Coxiella burnetii Inhibits Activation of Host Cell Apoptosis through a Mechanism That Involves Preventing Cytochrome c Release from Mitochondria

Abstract

Coxiella burnetii is an obligate intracellular pathogen and the etiological agent of the human disease Q fever. C. burnetii infects mammalian cells and then remodels the membrane-bound compartment in which it resides into a unique lysosome-derived organelle that supports bacterial multiplication. To gain insight into the mechanisms by which C. burnetii is able to multiply intracellularly, we examined the ability of host cells to respond to signals that normally induce apoptosis. Our data show that mammalian cells infected with C. burnetii are resistant to apoptosis induced by staurosporine and UV light. C. burnetii infection prevented caspase 3/7 activation and limited fragmentation of the host cell nucleus in response to agonists that induce apoptosis. Inhibition of bacterial protein synthesis reduced the antiapoptotic effect that C. burnetii exerted on infected host cells. Inhibition of apoptosis in C. burnetii-infected cells did not correlate with the degradation of proapoptotic BH3-only proteins involved in activation of the intrinsic cell death pathway; however, cytochrome c release from mitochondria was diminished in cells infected with C. burnetii upon induction of apoptosis. These data indicate that C. burnetii can interfere with the intrinsic cell death pathway during infection by producing proteins that either directly or indirectly prevent release of cytochrome c from mitochondria. It is likely that inhibition of apoptosis by C. burnetii represents an important virulence property that allows this obligate intracellular pathogen to maintain host cell viability despite inducing stress that would normally activate the intrinsic death pathway.

Q fever is a worldwide zoonotic disease caused by the obligate intracellular pathogen Coxiella burnetii (43). This small gram-negative bacterium is classified in the γ subdivision of the proteobacteria. C. burnetii is typically transmitted to humans by inhalation of infectious material from domestic livestock, and infection by as few as 10 bacteria can result in disease (7). Most animal infections are asymptomatic; however, abortion and infertility are economically important clinical manifestations of C. burnetii infections of livestock (5). Both an acute form and a chronic form of disease are observed in humans infected with C. burnetii. Acute Q fever usually presents as a self-limiting flu-like illness that can sometimes result in atypical pneumonia or hepatitis. Bacterial endocarditis is a clinical feature that is observed in patients with chronic C. burnetii infection. Other presentations of chronic infection include vascular infection, bone infection, and chronic hepatitis (43).

Phase variants of C. burnetii with reduced virulence can be obtained from infected animals and by ex vivo cultivation of the organism (25). C. burnetii phase I bacteria are serum resistant and infectious in animal models of disease, whereas phase II bacteria are serum sensitive and unable to cause disease in immune-competent animals (51). Although the significance of C. burnetii phase variation is not well understood, it is possible that an epigenetic switch between phase I bacteria and phase II bacteria could play an important role during infection. Phase II strains that are unable to revert back to highly virulent phase I organisms have been isolated (26). Of particular importance is a plaque-purified isolate of a C. burnetii Nine Mile phase II strain that contains a chromosomal deletion eliminating genes involved in modification of the C. burnetii lipopolysaccharide O antigen (30). Although this phase II strain is avirulent in animal models of disease, it retains the ability to infect and replicate in tissue culture cells, making it ideal for studying the cell biology of C. burnetii infection (42, 58).

After uptake by mammalian cells C. burnetii remains in a membrane-bound vacuole. This C. burnetii-containing vacuole initially appears to mature like phagosomes containing avirulent bacteria, undergoing fusion with endosomes and lysosomes, resulting in the formation of a phagolysosomal compartment (28). There is evidence that the C. burnetii-containing vacuole also interacts with autophagic vesicles, which intersect the endocytic pathway so that material inside the autophagosome can be delivered to lysosomes for degradation (23, 47). Although maturation along the endocytic pathway usually leads to digestion of internalized microbes in lysosomes (24), C. burnetii has the ability to modify this organelle into a spacious vacuole that permits bacterial replication (4, 8, 27, 31, 33).

