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Originally published In Press as doi:10.1074/jbc.M509404200 on January 25, 2006

J. Biol. Chem., Vol. 281, Issue 16, 11250-11259, April 21, 2006
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Helicobacter pylori Vacuolating Cytotoxin Induces Activation of the Proapoptotic Proteins Bax and Bak, Leading to Cytochrome c Release and Cell Death, Independent of Vacuolation*Formula

Eiki Yamasaki{ddagger}§, Akihiro Wada{ddagger}§1, Atsushi Kumatori, Ichiro Nakagawa||, Junko Funao||, Masaaki Nakayama§, Junzo Hisatsune§, Miyuki Kimura**, Joel Moss{ddagger}{ddagger}2, and Toshiya Hirayama§

From the {ddagger}PRESTO, Japan Science and Technology Corporation, Saitama 332-0012, Japan, the §Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan, the Division of Medical Science, Department of Disaster Prevention System, Faculty of Risk and Crisis Management, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan, the ||Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan, the **Department of Medical Technology, Faculty of Health Sciences, Okayama University Medical School, Okayama 700-8558, Japan, and the {ddagger}{ddagger}Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590

Received for publication, August 25, 2005 , and in revised form, December 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Helicobacter pylori vacuolating cytotoxin, VacA, which causes vacuolation of gastric epithelial cells and other types of cultured cells, is known to stimulate apoptosis via a mitochondria-dependent pathway. In the present study, we examined the mechanisms of VacA-induced mitochondrial damage. Intracellular VacA localization was monitored by immunostaining and confocal microscopy; in AZ-521 cells in which cytochrome c release was stimulated, most of VacA was localized to vacuoles rather than mitochondria. VacA reduced the membrane potential of isolated mitochondria without inducing cytochrome c release, suggesting that it did not act directly to induce cytochrome c release from mitochondria and that in intact cells, VacA-induced cytochrome c release involved apoptosis-related factor(s), such as a proapoptotic Bcl-2 family protein. In agreement, flow cyto-metric analyses using antibodies specific for activated Bax revealed that intracellular Bax was activated by VacA in a concentration- and time-dependent manner. Using active form-specific antibodies, we also observed that the Bcl-2 family protein, Bak, was activated. By confocal microscopy, Bax and Bak were activated in AZ-521 cells in which cyto-chrome c release was induced by VacA. In addition, small interfering RNA-induced silencing of the bax gene resulted in reduction of VacA-stimulated cytochrome c release, consistent with a contribution of VacA-induced Bax activation to cytochrome c release. NH4Cl enhanced both VacA-induced vacuolation and Bax activation, whereas Bax activation was not inhibited by bafilomycin A1, which inhibited vacuolation caused by VacA. These results suggest that VacA acts through different signaling pathways to induce apoptosis via Bax activation, independent of vacuolation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Persistent Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. The vacuolating cytotoxin, VacA, is one of the major pathogenic products of H. pylori and induces vacuoles within various types of cultured cells, not only gastric epithelial cells. Epidemiological studies and animal experiments showed that VacA is a major virulence factor associated with gastric mucosal damage (1-6). The 140-kDa VacA precursor is cleaved during secretion from the bacterium, generating the 87-95-kDa mature toxin (7). The mature toxin has amino-terminal 34-37-kDa (p37) and carboxy-terminal 58-kDa (p58) domains. It is believed that p58 is responsible for VacA binding to cells (8). Vacuolating activity resides in the amino-terminal region of VacA, which includes a large portion of the p58 domain (9, 10). Exposure of VacA to acidic conditions resulted in its association with planar lipid bilayers and formation of membrane-associated hexamers (11). These hexamers act as anion-selective, voltage-dependent channels (12, 13). It is believed that the VacA channel damages susceptible cells by inducing changes in the osmotic balance across certain membranes (including plasma and vacuolar membranes).

