Abstract
Coxiella burnetii is a Gram-negative obligate intracellular bacterium. After internalization, this bacterium replicates in a large parasitophorous vacuole that has features of both phagolysosomes and autophagosomal compartments. We have previously demonstrated that early after internalization Coxiella phagosomes interact with both the endocytic and the autophagic pathways. In this report, we present evidence that the Coxiella-replicative vacuoles (CRVs) also interact with the secretory pathway. Rab1b is a small GTPase responsible for the anterograde transport between the endoplasmic reticulum and the Golgi apparatus. We present evidence that Rab1b is recruited to the CRV at later infection times (i.e., after 6 h of infection). Interestingly, knockdown of Rab1b altered vacuole growth, indicating that this protein was required for the proper biogenesis of the CRV. In addition, overexpression of the active GTPase-defective mutant (GFP-Rab1b Q67L) affected the development of the Coxiella-replicative compartment inhibiting bacterial growth. On the other hand, disruption of the secretory pathway by brefeldin A treatment or by overexpression of Sar1 T39N, a defective dominant-negative mutant of Sar1, affected the typical spaciousness of the CRVs. Taken together, our results show for the first time that the Coxiella-replicative niche also intercepts the early secretory pathway.
Coxiella burnetii, the etiologic agent of Q fever in humans, is an airborne, Gram-negative bacterium, which develops and multiplies in large, acidified, hydrolase-rich vacuoles with phagolysosome-like characteristics. These Coxiella-replicative vacuoles (CRVs) are highly fusogenic and capable of interacting with both the endocytic and the phagocytic pathways (16). We have previously shown that the parasitophorous vacuoles also have the hallmarks of an autophagosomal compartment (4) and that autophagy induction is beneficial for the replication and survival of Coxiella (6, 13). Interestingly, we have recently reported that the interaction with the autophagic pathway takes place early after infection (i.e., few minutes) and that the autophagic protein LC3 is actively recruited by Coxiella to its phagosomes long before the formation of the CRV (30). Therefore, since the bacterium-customized replicative vacuole that shelter Coxiella also displays lysosomal markers such as Lamp1 proteins and lysosomal enzymes (16), we infer that C. burnetii resides in an autophagolysosome-like compartment.
Rab proteins are small GTPases that function as molecular switches regulating important steps in membrane trafficking such as budding, microtubule-mediated transport, and tethering of the vesicle with the target compartment prior to fusion (49). These proteins serve as scaffolds recruiting different downstream effector molecules to integrate vesicular transport and signaling (33, 49). More than 70 Rab and Rab-like proteins have been identified in humans, and the function of many of them has been determined (reviewed in reference 34). In general, each Rab protein is enriched in a specific compartment, and although several Rab proteins can be present in the same organelle, it is believed that they occupy discrete membrane domains. For instance, Rab5 is present in early endosomes, whereas Rab7 is enriched in late endocytic/lysosomal compartments.
We have recently demonstrated that Coxiella-containing phagosomes acquire Rab5 and Rab7 sequentially at early times after infection (30). Rab proteins are known to cycle between a GTP-bound active conformation and a GDP-bound inactive conformation. Dominant-negative mutants that preferentially remain in the GDP inactive estate have been generated for most of the Rab proteins. In our previous studies we have observed that overexpression of a dominant-negative mutant form of Rab5, but not of Rab7, hampered Coxiella entry, whereas both Rab5 and Rab7 dominant-negative mutants inhibited replicative vacuole formation, indicating that interaction with the endocytic pathway is a critical event for the biogenesis of the Coxiella vacuole (30).