It is not known how C. burnetii modifies the lysosomes, but one of the interesting properties of the C. burnetii-occupied vacuole is its unique capacity to undergo fusion with other secondary lysosomes in the cell to generate spacious lysosome-derived vacuoles (3, 27, 34). Generation of these spacious lysosomes is a process that requires continual synthesis of C. burnetii proteins (35), suggesting that bacterial proteins may directly affect the maturation of the C. burnetii-occupied vacuole. A type IV-like secretion system that is functionally related to the Dot/Icm system of Legionella pneumophila is thought to play a role in modulating host cellular processes by delivering bacterial effector proteins into the host cell (58, 60). In addition to modulating membrane traffic, it is predicted that effector proteins might also modulate processes important for cell survival, which would ensure that host cells remain viable during intracellular infection by C. burnetii (52).

Apoptosis is a programmed cell death pathway that is crucial for immune system maintenance and removal of damaged or infected cells (11). The two main pathways leading to apoptosis are the intrinsic and extrinsic pathways. The extrinsic pathway involves ligand binding to death receptors and adaptor proteins, which activate caspases (cysteinyl aspartate proteases). The intrinsic pathway involves activation of the Bax group of proteins. Activation of Bax and Bak is regulated by the prosurvival Bcl-2-like protein family members and by the proapoptotic BH3-only family members (50, 55). Once activated, Bax and Bak oligomerize and permeabilize the mitochondrial membrane, resulting in the release of cytochrome c, which causes caspase activation though activation of the apoptosome (2). Activated caspases mediate both cell death and inflammation through cleavage of specific cellular substrates. During apoptosis, the caspases are sequentially activated. First, one of the initiator caspases (caspase 8 or 9) is activated, which leads to the activation of a group of effector caspases (caspases 3, 6, and 7). Caspase 3, a key downstream effector of apoptosis, initiates robust proteolysis, and frees a dedicated DNase that fragments chromatin (15, 17).

Several pathogens have been shown to elicit a proapoptotic response during infection (21, 44, 61), while other pathogens appear to inhibit apoptosis (11). For obligate intracellular pathogens, such as C. burnetii, inhibition of apoptosis may be important, as it may enable bacteria that replicate slowly to establish a productive infection. The intracellular pathogens that have been shown to interfere with apoptosis include Chlamydia pneumoniae, Rickettsia rickettsii, and Toxoplasma gondii. C. pneumoniae prevents apoptosis by a process that involves degrading proapoptotic BH3-only proteins (19), R. rickettsii is thought to inhibit apoptosis by induction of genes encoding antiapoptotic proteins through NF-κB-mediated events (13), and T. gondii has been shown to interfere with multiple steps important for activation of the intrinsic cell death pathway (12).

Here we examine whether the intracellular pathogen C. burnetii has the ability to manipulate the host apoptotic pathway. To answer this question, Chinese hamster ovary (CHO) and HeLa cells were used to study interactions between C. burnetii and mammalian cells in vitro. These well-established epithelial cell lines have been used extensively for in vitro studies aimed at understanding how C. burnetii can alter cellular functions to establish an organelle that permits replication in mammalian hosts (23, 32, 57). Our results indicate that C. burnetii is able to prevent the induction of apoptosis induced by different stimuli. Mechanistically, inhibition of apoptosis by C. burnetii involves preventing cytochrome c release from mitochondria, which is a crucial event important for many proapoptotic signaling pathways.

MATERIALS AND METHODS

Reagents, cell lines, and bacterial strains.