VacA has been reported to induce apoptosis (14). VacA purified from H. pylori culture medium and recombinant VacA caused fragmentation of nuclear DNA in AGS gastric epithelial cells (15, 16). Another study showed that microinjection of DNA encoding VacA-green fluorescent protein (GFP)3 fusion protein or p37 fragment-GFP fusion protein induced apoptosis of Hep-2 cells (17), and transfection of HeLa cells with these DNAs resulted in the release of cytochrome c from mitochondria plus activation of caspase 3. Willhite et al. (18) and Nakayama et al. (19) also reported that VacA purified from H. pylori culture medium induced cytochrome c release in HeLa and AZ-521 gastric epithelial cells, suggesting that VacA induced apoptosis via a mitochondria-dependent pathway. VacA also caused mitochondrial damage, leading to a decrease in mitochondrial membrane potential (20, 21). The reduction in mitochondrial membrane potential occurred substantially before and at a lower concentration of VacA than did cytochrome c release. By confocal microscopy in HeLa cells, exogenously added full-length VacA and intracellularly expressed p37, but not p58, localized to mitochondria (14, 17, 21). By using a mutant that was deficient in VacA channel activity or a VacA channel inhibitor, it was shown that VacA channel activity was necessary for both cytochrome c release and the decrease in mitochondrial membrane potential (21), consistent with a model in which VacA channel activity is critical for mitochondrial damage. A recombinant VacA fragment (VacA418-799), which corresponds to the mid-region of the p58 domain, inhibited cell growth and induced apoptosis of AGS cells (22). Moreover, VacA418-799 increased the expression of several apoptosis-related proteins, including proapoptotic proteins. Some pro-apoptotic Bcl2 family proteins play a central role in inducing apoptosis via a mitochondria-dependent pathway and regulate mitochondrial membrane permeabilization and cytochrome c release (23).

To elucidate the mechanism of VacA-induced cytochrome c release and apoptosis, we compared VacA action on intracellular and isolated mitochondria and examined the role of activation of proapoptotic Bcl2 family proteins (Bax and Bak) in VacA-induced cytochrome c release. Here we report that even in cells in which cytochrome c release was induced, VacA was localized to vacuoles rather than mitochondria. In addition, VacA did not induce cytochrome c release from isolated mitochondria. VacA, however, stimulated activation of Bax and Bak in AZ-521 cells, in a process that was not affected by the inhibition of vacuolation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Monoclonal anti-Bax antibody (Clone 3) and mouse anti-cytochrome c antibody (7H8.2C12 and 6H2.B4) were purchased from BD Bioscience. Monoclonal antibody to mtHSP70 (JG1) was purchased from Alexis Biochemicals. Polyclonal antibodies to poly(ADP-ribose)polymerase (PARP) were purchased from Cell Signaling Technology. Rabbit anti-Bax-NT and anti-Bak-NT antibodies were purchased from Upstate Cell Signaling Solutions. 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), Mitotracker RED CM XRos, and species-specific Alexa fluor 633-, 488-, and 564-conjugated secondary antibodies were purchased from Molecular Probes. Recombinant adenovirus carrying a GFP-conjugated Rab7 (GFP-Rab7) expression cassette (Venus-Rab7) was obtained from Dr. I. Nakagawa (Osaka University, Osaka, Japan).

Cell Culture
HeLa cells and AZ-521 human gastric adenocarcinoma cells (Culture Collection of Health Science Resource Bank, Japan Health Science Fundamental) were grown in Eagle's minimal essential medium (EMEM) containing 10% fetal calf serum under 5% CO2 at 37 °C.

VacA Preparation
VacA was purified from H. pylori ATCC49503 strain culture supernatant using anti-VacA by our published procedure (24). VacA was acid-activated during purification, and its concentrations were calculated based on monomeric forms.

Assay for Vacuolating Activity
Vacuolation was quantified by neutral red accumulation as described by Cover et al. (25). The cells were incubated with freshly prepared 0.05% neutral red in phosphate-buffered saline (PBS) containing 0.3% bovine serum albumin and then washed three times with PBS containing 0.3% bovine serum albumin. After the addition of 70% ethanol containing 0.4% HCl, absorbance at 540 nm (Abs 540 nm) was measured.

