-type L. pneumophila lacks LAMP-1 at both 15 min and 8 h (A). In contrast, avirulent L. pneumophila phagosomes have a modest level of LAMP-1 at 15 min and stain intensely for LAMP-1 at 6 h. Phagosomes containing live M. tuberculosis have very little LAMP-1, whereas phagosomes containing heat-killed M. tuberculosis and latex beads stain intensely for LAMP-1 at both 2 h and 2 days (B). The nuclear membrane and plasma membrane have negligible staining for LAMP-1 and serve as internal negative controls (A and B).

FIG. 4

Quantitation of Rab5c immunogold staining in HeLa-Rab5c cells infected with wild-type or avirulent L. pneumophila. HeLa-Rab5c cells were coincubated with wild-type or avirulent L. pneumophila for 15 or 30 min and either fixed immediately or washed extensively, incubated for an additional 30 min to 8 h, and then fixed. After fixation, all cells were processed for cryoimmunoelectron microscopy, and Rab5c immunogold particles were enumerated on phagosomal, nuclear, and plasma membranes. Data shown are the means and standard deviations of gold counts on at least 20 cells (each with at least one phagosome) on each of at least three electron microscopy grids. (Left) At 15 min, Rab5c is scarce on wild-type L. pneumophila phagosomes but present on phagosomes containing avirulent L. pneumophila. Subsequently, Rab5c is absent or scarce on wild-type and avirulent L. pneumophila phagosomes. Rab5c is scarce on nuclear membranes and plasma membranes at all time points examined. (Right) As a control, Rab5c staining in the cytoplasm of the HeLa cells was quantitated and found to be comparable in the cells containing wild-type or avirulent L. pneumophila at all time points.

FIG. 5

Distribution of staining for Rab5c in HeLa-Rab5c cells fixed immediately after a 15-min coincubation with either wild-type or avirulent L. pneumophila. HeLa-Rab5c cells were coincubated with wild-type (A) or avirulent (B) L. pneumophila for 15 min, fixed, and processed for cryoimmunoelectron microscopy. The number of gold particles per micrometer of membrane on phagosomal, plasma, and nuclear membranes was enumerated. Data shown are the means ± standard deviations of the distributions from two separate experiments.

FIG. 6

Phagosomes containing wild-type L. pneumophila but not avirulent L. pneumophila exclude Rab5c. Suspensions of wild-type (A) or avirulent (B and C) L. pneumophila were spun down onto monolayers of pTRE/rab5c-HeLa Tet-off cells at 4°C, incubated at 37°C for 15 min, and either fixed immediately (A and B) or washed, incubated in fresh culture medium for 6 h, and then fixed (C). Cells were processed for cryoimmunoelectron microscopy. Rab5c has been stained using 15-nm gold particles (arrowheads), and L. pneumophila LPS has been stained using 5-nm gold particles (arrows). Rab5c is absent from the wild-type L. pneumophila phagosome (A), despite the presence of Rab5c immunogold staining on vesicles adjacent to the phagosome. Rab5c is present on the avirulent L. pneumophila phagosome at 15 min (B) but is absent by 6 h (C). Magnifications, ×72,827 (A), ×38,235 (B), and ×50,664 (C).

FIG. 7

Quantitation of Rab5c immunogold staining in HeLa-Rab5c cells infected with live or heat-killed M. tuberculosis. (A) Suspension of live or heat-killed M. tuberculosis together with 1-μm-diameter latex beads were centrifuged at 4°C onto monolayers of HeLa-Rab5c cells expressing Rab5c, incubated for 2 h at 37°C, and either fixed immediately (2-h time point) or washed extensively and incubated in fresh culture medium for 1 to 2 days and then fixed (1- or 2-day time point). The cells were processed for cryoimmunoelectron microscopy and stained for Rab5c. Rab5c-bound immunogold particles were enumerated on phagosomal, nuclear, and plasma membranes. Data shown are the means and standard deviations of gold counts on at least 20 cells (each with at least one phagosome) on each of at least three electron microscopy grids. (Left) Rab5c is present on phagosomes containing live M. tuberculosis at all time points but is scarce on phagosomes containing either heat-killed M. tuberculosis or latex beads, nuclear membranes, and plasma membranes. (Right) As a control, Rab5c staining in the cytoplasm of the HeLa cells was quantitated and found to be comparable in cells containing live or heat-killed M. tuberculosis. The level of staining in the cytoplasm at 2 h is somewhat less than that at 1 to 2 days, due to a shorter tetracycline-free induction period. (B) The distribution of staining for Rab5c in HeLa-Rab5c cells at the 2-day time point after coincubation with live M. tuberculosis and latex beads is shown (a total of 88 live M. tuberculosis phagosomes and 76 latex bead phagosomes were examined).