Unless otherwise noted, chemicals were purchased from Sigma (St. Louis, MO). Gel/Mount was obtained from biomeda (Foster City, CA). Complete protease inhibitor cocktail mixture was obtained from Roche (Mannheim, Germany). Cell lines were cultured at 37°C in 5% CO₂. CHO cells were grown in minimal essential medium alpha (Gibco, Carlsbad, CA) containing 5% heat-inactivated fetal bovine serum (Gibco). HeLa cells were grown in Dulbecco modified Eagle medium containing 5% fetal bovine serum. A plaque-purified isolate of the C. burnetii phase II Nine Mile strain was generously provided by T. Hackstadt of the Rocky Mountain Laboratories in Hamilton, MT, and was propagated in CHO cells. Infective C. burnetii was obtained by hypoosmotic lysis of infected CHO cells as described previously (59). After incubation of either CHO or HeLa cells with infective C. burnetii for 2 h at 37°C in 5% CO₂, the medium was removed and the cells were washed three times with phosphate-buffered saline (PBS) before fresh medium was added to remove noninternalized bacteria. C. burnetii-infected cells were cultivated at 37°C in 5% CO₂ and were passaged every 3 days by diluting cultured cells 1:3 into fresh medium. After the first passage, 90 to 95% of the cultured cells contained vacuoles with replicating C. burnetii, as determined by staining fixed cells with 4′,6-diamidino-2-phenyindole (DAPI) and examining cells by fluorescence microscopy, and this level of infection was maintained upon further passaging. C. burnetii-infected cells were discarded 60 days postinfection. All experiments involving C. burnetii-infected cells were conducted 24 h after cells were diluted 1:3 into fresh culture medium.

Apoptosis assays. (i) Nuclear fragmentation assay.

CHO cells, either uninfected or infected with C. burnetii, were seeded on coverslips in a 24-well plate at a concentration of 2 × 10⁴ cells per well. After incubation with staurosporine (0.5 μg/ml) for 5 h, the cells were fixed with 3% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with ice-cold methanol for 30 s, quenched with 50 mM NH₄Cl in PBS for 15 min at room temperature, and stained with DAPI (0.1 μg/ml) in PBS for 7 min. The stained cells were mounted onto slides using Gel/Mount (biomeda). The nuclear morphology of infected cells was determined using a Nikon TE-200 inverted microscope (Tokyo, Japan).

(ii) TUNEL assay.

HeLa cells, uninfected or infected with C. burnetii, were washed with PBS and exposed to UV light (500 J/m²) in a transilluminator box (Stratagene, La Jolla, CA). After fresh medium was added, cells were incubated for 4 h at 37°C in 5% CO₂. The cells were fixed with 2% paraformaldehyde in PBS, permeabilized using 0.1% Triton X-100 in PBS, and incubated with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture using the manufacturer's protocol (Roche). The cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA), and the data were evaluated with CellQuest software (Becton Dickinson).

(iii) Determining caspase 3/7 activity.

CHO cells, either uninfected or infected with C. burnetii, were incubated with staurosporine (0.5 μg/ml) for 6 h. The cells were washed with PBS and lysed with a lysis buffer (10 mM HEPES [pH 7.4], 50 mM NaCl, 2 mM MgCl₂, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin) for 20 min on ice. After three freeze-thaw cycles, the lysate was clarified by high-speed centrifugation (15 min, 16,000 × g, 4°C), aliquoted, and stored at −80°C until it was assayed. The total protein concentration was determined using a DC protein assay kit (Pierce, Rockford, IL). For the caspase 3/7 enzymatic assay, samples were quickly thawed, and an equal amount of protein was incubated with the chromophore-labeled substrate acetyl-DEVD-p-nitroanilide in 100 μl of buffer containing 50 mM HEPES (pH 7.4), 0.2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 20% glycerol, 2 mM EDTA, and 10 mM dithiothreitol for 1 h at 37°C. As a control for caspase specificity, the inhibitor N-acetyl-Asp-Glu-Val-Asp-al was incubated with the sample for 30 min at 30°C prior to incubation with the substrate. The cleavage of the chromophore p-nitroanilide from the tetrapeptide was measured by spectrophotometry at 405 nm.

(iv) Cytochrome c release.

CHO cells, either uninfected or infected with C. burnetii, were incubated with staurosporine (0.5 μg/ml) for 6 h. The cells were washed with ice-cold PBS, lysed with a lysis buffer (250 mM sucrose, 20 mM HEPES [pH 7.5], 1.5 mM MgCl₂, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1× protease inhibitor cocktail) for 10 min on ice. The cells were homogenized with 15 strokes in a Dounce homogenizer and centrifuged (750 × g, 10 min, 4°C) to remove the nuclei. The supernatant was centrifuged (16,000 × g, 25 min, 4°C) to separate the cytosolic fraction (supernatant) from the mitochondrial fraction (pellet). The total protein concentration was determined using a DC protein assay kit (Pierce). Proteins (5 μg) from the cytosolic fraction of each sample were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with anti-cytochrome c antibody (catalog no. 556433; BD Pharmingen, San Jose, CA) or with anti-actin antibody (catalog no. A2066; Sigma) to determine protein loading.