Isolation of Mitochondria from HeLa Cells
Mitochondria were isolated from HeLa cells as described previously (26). In brief, the HeLa cells were grown overnight, harvested, washed twice in PBS, dispersed in isolation buffer (20 mM Hepes-KOH, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 250 mM sucrose) supplemented with 1x complete protease inhibition mixture (Roche Applied Sciences). After chilling on ice for 10 min, the cells were disrupted by 50 strokes of a tight fitting Dounce tissue grinder. The homogenate was centrifuged at 700 x g for 10 min at 4 °C to remove unbroken cells and nuclei. The mitochondria-enriched fraction was then pelleted by centrifugation at 10,000 x g for 20 min at 4 °C. The mitochondrial pellet was rinsed twice in SEM buffer (250 mM sucrose, 5 mM EDTA, and 10 mM MOPS-KOH, pH 7.4), immediately suspended in SEM buffer, and incubated with VacA as described.

Measurement of Mitochondrial Membrane Potential and Cytochrome c Release from Isolated Mitochondria
Isolated mitochondria, at a final concentration of 0.1 mg protein/ml, were incubated with VacA (720 nM) or CaCl2 (250 mM) in SEM buffer supplemented with 5 mM NH4Cl. After centrifugation (15,000 x g, 10 min, 4 °C) of VacA-treated mitochondria, supernatant was transferred to a fresh tube. Proteins in samples of pellet (mitochondria) and supernatant were separated by SDS-PAGE in 15% gels and analyzed by immunoblotting with anti-cytochrome c antibody (7H8.2C12) as described previously (19). After incubation of isolated mitochondria (0.1 mg protein/ml) with VacA (360 or 720 nM), mitochondrial membrane potential was quantified using membrane potential sensitive reagent (JC-1) as described previously (27). JC-1 forms aggregates (J-aggregate) in polarized mitochondria, resulting in a green-orange emission of 590 nm after excitation at 490 nm. J-aggregate formation, which reflects intensity of mitochondrial membrane potential, was evaluated by measurement of emission at 590 nm (excited at 490 nm).

Quantitative Measurement of Cytochrome c Release
Quantification of cytochrome c release was executed by flow cytometry as previously described (28). In brief, AZ-521 cells were seeded (1.0 x 106 cells in 4 ml of EMEM/dish) in 60-mm culture dishes (IWAKI, Chiba, Japan) and grown overnight, and then the cells were incubated with the indicated concentrations of VacA, harvested, treated with digitonin (0.5 mg/ml) in PBS with 100 mM KCl for 5 min on ice, fixed, and incubated in blocking buffer (0.1% Triton X-100, 3% bovine serum albumin in PBS) for 1 h on ice. After washing, the cells were incubated for 1 h on ice with anti-cytochrome c antibody (6H2.B4) in blocking buffer and then for 1 h on ice with species-specific Alexa fluor 488-conjugated secondary antibody in blocking buffer before analysis using FACScalibur (BD Bioscience) for detection of Alexa fluor 488 fluorescence levels. We regarded the cells with low fluorescence as having high cytoplasmic cytochrome c levels and those with high fluorescence as having intact mitochondria.

Transfection with Bax Small Interfering RNA (siRNA)
Use of RNA interference to lower Bax content was performed as previously reported (29, 30). In brief, AZ-521 cells were seeded (1.0 x 106 cells in 4 ml of EMEM/dish) in 60-mm culture dishes and grown overnight; 0.2 µM Bax silencer siRNA (Bax-siRNA) duplexes were introduced into the cells using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer's recommendations. The cording strand of the Bax-siRNA was 5'-AACAUGGAGCUGCAGAGGAUGAdTdT-3' (purchased from TAKARA BIO Inc.). Negative Control siRNA (NC-siRNA) purchased from B-Bridge International Inc. was used for nonspecific interference. Silencing of bax gene was determined by measuring of Bax protein expression at 24 h after transfection by Western blotting using anti-Bax-NT antibodies.


