Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner

Abstract

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.

FIG.1.

Localization of GFP-Rab GTPases in C. trachomatis serovar L2-infected HeLa cells. HeLa cells were transiently transfected with DNA containing the indicated GFP-tagged Rab GTPases (B to L) or with pEGFPC1 (A). At 24 h posttransfection, the cells were infected with C. trachomatis serovar L2, and at 18 h postinfection, the cells were fixed and viewed by LSCM. Arrowheads indicate chlamydial inclusions, as determined by indirect immunofluorescence staining with antichlamydial LPS (data not shown). Specific associations between GFP-Rab1A, GFP-Rab4 (GFP-Rab4A and GFP-Rab4B), GFP-Rab6 (GFP-Rab6A and GFP-Rab6B), and GFP-Rab11 (GFP-Rab11A and GFP-Rab11B) and the C. trachomatis serovar L2 inclusion were detected, as shown by rim-like fluorescence staining around the entire periphery of the inclusion. Bar, 10 μm.

FIG.2.

Localization of GFP-Rab GTPases in C. trachomatis serovar D-infected HeLa cells. HeLa cells were transiently transfected with DNA containing the indicated GFP-tagged Rab GTPases (B to L) or pEGFPC1 (A). At 24 h posttransfection, the cells were infected with C. trachomatis serovar D, and at 18 h postinfection, the cells were fixed and viewed by LSCM. Arrowheads indicate chlamydial inclusions, as determined by indirect immunofluorescence staining with antichlamydial LPS (data not shown). Specific associations between GFP-Rab1A, GFP-Rab4 (GFP-Rab4A and GFP-Rab4B), GFP-Rab6 (GFP-Rab6A and GFP-Rab6B), and GFP-Rab11 (GFP-Rab11A and GFP-Rab11B) and C. trachomatis serovar D inclusion were detected, as shown by rim-like fluorescence staining around the entire periphery of the inclusion. Bar, 10 μm.

FIG.3.

Localization of GFP-Rab GTPases in C. muridarum-infected HeLa cells. HeLa cells were transiently transfected with DNA containing the indicated GFP-tagged Rab GTPases (B to L) or pEGFPC1 (A). At 24 h posttransfection, the cells were infected with C. muridarum, and at 18 h postinfection, the cells were fixed and viewed by LSCM. Arrowheads indicate chlamydial inclusions, as determined by indirect immunofluorescence staining with antichlamydial LPS (data not shown). Specific associations between GFP-Rab1A, GFP-Rab4 (GFP-Rab4A and GFP-Rab4B), GFP-Rab10, and GFP-Rab11 (GFP-Rab11A and GFP-Rab11B) and the C. muridarum inclusion were detected, as shown by rim-like fluorescence staining around the entire periphery of the inclusion. Bar, 10 μm.

FIG.4.

Localization of GFP-Rab GTPases in C. pneumoniae AR39-infected HeLa cells. HeLa cells were transiently transfected with DNA containing the indicated GFP-tagged Rab GTPases (B to L) or pEGFPC1 (A). At 24 h posttransfection, the cells were infected with C. pneumoniae strain AR39, and at 44 h postinfection, the cells were fixed and viewed by LSCM. Arrowheads indicate chlamydial inclusions, as determined by indirect immunofluorescence staining with antichlamydial LPS (data not shown). Specific associations between GFP-Rab1A, GFP-Rab4 (GFP-Rab4A and GFP-Rab4B), GFP-Rab10, and GFP-Rab11 (GFP-Rab11A and GFP-Rab11B) and the C. pneumoniae inclusion were detected, as shown by rim-like fluorescence staining around the entire periphery of the inclusion. Bar, 10 μm.

FIG.5.

Association of GFP-Rab proteins with chlamydial inclusions is maintained in the absence of microtubules. Transiently transfected HeLa cells were mock infected (A, B, D, E, G, H, J, K, M, and N) or infected with C. trachomatis serovar L2 for 18 h (C, F, I, and L) or with C. pneumoniae for 44 h (O). Cells were treated with 20 μM nocodazole for 3 h to disassemble microtubules, fixed, permeabilized, and stained with antichlamydial LPS (data not shown). Although treatment with nocodazole disrupts the microtubule network and causes dispersal of recycling endosomes and the Golgi apparatus, GFP-Rab1, GFP-Rab4, GFP-Rab6, GFP-Rab11, and GFP-Rab10 remain associated with the inclusion. Arrowheads indicate inclusions. Bar, 5 μm.

