The Early Secretory Pathway Contributes to the Growth of the Coxiella-Replicative Niche^{▿ †}

Materials.

Minimum essential alpha medium (α-MEM) and Dulbecco modified Eagle medium (DMEM) were obtained from Gibco Laboratories (Invitrogen, Argentina) and fetal bovine serum (FBS) was obtained from NATOCOR S.RL, Córdoba, Argentina). LysoTracker and DQ-BSA were from Molecular Probes (Invitrogen). All other chemicals were from Sigma (Buenos Aires, Argentina). Plasmids encoding Rab1b-myc and their mutants were kindly provided by Cecilia Alvarez, CIBICI (UNC-CONICET)-Fac. Cs. Químicas, UNC, Córdoba. These plasmids were subcloned in the vector pEGFP in our laboratory. Rabbit polyclonal anti-Coxiella antibody was generously provided by Robert Heinzen (Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT). pIRES2-DsRed2 bicistronic vectors encoding Sar1, Sar1 H79G, and Sar1 T39N were a gift from Hirofumi Kai and Akiko Niibori (Faculty of Pharmaceutical Sciences, Kumamoto University, Japan). Rab1b and control siRNAs were obtained from BIONEER (Korea).

Cell culture.

Chinese hamster ovary cells (CHO), Vero cells, HeLa cells and Raw macrophages were grown on coverslips in α-MEM or DMEM supplemented with 15% FBS at 37°C in an atmosphere of 95% air and 5% CO₂ in 24-well plates to 80% confluence. Stably transfected CHO cells overexpressing EGFP-Rab1b and its respective mutants were grown in the same medium supplemented with 0.2 mg of Geneticin ml⁻¹. All antibiotics were removed 24 h before infection with Coxiella.

Cell transfection.

CHO, HeLa, and Raw cells were transfected with the plasmids (1 mg ml⁻¹) using the Lipofectamine 2000 reagent (Invitrogen) according to the instructions supplied by the manufacturer. Transfected cells were incubated for 24 h in α-MEM supplemented with 15% FBS in 24- and 12-well plates to 80% confluence. Stably transfected cells were generated by selection with 0.5 mg of Geneticin ml⁻¹. In some cases, the cells were subsequently cloned and maintained with 0.1 mg of Geneticin ml⁻¹. Cotransfection experiments were carried with Lipofectamine 2000 (Invitrogen).

Propagation of C. burnetii phase II.

The clone 4 phase II Nine Mile strain of C. burnetii bacteria, which is infective for cells but not for mammals, was provided by Ted Hackstadt (Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, MT) and handled in a biosafety level II facility (13a). Infective inocula were prepared as described previously (48a). Nonconfluent CHO cells were cultured in T75 flasks at 37°C in α-MEM supplemented with 5% FBS, 0.22 g of sodium bicarbonate liter⁻¹, and 20 mM HEPES (pH 7) (MfbH). Cultures were infected with C. burnetii phase II suspensions at 37°C in an air (CO₂) atmosphere. After 6 days, the cells were scrapped and passed 20 times through a 27-gauge needle connected to a syringe. Cell lysates were centrifuged at 1,800 × g for 10 min at 4°C. The supernatants were centrifuged at 25,000 × g for 30 min at 4°C, and pellets containing C. burnetii were resuspended in α-MEM and passed 10 times through a 27-gauge needle connected to a syringe. Afterward, the bacterial suspensions were divided into aliquots and kept frozen at −70°C.

Infection of cells with C. burnetii.

CHO and HeLa cells plated in T25 flasks were washed two to three times with phosphate-buffered saline (PBS) and detached by using trypsin-EDTA. After resuspension in α-MEM, cells were plated on coverslips distributed in 6-, 12-, or 24-well plates. For infection, a 50-μl aliquot of C. burnetii suspension was diluted with 10 ml of α-MEM, and 0.5 to 1 ml of this dilution was added per well. In all experiments, host cells were infected with C. burnetii at a multiplicity of infection (MOI) of ∼20. Cells were incubated for different periods of time at 37°C in an atmosphere of 95% air and 5% CO₂. The same protocol was used for the infection of CHO cells overexpressing pEGFP-Rab1b and the corresponding mutants.