Figure 1
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  		FIGURE 1.
VacA-induced cytochrome c release and cell death in AZ-521 cells. A, NH4Cl-dependent increase of cytochrome c release in AZ-521 cells. AZ-521 cells were grown overnight, and then the cells were incubated with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with or without NH4Cl (5 mM), and cytochrome c release was analyzed by flow cytometry as described under "Materials and Methods." The number of cells with intact mitochondria and cells exhibiting cytochrome c release were counted. The bars represent the percentages of cells with cytochrome c release. The data are the means ± S.D. of values from three experiments. B, time-dependent increase of cytochrome c release in AZ-521 cells by VacA. AZ-521 cells were grown overnight, and then the cells were incubated with or without 120 nM acid-activated VacA for indicated time in EMEM supplemented with NH4Cl (5 mM), and cytochrome c release was analyzed as described for A. The data are the means ± S.D. of values from three experiments. The S.D. value of the data without error bars is <3.0%. C, time-dependent increase of PARP cleavage in AZ-521 cells by VacA. AZ-521 cells were grown overnight and incubated with or without 120 nM acid-activated VacA as described for B, and the cell lysates were prepared. The cell lysates were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes, and full-length PARP (116 kDa) and cleaved PARP (89 kDa) were detected by incubation with anti-PARP antibodies. Equal loading was confirmed with anti-actin antibody. The data are representative of at least three experiments. D,NH4Cl-dependent increase in AZ-521 cell death. AZ-521 cells were grown overnight, and then the cells were incubated with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with or without NH4Cl (5 mM). Cell viability was assessed by MTS assay. The data are the means ± S.D. of values from three separate experiments with assays in triplicate. The S.D. value of the data without error bars is <0.01.

 
Quantitative Measurement of Bax Activation
AZ-521 cells were seeded (1.0 x 106 cells in 4 ml of EMEM/dish) in 60-mm culture dishes and grown overnight before incubation with various concentrations of VacA; quantification of Bax activation by flow cytometry using conformation-specific anti-Bax antibody (Clone 3) was performed as described previously (31).

MTS Assay for Cell Viability
AZ-521 cells were seeded in 96-well culture plates (2.0 x 104 cells in 0.2 ml of EMEM/well) and grown overnight before incubation with or without VacA (120 nM). Bioreduction of tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) into a formazan product as an indicator of cell viability was quantified by absorbance at 490 nm (Abs 490 nm) using a CellTiter 96 AQueous nonradioactive cell proliferation assay kit (Promega) according to the manufacturer's instructions.

Immunocytochemistry and Confocal Analysis
Analysis of VacA Localization—For detection of VacA in mitochondria, AZ-521 cells were seeded (5.0 x 104 cells in 0.2 ml of EMEM/well) and grown overnight on 8-well chamber microscope slides (Nalge Nunc); then the cells were incubated with VacA (120 nM) before incubation with 2 µM Mitotracker RED CMXRos for 15 min at 37 °C, washing with PBS, and fixation with 2% paraformaldehyde in PBS, and then the cells were permeabilized for 5 min at room temperature with 0.1% Triton X-100 in PBS and blocked with 1.5% Block Ace (Snow-Brand, Tokyo, Japan) in PBS. The cells were incubated with anti-VacA and anti-cytochrome c (6H2.B4) antibodies diluted in blocking buffer (0.4% Block Ace in PBS) for 1 h at room temperature, before incubation with species-specific Alexa fluor 488-conjugated secondary antibody (for anti-VacA antibodies) and species-specific Alexa fluor 633-conjugated secondary antibody (for anti-cytochrome c antibody) diluted in blocking buffer for 1 h at room temperature in the dark. For detection of VacA in vacuoles, AZ-521 cells were seeded (2.0 x 104 cells in 0.2 ml of EMEM/well) and grown overnight on 8-well chamber microscope slides before infection with Venus-Rab7; 24 h later, they were treated with VacA (120 nM), fixed with 2% paraformaldehyde in PBS, permeabilized for 5 min at room temperature with 0.1% Triton X-100 in PBS and blocked with 1.5% Block Ace in PBS. The cells were incubated with anti-VacA and anti-cytochrome c (6H2.B4) antibodies diluted in blocking buffer for 1 h at room temperature, before incubation with species-specific Alexa fluor 546-conjugated secondary antibody (for anti-VacA antibodies) and species-specific Alexa fluor 633-conjugated secondary antibody (for anti-cytochrome c antibody), diluted in blocking buffer, for 1 h at room temperature in the dark. The images were captured by confocal laser microscopy (model TSC SP2; Leica, Heidelberg, Germany) independently with the 488-, 543-, and 633-nm lines of lasers used for excitation of Alexa fluor 488 (Ar/Kr laser, 488 nm), GFP-Rab7 protein (Ar/Kr laser, 488 nm), Mitotracker RED CMXRos (Green He-Ne laser, 543 nm), and Alexa fluor 633 (Red He-Ne laser, 633 nm), respectively.