Rab1 is a member of the Rab family that preferentially localizes at the endoplasmic reticulum (ER)-Golgi intermediate compartment (12, 32). It has been reported that Rab1 regulates anterograde transport between ER-Golgi compartments (25, 27), mediating the docking of ER-derived vesicles with the cis-Golgi compartment (27). Rab1 has been shown to interact with the Golgi membrane proteins GM130 and GRASP (25) contributing to the docking process. The intracellular pathogen Legionella pneumophila, after internalization, recruits early secretory vesicles from the host cell delaying the fusion with the lysosomal compartment (17, 21). Interestingly, Rab1b was recruited to the Legionella-containing vacuole (LCV) within minutes after internalization, and this Rab1b recruitment was important for the subsequent interaction of ER-derived vesicles with the LCV and for Legionella intracellular growth (22). In the present study, we show that at later infection times Rab1b decorates the Coxiella-containing vacuole, suggesting that this compartment intercepts with the early secretory pathway. Knockdown of Rab1b or overexpression of a Rab1 dominant-negative mutant affected vacuole growth, suggesting that this protein was involved in the biogenesis of the CRV. In addition, we present evidence that disruption of the secretory pathway by brefeldin A treatment or by overexpressing a dominant-negative mutant of Sar1 alters the formation and maintenance of the single large and spacious Coxiella vacuole. Interestingly, overexpression of the green fluorescent protein (GFP)-Rab1b GTPase-defective mutant affected the biogenesis of the CRV. Our results indicate that this mutant altered the normal growth of the Coxiella vacuole, at least in part, by changing its fusogenic capacity with endocytic compartments. Taken together, our results show the functional consequences of altering the secretory pathway on the Coxiella- replicative niche formation.
MATERIALS AND METHODS
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).
RESULTS
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.
FIG. 1.
Kinetic of infection of C. burnetii under Rab1b overexpression. Stably transfected CHO cells overexpressing EGFP alone, EGFP-Rab1b wt, or the GTPase-defective mutant EGFP-Rab1b Q67L were infected with C. burnetii. The cells were fixed at different infection times (10 min, 30 min, 1 h, 6 h, 12 h, and 24 h), subjected to indirect immunofluorescence using an antibody against C. burnetii (red), and visualized by confocal microscopy. The images show a typical Rab1b distribution in a perinuclear Golgi pattern; however, no colocalization of Rab1b (wt or mutant) with C. burnetii containing compartment was observed at early times (10, 30, or 60 min). Nevertheless, a clear recruitment of EGFP-Rab1b Q67L is observed at later infection times (6, 12, and 24 h). Bars, 10 μm.
FIG. 2.
Rab1b decorates the Coxiella-replicative niche at later times of infection. CHO cells stably overexpressing EGFP, EGFP-Rab1b wt, or the active mutant of Rab1 (EGFP-Rab1b Q67L) were infected for 48 h with C. burnetii. After fixation, cells were subjected to indirect immunofluorescence using an antibody against C. burnetii (red) and analyzed by confocal microscopy. As depicted in the insets, the large Coxiella-containing vacuole was strongly labeled by Rab1b Q67L and in a patchy pattern by Rab1b wt. Insets also show a yellow line scan in order to visualize the localization of the Rab1b wt and its defective dominant-active mutant (Q67L) in the vacuole membrane. Images are representative of at least three independent experiments. Bars, 10 μm.
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.
FIG. 3.
Overexpression of Rab1b N121I and knockdown of Rab1b affect CRV growth. (A) Outline of the experimental procedures applied in the different assays. (B) CHO cells were infected for 24 h with C. burnetii and subsequently transfected with pEGFP-Rab1 N121I or pEGFP (control). At 48 h postinfection the cells were fixed and subjected to indirect immunofluorescence to detect Coxiella using specific antibodies. Quantification of the vacuole diameter in cells overexpressing EGFP-Rab1b N121I was compared to the control condition (P ≤ 0.05). (C) HeLa cells were transfected with siRNA against Rab1b or an irrelevant siRNA as a negative control. After 48 h, cells were transfected for a second time, infected with C. burnetii, and cultured for an additional 72-h period to allow the development of the large CRV. Subsequently, the cells were fixed and subjected to indirect immunofluorescence for the detection of both Golgi protein GM130 and Coxiella by using specific antibodies. Images were captured by confocal microscopy. The panels show that in contrast to control cells, in cells treated with the siRNA against Rab1b the Golgi apparatus was disassembled (punctate distribution, arrowheads) in the majority of the cells, indicating that Rab1b was effectively depleted. Furthermore, the sizes of the Coxiella-vacuoles were markedly decreased in cells treated with the siRNA against Rab1b (yellow dashed line). (D) Quantification of the vacuole diameter in cells in which Rab1b was depleted compared to the control condition. The data represent the means ± the standard errors of the mean (SEM) of at least three independent experiments in which at least 200 vacuoles were scored in each experiment (P ≤ 0.001). The data represent the means ± the SEM of at least three independent experiments. (E) Western blot of the assay described in panel C and quantification of intensity of the Rab1 bands relative to actin. The data represent one of two independent experiments.