Confocal microscopy.

pEGFP-Rab1b-, pEGFP-, or pRFP-LC3-transfected CHO cells were analyzed by confocal microscopy using a Nikon C1 confocal microscope system or an Olympus FluoViewTM FV1000 confocal microscope (Olympus, Argentina), with the EZ-C1 software (Nikon, Japan) or the FV10-ASW (version 01.07.00.16) software, respectively. Images were processed by using Adobe CS3 (Adobe Systems).

Indirect immunofluorescence.

CHO, HeLa, and Raw cells were fixed with 3% paraformaldehyde solution in PBS for 10 min at room temperature, washed with PBS, and blocked with 50 mM NH₄Cl in PBS. Subsequently, cells were permeabilized with 1% saponin in PBS containing 1% bovine serum albumin (BSA), and then incubated with the primary antibody against Coxiella (1:1,000). Bound antibodies were detected by incubation with a secondary antibody conjugated with Texas Red or Cy5 (1:800; Jackson Immunoresearch Laboratories). Cells were mounted with Mowiol (plus Hoechst stain) and examined by confocal microscopy.

Measurement of the percentage of infected cells and the number and size of C. burnetii RVs.

At different times postinfection, the cells were fixed for 10 min in 3% paraformaldehyde. About 500 cells per coverslip (in triplicates) were scored for the presence or absence of large C. burnetii vacuoles using a confocal microscope (Nikon and Olympus) with a 60× objective lens. Infected cells were defined as those with at least one large parasitophorous vacuole (i.e., ≥2 μm) with clear identifiable bacteria inside. The size of the vacuoles was determined by a morphometric analysis using Metamorph and FV10-ASW software.

Endocytosis and phagocytosis assays.

CHO cells overexpressing EGFP, EGFP-Rab1b wild-type (wt), or Rab1b Q67L were plated at a confluence of 50 to 60% the day before the experiment. After 24 h, the cells were infected with C. burnetii. At 48 h postinfection, the cells were allowed to internalize heat-inactivated Staphylococcus aureus-rhodamine or 5 μg of dextran-rhodamine/ml to label the phagocytic and endocytic pathways, respectively. The 24-well plate was centrifuged for 10 min (1,500 rpm) in order to induce the contact of the S. aureus particles to the cell surface. After 1 h of incubation, the cells were washed three times with PBS, fixed with 3% paraformaldehyde for 12 min, and quenched with 50 mM NH₄Cl for 15 min. The cells were subjected to indirect immunofluorescence using a polyclonal antibody against C. burnetii. The glass slides were mounted using Mowiol and analyzed by confocal microscopy.

Phagocytosis of latex beads.

CHO cells overexpressing enhanced green fluorescent protein (EGFP), EGFP-Rab1b wt, or Rab1b Q67L were plated at a confluence of 50 to 60% the day before the experiment. The cells were allowed to internalize 4-μm latex beads (with an MOI of 10) for 24 h. The cells were then washed three times with PBS, fixed with 3% paraformaldehyde for 12 min, and quenched with 50 mM NH₄Cl for 15 min. Samples were mounted on glass slides using Mowiol and analyzed by confocal microscopy.

Fluorescent infectious FFU assay.

C. burnetii replication during its developmental cycle was quantified by using a replating fluorescent infectious focus-forming unit (FFU) assay (18). Confluent CHO cells stably overexpressing EGFP-Rab1b Q67L or EGFP (as a control) were grown in individual wells of a six-well culture dish. CHO cells were incubated with C. burnetii inocula for 48 h at 37°C. Infected cells were harvested by scraping and then disrupted by gentle sonication. Cell lysates with released C. burnetii were used to infect fresh confluent Vero cells in an individual well of a 24-well culture dish. Vero cells were incubated with cell lysates containing C. burnetii inoculum for 3 h at 37°C to allow internalization. Cells were washed several times to eliminate extracellular bacteria and incubated with fresh α-MEM (supplemented with 5% FBS). After 72 h of incubation, the cells were fixed, and the FFU were determined by indirect immunofluorescence using a polyclonal antibody against C. burnetii. FFU counts were scored by confocal microscopy in an Olympus FluoViewTM FV1000 confocal microscope with the FV10-ASW software.

siRNA silencing of Rab1b.