FIG. 6.

Association of GFP-Rab11A with the chlamydial inclusion is independent of microtubules. Transiently transfected HeLa cells expressing GFP-Rab11A were treated with nocodazole (D to F) or dimethyl sulfoxide (A to C) for 3 h prior to chlamydial infection to depolymerize microtubules. Cells were then infected with C. trachomatis serovar L2 at a multiplicity of infection of approximately 20. The cells were infected for 8 h in the presence of nocodazole, fixed in formaldehyde, permeabilized, labeled with antichlamydial LPS, and incubated with goat anti-mouse IgG conjugated to Alexa 594 (red). (A and D) GFP-Rab11A (green), (B and E) antichlamydial LPS (red), (C) panels A and B merged, (F) panels D and E merged. In the complete absence of microtubules, GFP-Rab11A is still recruited to individual chlamydia-containing vacuoles (D to F). The arrowhead indicates Golgi-localized chlamydial vacuoles, and arrows indicate examples of dispersed chlamydial vacuoles. Bar, 5 μm.

FIG. 7.

GFP-Rab11A partially colocalizes with Tfn-containing endosomes at the periphery of the chlamydial inclusion. Transiently transfected HeLa cells expressing GFP-Rab11A were infected with C. trachomatis serovar L2 for 18 h. Recycling endosomes were labeled with Alexa 568-labeled Tfn by serum starving cells for 2 h and incubated with Alexa 568-labeled Tfn for 60 min at 37°C. Cells were fixed, permeabilized, stained with antichlamydial LPS, and incubated with goat anti-mouse IgG conjugated to Cy5 (blue). In the inset in panel D, the arrowhead indicates Rab11A-positive Tfn-negative regions around the inclusion and the arrow indicates Rab11A-positive Tfn-positive regions. (A) GFP-Rab11A (green), (B) Alexa 568-labeled Tfn (red), (C) antichlamydial LPS (blue), (D) panels A, B, and C merged. Bar, 5 μm.

FIG. 8.

GFP-Rab11A partially colocalizes with IncG at the inclusion membrane. Transiently transfected HeLa cells expressing GFP-Rab11A were infected with C. trachomatis serovar L2 for 18 h. Cells were fixed, permeabilized, stained with polyclonal anti-C. trachomatis IncG, and incubated with goat anti-rabbit IgG conjugated to Alexa 594. GFP-Rab11A colocalizes with IncG at many regions along the inclusion membrane, as indicated by the yellow fluorescence, but there are regions that are IncG positive but Rab11A negative. In addition, Rab11A partially colocalizes to the IncG-laden fibers that stain and that extend from the inclusion membrane. The arrowheads indicate representative areas where GFP-Rab11A and IncG colocalize along the fibers. Two inclusions, at different focal planes, are visible within the cell, and each is indicated with an asterisk. (A) GFP-Rab11A (green), (B) IncG (red), and (C) panels A and B merged. Bar, 5 μm.

FIG.9.

Temporal analysis of GFP-Rab11A recruitment to C. trachomatis inclusions. HeLa cells transiently expressing GFP-Rab11A were infected at a multiplicity of infection of approximately 20. At different times postinfection (shown to the left of the figure), the cells were fixed and stained with antichlamydial LPS. The cells were then incubated with goat anti-mouse IgG conjugated to Alexa 594 (red) and viewed by LSCM. Beginning as early as 1 h postinfection, colocalization of EBs and GFP-Rab11A is observed. At each time point, insets show representative vacuoles colocalizing with GFP-Rab11A. (A, D, G, J, and M) GFP-Rab11A, (B, E, H, K, and N) antichlamydial LPS, (C, F, I, L, and O) GFP-Rab11A (green) and antichlamydial LPS (red) merged. Bars, 10 μm (O) and 2 μm (inset in panel O).