Figure 2
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  		FIGURE 2.
VacA localized to vacuoles, but not mitochondria, in VacA-treated AZ-521 cells in which cytochrome (cyto.) c release was stimulated. A and B, VacA localization in AZ-521 cells at 24 h. A, AZ-521 cells were grown overnight, and then the cells were incubated with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with NH4Cl (5 mM), and immunocytochemistry and confocal analysis were performed as described under "Materials and Methods." The cells were stained with anti-cytochrome c antibody (blue), Mitotracker RED (red), and anti-VacA antibodies (green). Right column, merged images of Mitotracker RED and VacA. B, AZ-521 cells were grown overnight before infection with Venus-Rab7 as described under "Materials and Methods." Twenty four hours after infection with Venus-Rab7, the cells were incubated with VacA (120 nM) as described for A. Expression and localization of GFP-Rab7 were analyzed by confocal microscopy (green). Localization of VacA (red) and cytochrome c (blue) was determined using indirect immunofluorescence methodology as described in A. Right column, merged images of GFP-Rab7 and VacA. C-E, AZ-521 cells were incubated with 120 nM acid-activated VacA for 12 h (C), 6h (D), and 3h(E) before immunocytochemistry and confocal analysis were performed as described above. Left panels, merged images of Mitotracker RED (red) and VacA (green). Right panels, merged images of GFP-Rab7 (green) and VacA (red). The data are representative of at least two experiments.

 
Analysis of Bax and Bak Activation and Cytochrome c Release—AZ-521 cells were seeded (5.0 x 104 cells in 0.2 ml of EMEM/well) and grown overnight on 8-well chamber microscope slides. Then the cells were incubated with VacA (120 nM) before fixation with 2% paraformaldehyde in PBS. After permeabilization for 5 min at room temperature with 0.1% Triton X-100 in PBS and blocking with 1.5% Block Ace in PBS, the cells were incubated for 1 h at room temperature with active form-specific anti-Bax or Bak antibodies (anti-Bax-NT antibodies and anti-Bak-NT antibodies, respectively), anti-cytochrome c antibody (6H2.B4), and/or anti-mtHSP70 (JG1) antibody (for mitochondrial marker), diluted in blocking buffer, and then incubated with Alexa fluor 488-conjugated secondary antibody against mouse IgG (for anti-cytochrome c antibody), Alexa fluor 546-conjugated secondary antibody against mouse IgG (for anti-mtHSP70 antibody), Alexa fluor 488-conjugated secondary antibody against rabbit IgG (for anti-Bax-NT and Bak-NT antibodies), and Alexa fluor 546-conjugated secondary antibody against rabbit IgG (for anti-Bax-NT and Bak-NT antibodies), diluted in blocking buffer, for 1 h at room temperature in the dark. The images were captured by confocal laser microscopy independently with the 488- and 543-nm lines of lasers used for excitation of Alexa fluor 488 (Ar/Kr laser, 488 nm) and Alexa fluor 546 (Green He-Ne laser, 543 nm), respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VacA Induced Cytochrome c Release and Death of Gastric Epithelial AZ-521 Cells—We confirmed that purified VacA induced cytochrome c release from mitochondria in gastric epithelial AZ-521 cells; the release was enhanced by the addition of 5 mM NH4Cl to the incubation medium (Fig. 1A). We also confirmed that cytochrome c release and PARP cleavage in AZ-521 cells were increased by VacA in a time-dependent manner (Fig. 1, B and C). In addition, VacA induced AZ-521 cell death, which was similarly enhanced by 5 mM NH4Cl (Fig. 1D).