Purified small interfering RNA (siRNA) against human Rab1b and control siRNA were purchased from BIONEER (Korea). Confluent HeLa cells were transfected with siRNA Rab1b or with control siRNA, both prepared at a final concentration of 20 nM in 400 ml of α-MEM without serum and with Lipofectamine 2000 reagent. The mix was added to a 24-well culture dish. After 5 h, the transfection mix was replaced by fresh α-MEM with 10% FBS. At 48 h posttransfection, the medium was removed, and the same transfection protocol with the corresponding siRNA was applied again (second hit). When the second transfection mix was removed, the HeLa cells were infected with C. burnetii for 72 h at 37°C.

Western blotting.

HeLa cells were washed twice, scraped, and resuspended in sample buffer containing 1% 2-mercaptoethanol and then sonicated for 10 min at 4°C. For Western blot analysis, protein extracts were subjected to electrophoresis in SDS-12.5% PAGE gels and transferred to a Hybond-enhanced chemiluminescence nitrocellulose membrane (Amersham GE, Buenos Aires, Argentina). Membranes were blocked with blotto (PBS with 5% nonfat milk and 0.1% Tween 20) for 1 h at room temperature. Subsequently, the membranes were washed twice with 0.1% PBS-Tween 20 and incubated with the primary antibody. To detect Rab1, membranes were incubated overnight at 4°C with rabbit anti-Rab1 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA), washed, and incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Immunoresearch Laboratories) at a final dilution of 1:10,000. Equivalent protein loadings were confirmed by incubating membranes with a mouse anti-actin antibody (1:300) for 1 h at room temperature. The bands were visualized using the ECL reagent from GE. Images of the bands were obtained by using a LAS-4000 luminescent image analyzer (Fujifilm).

Rab1b decorates the Coxiella-replicative compartment at later infection times.

As indicated in the introduction, Legionella-containing vacuoles recruit Rab1b within few minutes after internalization (8). Given certain similarities between the intracellular behavior of Legionella and Coxiella, we decided to analyze whether this Rab protein could be recruited to the Coxiella phagosomes. For this purpose, stably transfected CHO cells overexpressing EGFP-Rab1b wt or the GTPase defective mutant EGFP-Rab1b Q67L were infected with C. burnetii. Cells overexpressing EGFP alone were used as control. The cells were fixed at different infection times (from 10 min to 24 h) and subjected to immunofluorescence using an antibody against C. burnetii. As shown in the confocal images, Rab1b presented a typical perinuclear Golgi distribution (Fig. 1). However, no colocalization between Coxiella and Rab1b (wt or mutant) was observed between 10 and 60 min after the uptake. By 6 or 12 h postinfection, only in cells overexpressing the mutant Rab1b Q67L were some of the incipient Coxiella-containing vacuoles decorated by this protein. This colocalization was more evident by 24 h postinfection, and at this time point it was also visible in cells expressing the wt protein. As shown in Fig. 2, by 48 h postinfection, when the large and spacious Coxiella niche is generated, the vacuoles were clearly decorated by Rab1b Q67L, whereas in cells overexpressing Rab1b wt, the protein, although present, presented a more discontinuous distribution showing a patchy pattern (see Fig. 2, insets). The bottom panels show the quantification of the fluorescence intensity along the yellow line scan across the CRV depicted in the insets. The graphs show accumulation of Rab1b wt or Rab1b Q67L mutant (green) at the surrounding CRV membrane.