(v) Annexin V staining.

HeLa cells, either uninfected or infected with C. burnetii, were treated with chloramphenicol (20 μg/ml) for 16 h or not treated prior to exposure to UV light (500 J/m²) in a transilluminator box (Stratagene). After fresh medium with or without chloramphenicol was added, cells were incubated for 4 h at 37°C in 5% CO₂. The cells were washed with PBS, and 1 × 10⁵ cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in the dark according to the manufacturer's protocol (Annexin V-FITC apoptosis detection kit I; BD Bioscience). The percentages of apoptotic and necrotic cells were determined by flow cytometry using a FACSCalibur (Becton Dickinson), and the data were evaluated with CellQuest software (Becton Dickinson).

Immunoblot analysis.

A total of 7.5 × 10⁴ C. burnetii-infected or uninfected HeLa cells were added to each well of a six-well plate. Cells were treated with chloramphenicol (20 μg/ml) for 16 h or with staurosporine (0.5 μg/ml) for 5 h. Cells were lysed by adding 100 μl of 1× sample buffer (4% SDS, 20% glycerol, 120 mM Tris [pH 6.8], 0.01% bromophenol blue, 720 mM β-mercaptoethanol). After boiling for 5 min, the proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with antibodies specific for Bad (catalog no. 9292), Bax (catalog no. 2772), Bik (catalog no. 4592), Bim (catalog no. 4582), Bmf (catalog no. 4692), and Puma (catalog no. 4976) using the manufacturer's protocol (Cell Signaling, Danvers, MA) or Bcl-2 (catalog no. sc-7382; Santa Cruz, Santa Cruz, CA). Proteins were visualized using horseradish peroxidase-conjugated secondary antibodies (Zymed, South San Francisco, CA) and a chemiluminescence detection system (Perkin-Elmer, Boston, MA).

RESULTS

C. burnetii-infected cells are resistant to apoptosis induced by staurosporine and UV irradiation.

To determine whether C. burnetii modulates the host cell response to proapoptotic signals, HeLa cells that were either not infected or infected with C. burnetii were treated with UV light. DNA damage caused by UV irradiation is a well-characterized trigger for activation of the intrinsic cell death pathway (12, 37). Cellular responses were analyzed using the TUNEL assay, which measures DNA fragmentation, a key indicator of late-stage processes induced during apoptosis. Figure 1A shows that after treatment with UV light the number of HeLa cells in the uninfected samples that were TUNEL positive was significantly greater than the number of HeLa cells that were TUNEL positive in the C. burnetii-infected sample (P < 0.001). These data suggest that C. burnetii-infected cells are more resistant than uninfected cells to the induction of cell death by UV light.

FIG. 1.

C. burnetii-infected cells are resistant to UV light- and staurosporine-induced apoptosis. (A) HeLa cells, uninfected or infected with C. burnetii, were exposed to UV light (500 J/m²). The medium was replaced, and the cells were incubated for 4 h at 37°C in 5% CO₂. After fixation and permeabilization the cells were labeled with a TUNEL reaction mixture according to the manufacturer's protocol (in situ cell death detection kit; Roche). The percentage of apoptotic cells was determined by flow cytometry. The data are the results from three independent experiments. (B) CHO cells, uninfected or infected with C. burnetii, were seeded on coverslips in 24-well plates. After incubation with staurosporine (0.5 μg/ml) for 6 h at 37°C in 5% CO₂, the cells were fixed, the nuclei were stained with DAPI, and the nuclear morphology was scored as normal or fragmented (apoptotic). The percentage of cells with apoptotic nuclear morphology was calculated (200 nuclei per sample from three independent experiments were counted).