FIG. 8

M. tuberculosis phagosomes in HeLa-Rab5c cells stain positively for Rab5c. HeLa-Rab5c cells were maintained and expanded in the presence of tetracycline (5 μg/ml). One day prior to infection with M. tuberculosis, tetracycline was removed from the culture medium to induce Rab5c expression. The HeLa cells were coincubated for 2 h with M. tuberculosis using an MOI of 400:1. Nonadherent bacteria and beads were washed away, and the monolayers were incubated for 2 additional days. Monolayers were fixed and processed for cryoimmunoelectron microscopy. Rab5c was stained with 15-nm immunogold particles (arrowheads) and is abundant on the M. tuberculosis phagosomal membrane. Mycobacterial LAM was stained with 5-nm gold particles and is present on the mycobacterial cell wall (arrows). Nu, nucleus. Magnification, ×55,700.

FIG. 9

Quantitation of Rab5c and LAMP-1 immunogold staining in HeLa-Rab5c Q79L cells infected with L. pneumophila or M. tuberculosis. Monolayers of HeLa cells expressing Rab5c Q79L were coincubated with L. pneumophila or with M. tuberculosis and latex beads, fixed, and processed for immunoelectron microscopy after 30-min or 2-h incubations (respectively). The number of Rab5c-bound (A) and LAMP-1-bound (B) immunogold particles was enumerated on phagosomes, plasma membranes, and nuclear membranes. In the case of the _M. tuberculosis_-infected cells, vacuoles that contained only latex beads were scored as latex bead phagosomes. Vacuoles that contained both latex beads and M. tuberculosis were scored as M. tuberculosis phagosomes. Latex bead phagosomes were not scored for the L. pneumophila infected cells, due to inadequate uptake in the 30 min of coincubation. Data shown are the means and standard deviations of gold counts on at least 20 cells (each with at least one phagosome) on each of at least two electron microscopy grids.

FIG. 10

The phenotype of M. tuberculosis phagosomes and avirulent L. pneumophila phagosomes but not wild-type L. pneumophila phagosomes is altered by expression of the constitutively active Rab5c Q79L mutant. Suspensions of wild-type L. pneumophila (A), avirulent L. pneumophila (B), or M. tuberculosis and latex beads (D) were added to monolayers of HeLa cells expressing Rab5c Q79L and centrifuged at 1,160 × g for 20 min at 4°C, incubated at 37°C for either 30 min (L. pneumophila [A and B]) or 2 h (M. tuberculosis [D]), fixed, and processed for cryoimmunoelectron microscopy. Rab5c was stained with 15-nm immunogold particles (large arrowheads), LAMP-1 was stained with 10-nm immunogold particles (small arrowheads), and L. pneumophila LPS (A and B) or mycobacterial LAM (C and D) was stained with 5-nm immunogold particles (arrows). (A) Wild-type L. pneumophila resides in a morphologically tight phagosome that lacks immunogold staining for Rab5c and LAMP-1, which are present on an adjacent large vacuole (∗). (B) A large vacuole contains numerous avirulent L. pneumophila and stains positive for both Rab5c and LAMP-1. (C and D) M. tuberculosis resides in large vacuoles that stain positively for Rab5c and for LAMP-1. M. tuberculosis often shares the large vacuole with latex beads (D). Nu, nucleus. Magnifications, ×37,310 (A), ×37,310 (B), ×37,310 (C), and ×42,770 (D).

FIG. 10

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Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate - PubMed (original) (raw)