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.
FIG. 4.
BFA treatment or overexpression of a dominant-negative mutant of Sar1 alter CRVs growth. CHO cells transiently overexpressing EGFP were infected with C. burnetii. After 48 h of continuous infection, the cells were treated with BFA for 2 or 6 h. After treatment, the cells were fixed and subjected to indirect immunofluorescence with specific antibodies against C. burnetii and GM 130 as a Golgi marker. (A) The images show smaller CRVs (blue) under BFA treatment (6 h) compared to untreated control cells (yellow dashed line). A partial disassembled Golgi (red) is shown in the lower panels (arrowheads). (B) Quantification of the vacuole size (10 μm) of the experiment presented in panel A (P ≤ 0.05). (C) CHO cells infected with C. burnetii for 24 h were transfected with pIRES-DsRed Sar1 T39N. After fixation, the cells were subjected to immunofluorescence using antibodies against C. burnetii (blue) and GM130 as a Golgi marker (green). A Golgi apparatus totally disassembled is shown (arrowheads). The figure shows the reduction in the CRV size (yellow dashed line) under Sar1 T39N overexpression (white dashed line). (D) A bar graph shows a quantification of the >10-μm CRVs in control or Sar1 T39N-overexpressing cells. The data represent the means ± the SEM of at least three independent experiments (P ≤ 0.001). Bars, 15 μm.
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.
FIG. 5.
Overexpression of Rab1b Q67L alters the infection profile of C. burnetii. CHO cells transfected with pEGFP plasmids encoding Rab1b wt or the active mutant Rab1b Q67L were infected with C. burnetii for 48 or 72 h (see Materials and Methods), subjected to indirect immunofluorescence to detect the bacteria, and analyzed by confocal microscopy. (A) Confocal images showing the typical formation of one or two large vacuoles containing C. burnetii (yellow dashed line) in CHO cells overexpressing EGFP alone or EGFP-Rab1b wt. In contrast, in cells overexpressing EGFP-Rab1b Q67L, the Coxiella-containing vacuoles are smaller than those of the control condition but are present in large numbers (arrowheads). (B) Quantification of the number of vacuoles per cell at 48 and 72 h of infection from the experiment presented in panel A. (C) Quantification of the size of vacuoles at 48 h postinfection from the experiment presented in panel A. The data represent the means ± the SEM of at least three independent experiments. (D) CHO cells stably overexpressing EGFP (as control) or EGFP-Rab1b Q67L were incubated with C. burnetii. After 48 h of infection, the cells were lysed. Cell lysates with released C. burnetii were used to infect Vero cells. After 72 h of incubation (chase), cells were fixed, and the fluorescent infectious FFU were determined by indirect immunofluorescence, and FFU counts were obtained using confocal microscopy. The data represent the means ± the SEM of at least three independent experiments where at least 200 cells were scored in each experiment (P ≤ 0.05). Bars, 15 μm.
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.
FIG. 6.