Next, uninfected and C. burnetii-infected CHO cells were compared after treatment with staurosporine, a broad protein kinase inhibitor that is a potent inducer of the intrinsic cell death pathway. After treatment with staurosporine there was a significant increase in the number of uninfected CHO cells showing extensive nuclear fragmentation (P = 0.015), as revealed by DAPI staining, whereas CHO cells infected with C. burnetii showed no significant increase in nuclear fragmentation (P = 0.82) (Fig. 1B). Similar results were obtained when uninfected and C. burnetii-infected HeLa cells were treated with staurosporine (data not shown). These results clearly indicate that C. burnetii infection of both HeLa and CHO cells correlates with a defect in the ability of these cells to undergo apoptosis in response to agents that activate the intrinsic cell death pathway.

C. burnetii infection does not alter steady-state levels of proteins that regulate apoptosis.

Proteins that regulate the intrinsic cell death pathway include inhibitors of apoptosis (Bcl-2-like proteins), inducers of apoptosis (Bax and Bak), and triggers of apoptosis (BH3-only proteins). An apoptotic stimulus usually activates one or several BH3-only proteins that in turn activate Bax/Bak by an unknown mechanism. Active Bax/Bak then triggers the release of cytochrome c from mitochondria, which activates the apoptosome (10). Given that Chlamydia and Toxoplasma modulate the intrinsic cell death pathway by a process that involves degradation of BH3-only proteins (12, 19), we examined whether C. burnetii infection alters the steady-state levels of BH3-only proteins. Immunoblot analysis of uninfected and C. burnetii-infected HeLa cells revealed no obvious differences in steady-state levels of the BH3-only proteins Bad, Bik, Bim, Bmf, and Puma (Fig. 2). Additionally, no differences in the steady-state levels of Bcl-2 or Bax were observed, and the steady-state levels of these regulators of apoptosis were not altered by treatment of the uninfected or infected cells with staurosporine or when bacterial translation was blocked by chloramphenicol (56). These data suggest that C. burnetii-mediated inhibition of apoptosis does not involve alterations in the steady-state levels of these regulators of the intrinsic cell death pathway.

FIG. 2.

C. burnetii infection does not alter steady-state levels of apoptotic regulators. HeLa cells, uninfected (uninfect) or infected with C. burnetii (C.b. infect) were treated with chloramphenicol (CM) (20 μg/ml) for 16 h at 37°C in 5% CO₂ or were incubated with staurosporine (Stauro) (0.5 μg/ml) for 6 h at 37°C in 5% CO₂. Total protein extracts from 2 × 10⁴ cells were separated by SDS-PAGE, and immunoblots were probed with antibodies against Bad, Bax, Bcl-2, Bik, Bim, Bmf, and Puma, as well as β-actin (as a loading control). The results of one representative experiment of two independent experiments in which similar results were obtained are shown.

Activation of caspase 3/7 is inhibited by C. burnetii infection.

Both staurosporine and UV light activate the intrinsic cell death pathway. Induction of the intrinsic pathway involves caspase 9-mediated activation of the effector caspases 3 and 7 (39, 49). Following activation of both the intrinsic and extrinsic cell death pathways, caspases 3 and 7 inactivate the DNA repair enzyme poly(ADP-ribose) polymerase and activate caspase-activated DNase, thereby inducing DNA fragmentation. To further define the mechanism by which C. burnetii infection inhibits cell death, caspase 3/7 activity was measured in cells after induction of apoptosis. Caspase 3/7 activity was determined in cell lysates by measuring cleavage of a chromophore-labeled substrate that contains a DEVD motif that can be cleaved by caspase 3/7. After incubation with staurosporine, C. burnetii-infected and uninfected CHO cells were assayed for caspase 3/7 activity. As shown in Fig. 3, caspase 3/7 activity was lower in staurosporine-treated cells infected with C. burnetii than in uninfected cells (P = 0.05). To verify that the proteolytic activity was caspase 3/7 dependent, samples were incubated with the caspase 3/7 inhibitor Ac-DEVD-CHO prior to incubation with the substrate, which reduced the signal level to the background level (data not shown), indicating that the activity being measured was dependent on the caspase 3/7 function. These data indicate that C. burnetii infection of host cells limits the activation of effector caspases upon induction of apoptosis.