VacA Localization in AZ-521 Cells in Which It Induced Cytochrome c Release—Intracellularly overexpressed full-length VacA or amino-terminal 37-kDa fragment with GFP at the amino terminus was localized to mitochondria (17, 21). In another study, purified toxin applied to intact cells was bound in VacA-induced cytoplasmic vacuoles (32, 33). We evaluated VacA localization in AZ-521 cells in which cytochrome c release was induced (Fig. 2A). In AZ-521 cells incubated with VacA, cytochrome c was dispersed throughout the cytoplasm. In contrast, in cells without VacA treatment, cytochrome c appeared to be associated with mitochondria. To determine whether VacA was associated with mitochondria when cytochrome c was released, we used anti-cytochrome c and anti-VacA antibodies with Mitotracker RED (Fig. 2A). Even in cells that exhibited cytochrome c release, most of the VacA was not associated with mitochondria. Consequently, we considered the possibility that VacA localized to VacA-induced cytoplasmic vacuoles. Membranes of VacA-induced vacuoles reacted with antibodies against proteins identified in late endosomal compartments, such as the small GTPase Rab7 (32, 34). We used GFP-conjugated Rab7 (GFP-Rab7) as a vacuolar marker. VacA induced cytochrome c release in AZ-521 cells overexpressing GFP-Rab7, just as it did in wild-type AZ-521 cells (Fig. 2B). By confocal microscopy, most of VacA appeared to be colocalized with GFP-Rab7 in cells exhibiting cytochrome c release. Furthermore, we examined VacA localization in AZ-521 cells at the early time points. In cells incubated with VacA for 12 h, which is the time point when cytochrome c release was observed in ~50% of cells (Fig. 1B), most of VacA was co-localized with GFP-Rab-7 rather than Mitotracker RED (Fig. 2C). In addition, in cells incubated with VacA for 3 or 6 h, which were time points prior to optimal cytochrome c release, most of VacA was co-localized with GFP-Rab-7 rather than Mitotracker RED (Fig. 2, D and E). We have also observed nonmitochondrial localization in HeLa cells by immunostaining and confocal analysis with anti-VacA antibodies and antibody against mitochondrial marker protein, mitochondrial HSP70, using similar experimental conditions (data not shown). From these data, it is likely that, when VacA had induced cytochrome c release, most of the toxin did not localize to mitochondria but rather to vacuoles. Nonmitochondrial localization of VacA suggested the involvement of an intracellular factor(s) in VacA-induced cytochrome c release.


Figure 3
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  		FIGURE 3.
VacA inhibited function of isolated mitochondria but did not induce cytochrome c release from isolated mitochondria. A, reduction of isolated mitochondrial membrane potential by VacA. Mitochondria (0.1 mg protein/ml) isolated from HeLa cells were incubated with acid-activated VacA (360 and 720 nM), with heat-inactivated VacA (720 nM), or without VacA for 10 min, and then the mitochondrial membrane potentials were assessed by JC-1 assay as described under "Materials and Methods." The emission intensity at 590 nm by J-aggregate was quantified; high intensity indicates active, polarized mitochondria. The value of samples without VacA treatment was set at 100%. The data are the means ± S.D. of values from three separate experiments. The S.D. value of the data without error bars is <3.0%. B, VacA did not induce cytochrome c release from isolated mitochondria. Mitochondria (0.1 mg protein/ml) isolated from HeLa cells were incubated with acid-activated VacA (720 nM), with heat-inactivated VacA (720 nM), or without VacA for indicated time in SEM buffer supplemented with 5 mM NH4Cl, and then mitochondria were pelleted. Pellets (ppt; mitochondria) and supernatants (sup) were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes, which were incubated with anti-cytochrome c antibody. CaCl2 (250 mM) was used as a positive control for cytochrome c release from isolated mitochondria. The data are representative of at least two experiments. conc., concentration

 
VacA Induced Dysfunction of Isolated Mitochondria, but Not Cytochrome c Release—Although we had found that, even in cells exhibiting cytochrome c release, most of the VacA was localized to vacuoles rather than mitochondria, VacA localization to the mitochondria has been reported under certain conditions (21). Importation of VacA synthesized in reticulocyte lysate into isolated yeast mitochondria was also reported (17). Accordingly, only a small fraction of intracellular VacA might be sufficient to stimulate cytochrome c release. We therefore investigated whether VacA caused cytochrome c release from isolated mitochondria. Isolated mitochondria were incubated with purified toxin, and mitochondrial membrane potential was assessed using the JC-1 assay (Fig. 3A). VacA led to the reduction of mitochondrial JC-1 aggregating activity, whereas no reduction was observed with heat-inactivated VacA, indicating that VacA could affect isolated mitochondrial membrane potential.