Coxiella-containing compartments labeled with Rab1b Q67L retain its acidic and degradative characteristics and are also labeled by LC3. CHO cells stably overexpressing EGFP, EGFP-Rab1wt, or EGFP-Rab1b Q67L were infected with C. burnetii. At 48 h of infection, the cells were incubated for 30 min with the acidotropic probe LysoTracker or DQ-BSA for 6 h. After fixation, the cells were examined by indirect immunofluorescence with a specific antibody against C. burnetii and analyzed by confocal microscopy. (A) The insets show the colocalization of LysoTracker (red) and C. burnetii (blue) inside the Coxiella vacuole in all cell types, suggesting that these compartments preserve the acidic properties typical of the Coxiella-replicative niche. (B) Confocal images show the colocalization of C. burnetii (blue) and DQ-BSA (red), indicating the degradative characteristics of the vacuoles. Bars, 20 μm. (C) Quantification of the percentage of colocalization of C. burnetii and LysoTracker. (D) Quantification of the percentage of colocalization of C. burnetii and DQ-BSA. A total of 50 cells were counted in each condition. (E) CHO cells stably overexpressing EGFP-Rab1b Q67L were transiently transfected with pRFP-LC3. At 24 h posttransfection, the cells were infected with C. burnetii for 72 h. Cells were fixed, analyzed by indirect immunofluorescence, and examined by confocal microscopy. Insets show the colocalization of Rab1b Q67L (green) and LC3 (red) at the membrane of the vacuoles containing Coxiella (blue). This result suggests that the interaction with the autophagic pathway is maintained under overexpression of Rab1b Q67L. Bars, 15 μm.
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).
FIG. 7.
Overexpression of Rab1b Q67L alters the fusogenic and replicative properties of the C. burnetii compartments. CHO cells stably overexpressing EGFP alone as a control or EGFP-Rab1b Q67L were infected with C. burnetii. At 48 h postinfection, the cells were incubated for 1 h with heat-inactivated Staphylococcus aureus-rhodamine or with 5 μg of dextran-rhodamine/ml to label the phagocytic and endocytic pathways, respectively. (A) The upper panels show colocalization (see inset) of C. burnetii (blue) and S. aureus-rhodamine (red). In contrast, no colocalization of heat-inactivated S. aureus-rhodamine, and the bacterium is observed in the vacuoles of cells overexpressing Rab1b Q67L (lower panels), suggesting that these compartments have its fusogenic properties with the phagocytic pathway altered. (B) The upper panels show that in CHO cells overexpressing EGFP the majority of the Coxiella-vacuoles contain the endocytic marker dextran- rhodamine (red), whereas the absence of colocalization is observed in cells overexpressing Rab1b Q67L (lower panels), suggesting that the fusogenic capacity with the endocytic pathway was altered. Bars, 20 μm. (C) Quantification of the percentage of colocalization of C. burnetii and heat-inactivated Staphylococcus aureus-rhodamine or dextran-rhodamine. A total of 50 cells were counted in each condition.
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).
FIG. 8.
Overexpression of a dominant-negative mutant of Sar1 prevents the recruitment of Rab1b Q67L to the C. burnetii vacuole membrane. CHO cells infected with C. burnetii for 24 h were cotransfected with pEGFP-Rab1b Q67L and pIRES-DsRed Sar1 T39N, pEGFP-Rab1b Q67L, and pIRES-DsRed Sar1 H79G or individually transfected with pEGFP, pIRES-DsRed Sar1 T39N, pIRES-DsRed Sar1 H79G, or pEGFP-Rab1b Q67L as controls. At 24 h posttransfection, the cells were fixed and subjected to indirect immunofluorescence with specific antibodies against C. burnetii (blue) and GM130 (red). The images were analyzed by confocal microscopy. (A) The insets in the left panels show clearly the recruitment of Rab1b Q67L to the vacuole membrane. In contrast, no recruitment of Rab1b Q67L to the CRV (see the inset) is observed under overexpression of Sar1 T39N (dashed delineated cell). The middle panels show the different diameters of the vacuoles under Rab1b overexpression, coexpression of Rab1b Q67L and Sar1 T39N (dashed delineated cell), or untransfected cells (arrows). A disassembled Golgi apparatus is showing cells overexpressing Sar1 T39N in contrast with an untransfected cell (arrowheads). (B) Quantification of the size of vacuoles at 48 h of infection from the experiment presented in panel A. The data represent the means ± the SEM of at least three independent experiments (P ≤ 0.001). (C) Quantification of the vacuole size at 48 h postinfection from cells overexpressing EGFP, EGFP-Rab1b Q67L, or DS-Red-Sar1 H69G or coexpressing Rab1b Q67L and DsRed H69G. The data represent the means ± the SEM of at least three independent experiments (P ≤ 0.001). Bars, 15 μm.