The results point out that at later infection times the Coxiella-replicative niche recruits Rab1b or interacts with a Rab1b-labeled compartment. In order to demonstrate the specificity of the Rab1 recruitment to the Coxiella vacuole, we analyzed the interaction with Rab11, another member of the Rab family of GTPases. Rab11, is a well-known marker for recycling endosomes (RE) and plays a key role in regulating the transport of vesicles/proteins from REs and early endosomes to the trans-Golgi network and plasma membrane through recycling pathways (28, 41, 46). In contrast to Rab1, no colocalization with either Rab11wt or the GTPase-deficient mutant (Rab11Q70L) was observed (see Fig. S1A in the supplemental material), even at more than 48 h postinfection, suggesting that the presence of Rab1 on the vacuole was not just due to overexpression of any Rab protein. In addition, with the purpose of demonstrating that the recruitment of Rab1 wt or Q67L mutant was specific to the Coxiella-vacuole, CHO cells overexpressing EGFP, EGFP-Rab1b wt, or EGFP-Rab1b Q67L were allowed to phagocytose latex beads for 24 h (see Materials and Methods). As expected, no EGFP-Rab1b wt or EGFP-Rab1b Q67L was found decorating the bead phagosomal membrane (see Fig. S1B in the supplemental material), indicating that Rab1 recruitment was limited to the pathogen-containing compartment.

In order to demonstrate that the association with Rab1 was also observed in another cell type, we next assessed the presence of Rab1 on the CRV using a macrophage cell line. Similar to the results obtained with CHO cells, in Raw macrophages EGFP-Rab1b Q67L was also recruited to the vacuole membrane since 6 h after infection, whereas in Rab1b wt-overexpressing cells the recruitment was only observed by 24 h postinfection (see Fig. S2 in the supplemental material). These results indicate that no major differences in the kinetics of Rab1 recruitment are observed between the two cell lines used (i.e., CHO and Raw macrophages). An interesting observation is that in both CHO and Raw macrophages overexpressing the GTPase-defective mutant (Rab1bQ67L) the infection pattern was different from that of cells overexpressing GFP alone or Rab1b wt. Instead of the one or two large spacious vacuole typically observed by 48 h postinfection, numerous but smaller vacuoles per cell were present, suggesting that the overexpression of this Rab1 mutant somehow alters the normal development of the Coxiella-vacuole.

Taken together, these results indicate that, unlike Legionella, Coxiella phagosomes do not interact with Rab1b at early times after internalization (i.e., 10 to 60 min), whereas at later times postinfection the CRV becomes decorated by Rab1, mainly when the protein is in its GTP-bound state (i.e., Rab1b Q67L mutant). The association of Rab1b with the limiting membrane of the Coxiella-vacuole remains even after 48 h, when the vacuole is fully developed.

Overexpression of Rab1b N121I or Rab1b silencing alter Coxiella-vacuole growth.

Given the observation that both EGFP-Rab1b wt and the constitutively active mutant (Rab1b Q67L) were present at the Coxiella-vacuole membrane, we decided to analyze the effect of overexpressing a dominant-negative mutant of Rab1b (Rab1b N121I) on CRV growth. The N121I mutation renders a Rab1 protein defective in binding to either GDP or GTP (i.e., nucleotide empty mutant). The N121I mutant presents a diffuse cellular pattern without associating with a clearly defined compartment (2). Expression of this mutant in cells blocks the exit of cargo vesicular stomatitis virus G protein from the ER and causes the complete disassembly of the Golgi apparatus. Because prolonged overexpression of this mutated protein is lethal to CHO cells, we applied a different experimental approach to assess the impact of Rab1b N121I on Coxiella-vacuole growth. As shown in Fig. 3 A (experimental procedure 1), CHO cells were infected for 24 h before the transfection process and fixed after 48 h of continuous infection. Measurements of the vacuole diameter under the overexpression of Rab1b N121I showed a decrease in the large vacuole population (>10 μm), in contrast to the control condition (Fig. 3B). These results indicate that a functional Rab1b is required for the normal growth of the CRV.