FIG. 3.

C. burnetii infection interferes with the activation of caspase 3/7 after induction of apoptosis with staurosporine. CHO cells, uninfected or infected with C. burnetii, were incubated with or without staurosporine (0.5 μg/ml) for 6 h at 37°C in 5% CO₂. The cells were washed and lysed. After centrifugation, caspase 3/7 activity was measured in supernatants and expressed in units (optical density at 405 nm/mg × h). The activity of induced uninfected cells was defined as 100, and the relative caspase 3/7 activities of the other samples are shown. The data are the results of three independent experiments.

C. burnetii infection of host cells inhibits cytochrome c release from mitochondria upon induction of the intrinsic cell death pathway.

To further define the mechanism by which C. burnetii infection can interfere with the intrinsic cell death pathway, we next investigated whether C. burnetii is able to inhibit mitochondrial signaling in response to proapoptotic stimuli by examining cytochrome c release. To measure the release of cytochrome c into the cytosol, CHO cells were separated into cytosolic and mitochondrial fractions. The levels of cytochrome c in the cytosolic and mitochodrial fractions were determined by immunoblot analysis. The mitochondrial fractions from uninfected and infected cells that were both uninduced and induced with staurosporine showed no difference in cytochrome c level, as determined by immunoblotting (data not shown). However, cytochrome c release into the cytosolic fraction was detected after stimulation of uninfected cells with staurosporine (Fig. 4). For cells infected with C. burnetii, however, there was no detectable release of cytochrome c following stimulation with staurosporine. These data indicate that C. burnetii infection interferes with the release of cytochrome c from host cell mitochondria in response to proapoptotic stimuli, which would explain how C. burnetii is able to prevent activation of caspase 3/7 and limit cell death.

FIG. 4.

C. burnetii infection blocks cytochrome c release following apoptosis induction with staurosporine. CHO cells, uninfected or infected with C. burnetii (C.b.), were incubated with staurosporine (Stauro) (0.5 μg/ml) for 6 h at 37°C in 5% CO₂. The cells were washed, lysed, homogenized, and centrifuged to separate the mitochondrial fraction from the cytosolic fraction. Protein (5 μg) from the cytosolic fraction of each sample was separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-cytochrome c antibody or with anti-β-actin as a control. The data are from a single experiment that was repeated three times independently with similar results.

C. burnetii protein synthesis is important for inhibition of host cell apoptosis.

We hypothesized that proteins produced by C. burnetii could mediate the inhibition of apoptosis directly, which would predict that a sustained antiapoptotic effect on cells might require continual synthesis of bacterial proteins. To test whether C. burnetii proteins potentially play a direct role in modulating host responses to proapoptotic signals, cells were treated with chloramphenicol to interfere with de novo protein synthesis by intracellular bacteria prior to induction of apoptosis by UV light. Apoptotic cells were labeled with fluorescent Annexin V, a protein that binds to phosphatidylserine exposed on the surface of cells in an early stage of apoptosis, and counterstained with PI, which labels necrotic cells that have lost plasma membrane integrity. Figure 5A shows that UV irradiation of uninfected HeLa cells resulted in an increase in the percentage of apoptotic cells (Annexin V-positive, PI-negative cells). Pretreatment of uninfected cells with chloramphenicol did not alter the percentage of apoptotic cells after UV induction. Compared to uninfected cells, a lower percentage of C. burnetii-infected cells showed signs of apoptosis following UV irradiation. However, when the infected cells were treated with chloramphenicol and then UV irradiated, the percentage of cells showing signs of apoptosis increased compared to the percentage of infected cells that were not treated with chloramphenicol. These data indicate that bacterial protein synthesis is important for sustained inhibition of apoptosis by C. burnetii.

FIG. 5.