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.
DISCUSSION
C. burnetii, like several other intracellular pathogens, replicates in a membrane-bound compartment within the host cell (reviewed in reference 45). After 48 to 72 h of infection, Coxiella generates a large replicative compartment (i.e., CRV) that in many cases occupies the whole cytoplasm, displacing the majority of the organelles and even the nucleus. It is assumed that for the generation of such a large vacuole the microorganism manipulates several intracellular trafficking pathways in order to obtain enough membrane. Cumulative evidence indicates that both the endocytic and the phagocytic pathways contribute to the development and expansion of the CRV (4, 13, 16, 30). In the present study we present the first evidence that the Coxiella-replicative niche also intercepts the early secretory pathway (see the model in Fig. 9). Our results indicate that the CRVs are not only decorated by the small GTPase Rab1b and its GTPase-defective mutant but also that the formation of the CRV requires a functional Rab1b, a protein involved in ER-to-Golgi and intra-Golgi trafficking (27, 32). This close relationship with the secretory pathway seems to facilitate expansion (see below) and contributes to the biogenesis of the Coxiella-customized niche, which presents particular features to facilitate bacterial growth.
FIG. 9.
Model showing the interaction among C. burnetii and the endocytic, autophagic, and secretory pathways during the vacuole development process. Upon internalization, bacterium phagosomes interact with the autophagic pathway (the protein LC3 is present on the vacuole membrane). These Coxiella-containing phagosomes also interact with degradative organelles such as the lysosomes. These fusion events contribute to generate a proper environment for the replication process. The secretory pathway through Rab1b-labeled vesicles likely contributes by supplying membrane to generate the spacious Coxiella-vacuole. The highly fusogenic properties of the Coxiella-phagosomes would contribute not only to the formation of the large replicative niche but also to the acquisition of key factors and nutrients to favor the transformation of the bacteria into the replication-competent form (i.e., large cell variant).
The interaction between the secretory pathway and a pathogen-containing compartment is not unique to Coxiella. Indeed, it is widely documented that the Legionella-containing vacuole (LCV), within 10 min of bacterial uptake recruits Rab1b and subsequently interacts with vesicles derived from the early secretory pathway (8, 22, 42). However, in contrast to findings with Legionella pneumophila, our results indicate that either in CHO cells or in Raw macrophages Rab1b is not recruited to the Coxiella phagosome at early times after internalization (i.e., 1 h uptake) but when the Coxiella has developed larger compartments generated after 12 to 24 h infection. In the case of Chlamydia trachomatis, the inclusion membrane is also decorated by Rab1b, as well as by Rab4 and Rab11 (7, 31), whereas neither Rab5 (early endosome marker) nor Rab7 (late endosome marker) were recruited to the inclusion membrane. On the contrary, we have previously shown that both Rab5 and Rab7 bind to the Coxiella phagosome at an early time after internalization (30), whereas no recruitment of Rab11 has been observed at any time during infection (see Fig. S1A in the supplemental material). Thus, all of these data indicate that each microorganism recruits a subset of Rab proteins to specifically interact with defined intracellular compartments, in a time window, in order to generate a self-tailored replicative niche (for a review, see reference 5).
As mentioned above, Rab1b is a small GTPase that recruits factors necessary for the tethering and fusion of vesicles derived from the ER/intermediate compartment with target membranes (1, 25). We hypothesize that Rab1b is anchored onto the CRV membrane to subsequently allow the tethering of vesicles derived from the early secretory pathway. These vesicles may contribute with membranes to generate the spacious CRV or, alternatively, they may carry key fusion proteins such as SNAREs, i.e., transmembrane proteins required for specific fusion processes (reviewed in reference 20). Indeed, in the case of Legionella it has been shown that the ER-derived vesicles transport the SNARE Sec22b to the LCV (22). To the best of our knowledge, no single SNARE molecule has been yet identified in the Coxiella-containing vacuole. Ongoing work in our laboratory is currently designed to establish some of the SNAREs molecules involved in Coxiella intracellular trafficking.