In order to confirm the requirement of Rab1b for the generation of a spacious Coxiella-replicative vacuole, a knockdown assay was performed. HeLa cells were transfected with a siRNA against human Rab1b or a control siRNA. A double-hit protocol, as depicted in Fig. 3A (experimental procedure 2), was applied to maintain the protein silencing effect during the entire infection process. HeLa cells were transfected twice with the siRNA during the 48-h period after cell seeding. Immediately after the second transfection, the cells were infected with C. burnetii and cultured for an additional 72 h to allow the growth of the large CRV. It has been demonstrated that Rab1b depletion results in ER-to-Golgi trafficking defect and disruption of the Golgi apparatus (19). Thus, in order to corroborate that Rab1b was effectively knocked down, the distribution of the Golgi marker GM130 was analyzed. The results of an indirect immunofluorescence assay for the detection of GM130 (a cis-Golgi protein) (red, arrowheads) and C. burnetii (green) are shown in Fig. 3C. As expected, in cells treated with the siRNA against Rab1b, the Golgi apparatus was disassembled. A disperse distribution of the Golgi apparatus was observed, in contrast to the normal perinuclear pattern observed under the control condition (Fig. 3C, arrowheads), indicating disruption of the organelle. A Western blot assay to detect Rab1 was also performed in order to verify the silencing of the protein (Fig. 3E). As shown in the figure, in cells treated with the siRNA against Rab1b the Coxiella-vacuoles (surrounded by a yellow dashed line) were smaller than in cells treated with the control siRNA (arrows). The vesicle size was quantified, and a marked decrease in the diameter of CRVs was clearly observed in cells without normal expression levels of Rab1b (Fig. 3D). These results indicate that Rab1b silencing affects vacuole growth, suggesting that this protein has a critical role in the biogenesis of the Coxiella-replicative niche.

Disruption of the secretory pathway alters the size of the spacious Coxiella-vacuole.

As indicated above, Rab1b N121I, a mutant that leads to disruption of the early secretory pathway with the consequent Golgi disassembly, altered CRV growth. To confirm the participation of the secretory pathway in the generation of the CRV, we used two different strategies. The first strategy involved the Ras-related protein ADP-ribosylation factor 1 (Arf1), which is a low-molecular-weight GTP-binding protein, that in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes (9). Brefeldin A (BFA) is a noncompetitive inhibitor of Arf1 activation. Cell treatment with BFA results in specific inhibition of the Arf1 guanine-nucleotide exchange factor (GEF) and in efficient and reversible blocking of membrane traffic at the Golgi apparatus (26). In order to analyze the effect of BFA on the maintenance of the spacious CRV, CHO cells were transfected with EGFP or EGFP-Rab1b Q67L, the GTPase-defective mutant of Rab1b. After transfection, cells were infected with C. burnetii for 48 h and incubated for 2 or 6 h in the presence or absence of 200 nM BFA. The cells were fixed and subjected to indirect immunofluorescence for the detection of both the Golgi protein GM130 and Coxiella using specific antibodies. As expected, after 2 h of BFA treatment, the Golgi apparatus showed a disperse pattern, indicating that this organelle has been disassembled (Fig. 4 A, lower panels, arrowheads). BFA caused a significant decrease in the CRV size compared to untreated cells (Fig. 4A, arrows pointing to a CRV encircled with a yellow line). The quantification of the CRV diameter is depicted in Fig. 4B. In cells overexpressing Rab1b Q67L and treated with BFA, the CRVs remained decorated with this Rab1b mutant (data not shown), and no differences in vacuole diameter were observed compared to untreated cells (i.e., no BFA). Our results are in agreement with previously published data indicating that overexpression of Rab1b Q67L conferred resistance to BFA (24). The reduction in vacuole size after BFA treatment indicates that a continuous provision of membranes from the ER is a requirement for the persistence of the spacious Coxiella vacuole.