We investigated whether VacA could induce cytochrome c release from isolated mitochondria (Fig. 3B). Although VacA induced dysfunction of isolated mitochondria within 10 min, cytochrome c release was not induced even after 60 min. Cytochrome c release, assessed by Western blot analysis of supernatants from isolated mitochondria, was increased by CaCl2, which is known to cause cytochrome c release from isolated mitochondria (35). These data showed that VacA disrupted function of isolated mitochondria but did not promote cytochrome c release. Accordingly, we hypothesized that VacA induction of cytochrome c release was not a direct effect on mitochondria and might require intermediary molecule(s).

Bax and Bak Are Activated in VacA-treated Cells—It has been suggested that in the several types of apoptosis, the proapoptotic Bcl-2 family proteins, such as Bax, are pivotal regulators of cytochrome c release from mitochondria (36). We therefore asked whether VacA-induced apoptosis involved Bax activation. Conformational changes in Bax, which facilitate its dimerization and translocation to the mitochondrial outer membrane, were observed in Bax-mediated apoptosis. In a previous study, the conformational states of Bax were distinguished using conformation-specific anti-Bax antibodies (31). We performed flow cytometric analysis using antibody specific for the active form of Bax (Clone 3) and Alexa 488-conjugated secondary antibody to compare VacA-treated and untreated cells (Fig. 4, A-F). Among cells incubated without VacA, ~14% were positive for antibodies specific for active Bax, i.e. were considered Baxact (Fig. 4A). After 6 h of exposure to 120 nM VacA, this percentage had not increased, but by 12 h it had reached ~37% and continued to rise thereafter to ~77% at 24 h (Fig. 4, B-E). In contrast, there was no increase in amount of Baxact after 24 h of exposure to heat-inactivated VacA (Fig. 4F). We confirmed that the increase in cells that reacted with antibodies specific for active Bax was not attributable to a greater amount of total Bax expression (Fig. 4G). These results indicated that Bax underwent a conformational change to an active form during incubation of AZ-521 cells with VacA. The time course of Bax activation in response to VacA paralleled that of cytochrome c release (Fig. 4H). The effects of exposure to VacA for 24 h on cytochrome c release and the percentage of Baxact cells were similarly VacA concentration-dependent (Fig. 4I).


Figure 4
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  		FIGURE 4.
VacA induced Bax activation in AZ-521 cells. A-F, Bax activation increased with time of exposure to VacA. AZ-521 cells were grown overnight as described under "Materials and Methods," and then the cells were incubated without VacA (A) or with 120 nM VacA (B-E) or heat-inactivated VacA (IA VacA) (F) for indicated time in EMEM supplemented with NH4Cl (5 mM). Intracellular flow cytometry was performed using conformation-specific, anti-Bax antibody (Clone 3) (solid line) or secondary antibody alone as negative controls (dashed line) as described under "Materials and Methods." In parentheses are the percentages of cells with activated Bax (Baxact). The data are representative of at least two experiments. G, Bax expression in VacA-treated AZ-521 cells. AZ-521 cells were grown overnight and incubated with or without 120 nM acid-activated VacA for indicated times as described above, and the cell lysates were prepared. The cell lysates were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes; Bax was detected by incubation with anti-Bax antibodies. Equal loading was confirmed with anti-actin antibody. The data are representative of at least three experiments. H and I, Bax activation is positively correlated with cytochrome c release in AZ-521 cells. AZ-521 cells were grown overnight, and then the cells were incubated with 120 nM acid-activated VacA for indicated times (H) or were incubated with indicated concentrations of acid-activated VacA for 24 h (I) in EMEM supplemented with NH4Cl (5 mM). Bax activation or cytochrome c release was analyzed as described above. The data are presented as percentages of the number of cells showing Bax activation or cytochrome c release. The data are the means ± S.D. of values from three separate experiments. The S.D. value of the data without error bars is <3.0%.

 
To confirm the contribution of Bax to VacA-induced cytochrome c release, we examined the effect of silencing of bax gene on VacA-induced cytochrome c release (Fig. 5). In cells in which Bax level was decreased by RNA interference with Bax-siRNA duplex transfection, the percentage of the cells with cytochrome c released by VacA was lower than that of the negative control siRNA (NC-siRNA)-transfected cells. These results indicate that Bax may play a central role in VacA-induced cytochrome c release.