We and others have demonstrated that the Coxiella-replicative compartment is highly fusogenic (4, 13, 16, 18, 30), and these fusogenic properties are likely important for the generation of the spacious CRV. We have observed that, in cells overexpressing the Rab1b mutant defective in GTPase activity (i.e., Rab1Q67L), the fusion capability of the CRVs with both endocytic and phagocytic compartments was significantly impaired. It is known that the dynamic association/dissociation of a Rab protein is essential for an efficient transport through a given pathway (38). As a compartment matures, the Rab composition in specific domains changes, thus the dissociation of a certain Rab protein would allow the recruitment of another one, as well as its interacting proteins (e.g., effectors), leading to new fusion and fission events favoring the maturation process. Therefore, it is likely that the irreversible association of the GTPase-deficient mutant of Rab1b to the CRV may hamper the recruitment of a critical Rab or other key factors required for fusion with endo/phagocytic compartments. These alterations in the fusion capability of the CRVs generated in cells overexpressing the mutant Q67L would also explain the increased vesicle number, albeit of smaller size, suggesting that homotypic fusion between Coxiella vacuoles is also impeded.
There are some interesting similarities between the results obtained with Legionella and Coxiella regarding the relationship of both pathogens with the autophagic and secretory pathways. It is well established that L. pneumophila exploits the autophagic pathway to generate an adequate replicative niche (10). At the ultrastructural level, the LCVs resemble nascent autophagosomes (36). In addition, the autophagic proteins Atg7 and LC3 transiently associate with the pathogen-containing vacuoles (3). Furthermore, amino acid depletion, which stimulates autophagy, increases the association of the bacteria with the ER and enhances bacterial growth. Besides, blocking the early secretory pathway with Brefeldin A diminish the colocalization of Atg7 and the LCV and prevents bacterial replication (3), indicating a dynamic interaction between both the secretory and autophagic pathways. Similarly, work from our laboratory indicates that the CRV is decorated by the autophagic protein LC3 (4). Physiological (i.e., starvation) or pharmacological (i.e., rapamycin treatment) induction of autophagy significantly enhances C. burnetii replication and viability, as determined by an FFU assay (13), which indicates that autophagy favors the biogenesis of the Coxiella-replicative niche. Our present results also indicate that the CRV recruits Rab1b and that adequate levels of this protein are critical for Coxiella replication since knockdown of this protein markedly reduces bacterial growth. Previous evidences indicate that the overexpression of Rab1b Q67L reverts the BFA effect (24). In agreement with this result, we observed that under BFA treatment the recruitment of Rab1b Q67L to the vacuole membrane was not altered. In contrast, Rab1b Q67L was not recruited to the CRV in cells overexpressing neither Sar1 T39N nor Sar1 H79G, indicating that vesicles produced by the Sar1/COPII system are necessary for transport of Rab1b to the CRV. This evidence agrees with the observation that in cells overexpressing the Sar1 H79G protein there is no tethering between ER vesicles and the LCV in L. pneumophila-infected cells (29). In summary, our results support the idea that the interaction with ER-derived vesicles is critical for the generation of the large CRV and sustain the hypothesis that the ER might be the source of autophagosome membranes (11, 40). Indeed, recently published work support this hypothesis since studies by electron tomography have revealed that both ER and the phagophore/isolation membranes are interconnected (14, 47). Moreover, we have recently found that autophagosome formation depends on the small GTPase Rab1b and functional ER exit sites (50). Our present results indicate that vesicles departing from ER exit sites intercept the Coxiella-replicative compartment. However, further studies are certainly needed to fully understand the molecular interplay between transport from the ER and the autophagic pathway in Coxiella-vacuole development.
Supplementary Material
Acknowledgments
We are grateful to Hirofumi Kai and Akiko Niibori (Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan) for providing the DrpIRES2DsRed2 bicistronic vectors encoding Sar1, Sar1 H79G, and Sar1 T39N. We thank Luis Mayorga and Walter Berón for critically reading of the manuscript. We also thank Alejandra Medero for technical assistance with tissue culture.
Editor: R. P. Morrison
Footnotes
Published ahead of print on 11 October 2010.
Supplemental material for this article may be found at http://iai.asm.org/.
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