Sar1 is a small GTPase involved in the formation of COPII-coated transport vesicles that bud from the endoplasmic reticulum (reviewed by (37). Overexpression of both Sar1 T39N, a GDP-restricted mutant of Sar1 (23), or Sar1 H79G, a GTP-restricted mutant, blocks ER exit sites disrupting the secretory pathway with the consequent disassembly of the Golgi apparatus (48). Thus, the second strategy consisted in disrupting the secretory pathway with Sar1 T39N. Under overexpression of Sar1 T39N, the protein GM130 is redistributed into punctate cytoplasmic structures, namely, Golgi remnants. When Sar1 T39N is overexpressed the COPII coat protein Sec13p is displaced from the ER exit sites (35), blocking ER-Golgi transport. To analyze the Sar1 T39N effect on the generation of the spacious CRV, CHO cells were infected with C. burnetii, according to the experimental procedure 1 described in Fig. 3A. At 24 h postinfection, the cells were transfected with a bicistronic plasmid encoding Sar1 T39N and the protein Ds-Red (pIRES-DsRed Sar1 T39N) to visualize the transfected cells. Subsequently, at 24 h posttransfection, the cells were fixed, subjected to immunofluorescence, and analyzed by confocal microscopy. Figure 4C (inset) shows two cells, one of them overexpressing Sar1 T39N (ds-red) with a disassembled Golgi apparatus (green) and an untransfected cell with the typical perinuclear Golgi distribution (see arrowheads). The lower panel shows that the size of the vacuoles (surrounded by a yellow dashed line) is smaller in Sar1 T39N-overexpressing cells than in control untransfected cells (arrows). Quantification of the CRV diameter is depicted in Fig. 4D, which shows that the percentage of larger vacuoles (>10 μm) was dramatically decreased under Sar1 T39N overexpression compared to the control condition. Taken together, both the BFA effect and the results obtained with the Sar1 mutant confirm that disruption of the early secretory pathway alters normal CRV growth.

In cells transfected with the GTPase-deficient mutant of Rab1b, the biogenesis of the C. burnetii-containing compartment is altered.

In general, at 48 h postinfection with C. burnetii a very large CRV vacuole is generated. At this infection time, it is very common to observe only one or two spacious vacuoles per cell, likely as a result of homotypic fusion events. An interesting observation from the images shown in Fig. 2 is that, apparently, in cells overexpressing the GTPase-defective mutant Rab1b Q67L the size of the CRVs was decreased, albeit with a concomitant increase in vesicle number. Published work has shown that expression of the constitutively active Q67L mutant has no detectable effect on ER-Golgi trafficking and Golgi structure (2, 39). Thus, the diminished size is not likely attributed to a deficient delivery of early secretory vesicles to the CRV. Therefore, we were interested in determining why the size and number of the CRVs were altered in Rab1b Q67L-expressing cells. To begin addressing this point, we first decided to carefully analyze both aspects (i.e., vesicle size and number) in cells transfected with pGFP-Rab1b wt or pGFP-Rab1b Q67L. Transfected cells were infected with C. burnetii for 48 or 72 h and subjected to immunofluorescence to detect the bacteria. As shown in Fig. 5 A, in control cells overexpressing EGFP alone the typical formation of one or two very large CRV containing C. burnetii is depicted (arrows). A similar infection pattern was observed in cells overexpressing EGFP-Rab1b wt. In contrast, numerous CRVs were present in cells overexpressing GFP-Rab1b Q67L at either 48 or 72 h postinfection. Both parameters, vesicle size and number, were quantified and, as indicated in Fig. 5B and C, a noticeable increase in the number of CRVs was clearly observed in cells overexpressing the Rab1b mutant with a marked decrease in vacuole size. These results indicate that overexpression of the GTPase-defective mutant of Rab1b alters the infection pattern of C. burnetii modifying the generation of the Coxiella-replicative compartment.