C. burnetii protein synthesis is important for host cell resistance to apoptosis. (A) HeLa cells, uninfected or infected with C. burnetii, were treated with chloramphenicol (CM) (20 μg/ml) for 16 h or left untreated prior to exposure to UV light (500J/m²). Fresh medium with or without chloramphenicol (20 μg/ml) was added. After 4 h of incubation at 37°C in 5% CO₂, the cells were washed and stained with Annexin V-FITC and PI according to the manufacturer's protocol (Annexin V-FITC apoptosis detection kit I; BD Bioscience) and analyzed by flow cytometry. The data are from one representative experiment of two independent experiments in which similar results were obtained. C.b., C. burnetii. (B) CHO cells, uninfected or infected with C. burnetii, were treated with chloramphenicol (20 μg/ml) for 16 h or left untreated prior to incubation with staurosporine (Stauro) (0.5 μg/ml) for 6 h. The cells were washed, lysed, homogenized, and centrifuged to separate the mitochondrial fraction from the cytosolic fraction. Protein (5 μg) from the cytosolic fraction of each sample was loaded on a SDS-PAGE gel, transferred to a PVDF membrane, and probed with anti-cytochrome c antibody or with anti-β-actin as a control. The results of one representative experiment of two independent experiments in which similar results were obtained are shown.

To confirm these results, uninfected or infected CHO cells were treated with chloramphenicol prior to the induction of apoptosis with staurosporine. Samples were then analyzed for the release of cytochrome c from mitochondria by immunoblotting. As shown in Fig. 5B, after induction of apoptosis with staurosporine, cytochrome c release was detected in the uninfected samples, whereas the C. burnetii-infected cells showed lower levels of cytochrome c release. Cytochrome c release was greater in staurosporine-treated cells infected with C. burnetii when bacterial protein synthesis was inhibited with chloramphenicol than it was in infected cells treated with staurosporine alone. These data indicate that bacterial protein synthesis is important for the C. burnetii-mediated inhibition of apoptosis. Taken together, these data provide evidence that a bacterial factor(s) mediates the antiapoptotic activity observed in C. burnetii-infected cells.

DISCUSSION

The survival of the obligate intracellular pathogen C. burnetii within host cells is dependent upon the ability of the bacterium to establish a phagolysosome-like compartment (28). C. burnetii proteins modulate membrane fusion events within the infected host cell, either directly or indirectly, to promote homotypic fusion of lysosomes, a process that generates a very spacious compartment that harbors a large number of replicating bacteria (14). Presumably, the process that establishes and maintains this unusual compartment places tremendous stress on the host cell; however, detrimental effects on host cell viability are not typically observed during C. burnetii infection, and cell cultures containing replicating C. burnetii can be continually passaged (46, 53). This has led to the suggestion that C. burnetii, like other obligate intracellular pathogens (12, 13, 18, 19), has developed mechanisms to promote host cell survival during intracellular infection. Here, we describe the ability of C. burnetii-infected cells to resist apoptosis. Infected cells showed significant protection against the induction of apoptosis by staurosporine and UV light treatment (Fig. 1A and 1B). Because host cell apoptosis can facilitate the killing of intracellular pathogens either by provoking an inflammatory response or by delivering the intracellular pathogens to professional phagocytes (18, 20), these data indicate that C. burnetii has evolved an antiapoptotic activity(s) to suppress host defenses in order to ensure bacterial survival and multiplication.

