Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics - PubMed (original) (raw)

Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics

Walter Berón et al. Infect Immun. 2002 Oct.

Abstract

The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

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Figures

FIG. 1.

The acidotropic probe LysoTracker red accumulates into vacuoles containing C. burnetii. HeLa cells were incubated with C. burnetii suspended in MfbH for 48 h at 37°C in an air atmosphere. Afterwards, _Coxiella_-infected HeLa cells were incubated for 15 min at room temperature with LysoTracker red and analyzed by phase-contrast (PC; left) and fluorescence (right) microscopy. Arrows, vacuoles containing C. burnetii (CV) loaded with LysoTracker red (top); arrowheads, clustering of small vacuoles labeled with LysoTracker red around the CV (bottom). N, nucleus.

FIG. 2.

_C. burnetii_-containing vacuole colocalizes with MDC and LC3, specific markers of the autophagic pathway. _Coxiella_-infected HeLa cells were incubated for 10 min at 37°C with MDC. After being washed, cells were analyzed by phase-contrast (PC; left) and fluorescence (right) microscopy. (A) Arrows, _Coxiella_-containing vacuoles (CV) loaded with MDC; arrowheads, clustering of small vacuoles labeled with MDC (bottom). N, nucleus. (B) Stably transfected CHO cells overexpressing EGFP-LC3 were infected with Coxiella as described in Materials and Methods. Cells were analyzed by phase-contrast (left) and fluorescence (right) microscopy. (C) _Coxiella_-infected HeLa cells were incubated for 20 min at 37°C with rhodamine 6G (Rh6G). After being washed, cells were analyzed by phase-contrast (left) and fluorescence (right) microscopy. Arrows show that the _Coxiella_-containing vacuoles are not decorated by the ER marker.

FIG. 3.

Rab7 decorates the _C. burnetii_-containing vacuole. _Coxiella_-infected HeLa cells were incubated for 1 h with recombinant Sindbis viruses encoding the GFP-Rab7wt fusion protein or active mutant GFP-Rab7Q67L. After overnight incubations at 37°C in an air atmosphere, cells were analyzed by phase-contrast (PC; left) and fluorescence (right) microscopy. GFP-Rab7wt and GFP-Rab7Q67L decorate large _Coxiella_-containing vacuoles (top and bottom, respectively).

FIG. 4.

The constitutively active form of Rab5 (Rab5Q79L) localizes with the _C. burnetii_-containing vacuole. _Coxiella_-infected HeLa cells were incubated for 1 h with recombinant Sindbis viruses encoding the GFP-Rab5wt fusion protein, negative mutant GFP-Rab5S34N, or active mutant GFP-Rab5Q79L. After overnight incubations at 37°C in an air atmosphere, cells were analyzed by phase-contrast (PC; left) and fluorescence (right) microscopy. _Coxiella_-containing vacuoles did not colocalize with GFP-Rab5wt and GFP-Rab5S34N proteins (top and middle, respectively). Interestingly, the _Coxiella_-containing vacuole membrane was strongly labeled by GFP-Rab5Q79L (bottom).

FIG. 5.

The dominant-negative Rab7 mutant does not localize with the Coxiella replication compartment. HeLa cells transfected with pEGFP plasmids encoding Rab7T22N, a dominant-negative mutant, or active mutant Rab7Q67L were incubated with C. burnetii under infection conditions (see Materials and Methods). Cells were analyzed by phase-contrast (PC; left) and fluorescence (right) microscopy. The active Rab7Q67L mutant clearly labels the _Coxiella_-containing vacuole membrane (arrows, top), whereas the inactive Rab7T22N mutant does not (arrows, bottom).

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Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics - PubMed (original) (raw)