Another important point was to determine whether the change in the typical infection pattern described above interfered with the replicative properties of C. burnetii. Therefore, with the purpose of determining this aspect, we performed a fluorescent infectious FFU assay (see Materials and Methods). CHO cells stably overexpressing EGFP (as control) or EGFP-Rab1b Q67L were infected with C. burnetii for 48 h. The cells were then harvested by scraping and disrupted by sonication. Afterward, Vero cells were incubated with the cell lysates containing C. burnetii for 3 h (uptake) at 37°C to allow internalization. After 72 h of incubation, the cells were fixed and subjected to indirect immunofluorescence, and FFU counts were determined by using confocal microscopy. As shown in Fig. 5D, there was a marked decrease (ca. 50%) in the bacterial replication of Rab1b Q67L compared to the control condition (EGFP) or to cells overexpressing GFP-Rab1wt (not shown).

In conclusion, these results show that the vacuole size was altered by overexpression of Rab1b Q67L and, more importantly, that the C. burnetii replication rate was affected.

The Coxiella-containing compartments generated in cells overexpressing the mutant Rab1b Q67L retain its acidic and degradative characteristics.

As indicated in the introduction, it is well known that the large vacuoles generated by C. burnetii are acidic and have features of phagolysosomes (reviewed in reference 15). Since in cells overexpressing the Rab1b Q67L mutant no generation of the large CRVs was observed, we were interested in determining whether the smaller vacuoles conserved the other features of normal CRVs. To assess whether these Coxiella-containing vesicles were indeed acidic, CHO cells stably overexpressing GFP, Rab1wt, or Rab1b Q67L were infected with Coxiella for 48 h and labeled with the acidotropic marker LysoTracker. As shown in Fig. 6 A, the Coxiella-containing vacuoles generated in all of the conditions were clearly labeled with LysoTracker (see insets), indicating that even in cells overexpressing the Q67L mutant the bacterium-containing compartments preserve the acidic properties typical of the Coxiella-replicative niche.

We next assessed whether these Coxiella-containing vacuoles had lysosomal features (i.e., hydrolytic compartment). For this purpose, infected cells were incubated for 6 h with DQ-BSA (Molecular Probes), a BSA derivative so heavily labeled that the fluorophore is self-quenched. Proteolysis of this compound results in dequenching and the release of brightly fluorescent fragments. Thus, the use of DQ-BSA is a valuable tool for the visualization of a proteolytic compartment. Similar to the control condition (i.e., cells overexpressing the vector alone), colocalization of Coxiella and DQ-BSA in a EGFP-Rab1b wt or Q67L-decorated vacuole is depicted in Fig. 6B, indicating the degradative characteristics of the vacuole. As shown in panels C and D, practically no differences were observed in the percentage of colocalization with LysoTracker or DQ-BSA and Coxiella-vacuole in cells overexpressing the different constructs.

We have previously demonstrated that the Coxiella-replicative niche is decorated by the protein LC3, a hallmark of autophagic vacuoles (4, 13). Furthermore, we have shown that autophagy induction by amino acid deprivation favors the development of the CRV (13) and that LC3 recruitment is actively modulated by Coxiella itself (30). Therefore, we were interested in determining whether the Coxiella-containing vacuoles generated in cells overexpressing the Rab1b mutant also conserved the capability of recruiting the autophagic protein LC3. Stably transfected CHO cells overexpressing EGFP-Rab1b Q67L were transiently transfected with pRFP-LC3. The cells were subsequently infected with C. burnetii for 72 h, subjected to immunofluorescence, and analyzed by confocal microscopy. In the insets in Fig. 6E, colocalization of Rab1b Q67L (green) and LC3 (red) at the membrane of the Coxiella-containing vacuoles (blue) is depicted, indicating that the interaction with the autophagy pathway is also maintained.

Taken together, our results suggest that despite the reduced vesicle size of the vacuoles that shelter Coxiella in cells overexpressing the mutant Rab1b Q67L, the vacuoles retain the acidic and autophagolysosome-like features of these compartments.

The fusogenic and replicative properties of the Coxiella compartments generated in cells overexpressing Rab1b Q67L are altered.