The mechanism by which C. burnetii infection inhibits host cell apoptosis was examined. Our data show that C. burnetii infection prevents the release of cytochrome c and the subsequent activation of caspase 3/7 after cell death is induced (Fig. 3 and 4). Release of cytochrome c from mitochondria is one of the main regulatory checkpoints in the intrinsic cell death signaling cascade (22, 36) and is necessary for the activation of effector caspases that mediate apoptosis (40). Several other intracellular pathogens that either inhibit or activate host cell apoptosis have been shown to modulate cytochrome c release from mitochondria following infection (9, 29). It is well established that release of cytochrome c is regulated by the activation of Bax and Bak, which in turn are regulated by Bcl-2-like proteins (antiapoptotic) and BH3-only proteins (proapoptotic) (15). Some pathogens have evolved mechanisms to interfere with the intrinsic cell death pathway by degrading BH3-only proteins. For instance, Chlamydia infection leads to degradation of the BH3-only proteins Bik, Puma, and Bim (16), and a T. gondii infection results in the degradation of the BH3-only protein Bad. Furthermore, it is known that UV irradiation activates the BH3-only proteins Bim and Bmf (38) and that staurosporine activates Puma (55). Based on immunoblot analysis, we were unable to observe any changes in steady-state levels of the BH3-only proteins Bad, Bik, Bim, Bmf, and Puma (Fig. 2), suggesting that C. burnetii infection does not promote the degradation of these proapoptotic regulators. Furthermore, we did not observe a change in the steady-state protein level of Bax or the prosurvival protein Bcl-2. Thus, in the cell lines that we have used, modulation of apoptosis by C. burnetii does not correlate with changes in the levels of proteins that regulate apoptosis by functioning upstream of cytochrome c release from mitochondria.

In agreement with our findings, Voth et al. have recently reported a block in apoptosis upon C. burnetii infection of macrophages (54). Interestingly, these authors demonstrated that C. burnetii infection results in upregulation of c-IAP2 and A1/bfl-1. Upregulation of A1/bfl-1, an antiapoptotic Bcl-2 family member, could contribute to the C. burnetii-mediated antiapoptotic response by a mechanism that involves preventing activation of specific BH3-only proteins, and upregulation of c-IAP2 could prevent activation of caspase 3. Both our data (Fig. 5A and B) and the work of Voth et al. revealed that the C. burnetii-mediated process that prevents activation of the intrinsic cell death pathway requires bacterial protein synthesis. Taken together, the data presented here and the data of Voth et al. demonstrate that C. burnetii has the ability to interfere with apoptosis in different cell types and after induction by many different stimuli.

It is likely that C. burnetii has evolved multiple mechanisms to ensure that inhibition of the intrinsic cell death pathway is maintained for the duration of infection. Upregulation of A1/bfl-1 and c-IAP2 likely contributes to the process by which cells become resistant to apoptosis following C. burnetii infection; however, it is unclear whether the increase in the expression of these factors observed after C. burnetii infection of cells is sufficient to block cytochrome c release after treatment of cells with either staurosporine or UV light. Additionally, the induction of antiapoptotic genes in macrophages is a common response that is triggered upon microbial activation of Toll-like receptors, a process that does not require sustained bacterial synthesis of proteins. Based on these data, we hypothesize that the ability of C. burnetii to modulate host cell survival is conferred through the specific biochemical activities of bacterial proteins produced during infection.

Although specific C. burnetii proteins that can inhibit host cell apoptosis were not identified in this study, putative effector proteins delivered into host cells by the Dot/Icm system could potentially be involved in modulating host cell survival during infection. In agreement with this hypothesis, multiple type IV secretion substrates involved in promoting antiapoptotic activities have been identified in the pathogens L. pneumophila and Bartonella spp. The SidF protein from L. pneumophila has been shown to contribute to apoptosis resistance in L. pneumophila-infected cells by interacting with and neutralizing the effects of BNIP3 and Bcl-rambo, two proapoptotic BH3 protein family members (6). Additionally, L. pneumophila has been shown to be important for the upregulation of genes encoding antiapoptotic factors through stimulating the activation of NF-κB (1, 41). The effector protein BepA from Bartonella can interfere with apoptosis by a process that involves increasing cellular cyclic AMP levels (48). Therefore, it can be speculated that a C. burnetii type IV secretion effector(s) is important for the inhibition of cytochrome c release, which is known to be regulated by the activation of the BH3-only proteins (45). Although we did not observe a difference in the steady-state levels of proapoptotic BH3-only proteins in C. burnetii-infected cells, these data do not exclude the possibility that a C. burnetii effector(s) interferes with the activation of these proteins though direct or indirect interactions. Future identification of C. burnetii effectors translocated into the host cell by the Dot/Icm system should provide further insight into the biochemical mechanisms by which this intracellular pathogen can make cells refractory to apoptosis.