As indicated above, the expression of the constitutively active Q67L mutant does not inhibit ER-Golgi transport (2, 39). Since the CRVs generated in Rab1b Q67L-expressing cells have a reduced vesicle size, we wondered whether the fusion capacity of the CRVs was altered. It has been previously demonstrated that molecules internalized by fluid-phase endocytosis are found in vacuoles containing C. burnetii (15) and that these vacuoles also fuse with other compartments of the phagocytic/endocytic system (43, 44). To analyze the fusion capacity of the CRVs generated in cells overexpressing the Rab1b mutant Q67L, CHO cells overexpressing EGFP alone (control) or EGFP-Rab1b Q67L were infected with C. burnetii for 48 h to allow the development of the Coxiella vacuole. Subsequently, cells were incubated for 1 h with heat-inactivated Staphylococcus aureus-rhodamine or dextran-rhodamine to label the phagocytic and endocytic pathways, respectively. After fixation, cells were labeled by immunofluorescence with a specific antibody against C. burnetii and analyzed by confocal microscopy. As shown in Fig. 7 A, upper panels, colocalization of S. aureus-rhodamine (red) and C. burnetii (blue) in cells overexpressing EGFP is clearly visualized in most of the CRVs (see also the quantification in Fig. 7C). In contrast, in CHO cells overexpressing EGFP-Rab1b Q67L very limited colocalization is observed (Fig. 7A, lower panels, and Fig. 7C), suggesting that these compartments have its fusogenic properties with the phagocytic pathway altered. Similar results were obtained when the endosomal compartment was loaded with rhodamine-dextran (Fig. 7B, lower panels), indicating that the fusogenic capacity of the CRVs with the endocytic pathway was also affected (see the quantification in Fig. 7C).

Taken together, these results suggest that the GTPase-defective mutant of Rab1b alters normal growth of the Coxiella vacuole by changing the fusogenic properties of this compartment, since fusion between the Coxiella vacuole and both the endocytic and the phagocytic pathways was hampered.

The recruitment of Rab1b Q67L to the Coxiella-vacuole membrane depends on a functional secretory pathway.

We have presented evidence that Rab1b Q67L is decorating the Coxiella-vacuole membrane (Fig. 1 and 2). We have also shown that vacuole growth requires a functional secretory pathway, since the overexpression of the negative mutant of Sar1 affected CRV enlargement. Therefore, our next aim was to elucidate whether the recruitment of the GTPase defective mutant was a process mediated by vesicular transport from the ER or whether the protein was directly recruited from the cytosol. For this purpose, we blocked the early secretory pathway by overexpressing the GDP-restricted mutant of Sar1 (Sar1 T39N) which strongly inhibits vesicle budding from the ER. Because the overexpression of Sar1 T39N is extremely toxic, cells were infected prior to the transfection procedure (see Fig. 3A, experimental procedure 1). At 24 h postinfection, cells were cotransfected with Rab1b Q67L and Sar1 T39N for 24 h. After fixation, cells were subjected to immunofluorescence and analyzed by confocal microscopy. As previously observed in cells expressing Rab1b Q67L alone, this mutant protein was recruited to the CRV (Fig. 8 A,). In contrast, in cells coexpressing the dominant-positive mutant of Rab1b (Q67L) and the dominant-negative mutant of Sar1 (T39N) the CRVs were not decorated with Rab1b Q67L (Fig. 8A, inset). As expected, Fig. 8A (arrowheads) also shows that the Golgi integrity is affected in cells overexpressing Sar1 T39N (delineated cell). Likewise, when the dominant-positive mutant of Sar1 (Sar1 H79G), which also inhibits the ER-Golgi transport, was coexpressed with Rab1b Q67L, the Coxiella vacuoles were not decorated by this Rab1b mutant (data not shown). Quantification of the CRVs size also revealed that, similar to the effect of Sar1 T39N, the GTPase-defective mutant Sar1 H79G also affected Coxiella-vacuole growth (Fig. 8B and C).

Taken together, these results highlight the important contribution of the secretory pathway to the biogenesis of the C. burnetii-replicative niche, since disruption of this essential pathway affects the enlargement of the vacuole. Moreover, the evidence also indicates that the recruitment of Rab1b Q67L to the vacuole membrane depends on a functional secretory pathway.