Figure 5
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  		FIGURE 5.
Effect of Bax silencing on VacA-induced cytochrome c release from mitochondria in AZ-521 cells. AZ-521 cells were grown overnight, and silencing of bax gene was performed with Bax-siRNA or NC-siRNA as described under "Materialsand Methods." 24 h after transfection, the cells were incubated with acid-activated VacA (120 nM) for 12 h in EMEM supplemented with NH4Cl (5 mM). A, cytochrome c release was analyzed as described in the legend to Fig. 1. The data are presented as percentages of the number of cells showing cytochrome c release. The data are the means ± S.D. of values from three separate experiments. The S.D. value of the data without error bars is <2.0%. B, reduction of Bax protein level was confirmed by Western blotting with anti-Bax-NT antibodies as described under "Materials and Methods." The data are representative of at least two experiments. NC-siRNA transfection did not affect both Bax level and cytochrome c release by VacA (data not shown)

 


Figure 6
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  		FIGURE 6.
Activated Bax and Bak in AZ-521 cells in which VacA induced cytochrome (cyto.) c release. AZ-521 cells were grown overnight on 8-well chamber microscope slides as described under "Materials and Methods," and then the cells were incubated with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with NH4Cl (5 mM) before immunocytochemistry and confocal analysis were performed. A and B, the cells were incubated with active-form-specific anti-Bax antibodies (Bax-NT) and anti-mtHSP70 antibody (A) or antibody against cytochrome c (B). In the case of A, merged images of Bax and mtHSP70 are on the right. C and D, the cells were incubated with active-form-specific anti-Bak (Bak-NT) and anti-mtHSP70 antibodies (C) or antibody against cytochrome c (D). In the case of C, merged images of Bak and mtHSP70 are on the right. The data are representative of at least two experiments.

 
It had been reported that activated Bax was associated with intracellular membranes, principally the mitochondrial outer membrane (36). We evaluated the subcellular distribution of activated Bax in VacA-treated AZ-521 cells using active form-specific anti-Bax antibodies and confocal microscopy (Fig. 6). The numbers of antibody-reactive cells were increased by incubation with VacA. Reaction with antibody against mitochondrial HSP 70 (mtHSP 70) revealed that activated Bax in VacA-treated gastric cells was localized to mitochondria (Fig. 6A). Double staining with active form-specific anti-Bax and anti-cytochrome c antibodies revealed that in the cells in which cytochrome c was released, Bax activation was also induced (Fig. 6B). In addition, we also observed Bak activation by VacA using conformation-specific anti-Bak antibodies (Fig. 6, C and D), indicating that Bak, another Bcl-2 homolog, is also a regulator of VacA-induced cytochrome c release.


Figure 7
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  		FIGURE 7.
VacA-induced Bax activation was enhanced by NH4Cl. AZ-521 cells were grown overnight in 60-mm culture dishes as described under "Materials and Methods," and then the cells were incubated with 120 nM acid-activated VacA for 24 h in EMEM supplemented without (A) or with (B) NH4Cl (5 mM), and Bax activation was analyzed as described in Fig. 4. The data are representative of at least three experiments. In parentheses are the percentages ± S.D. of values of cells that are Baxact from three separate experiments.

 
NH4Cl Enhanced VacA-induced Bax Activation—VacA effects, including cell vacuolation and cytochrome c release from mitochondria, were enhanced by the addition of NH4Cl to the incubation medium (Fig. 1A and Ref. 37). We therefore investigated whether NH4Cl also accelerated VacA-induced Bax activation. Flow cytometric quantification showed that the percentage of Baxact cells after incubation with VacA for 24 h without NH4Cl was lower than that in medium with NH4Cl (Fig. 7). Thus, VacA effects on Bax activation, as well as those on cytochrome c release and cell death, were enhanced by NH4Cl, consistent with the conclusion that Bax may play a central role in VacA-induced apoptosis.

VacA-induced Bax Activation Was Not Blocked by Inhibition of Cell Vacuolation—Intracellular expression of VacA was reported to cause apoptosis without vacuolation (16, 38), indicating that apoptosis could be independent of vacuolation when VacA was expressed intracellularly. To explore whether VacA-induced Bax activation was required for vacuolation, the effects of a vacuolation inhibitor on VacA-induced Bax activation and cell death were investigated. VacA-induced vacuolation depends on the activity of vacuolar-type ATPase proton pumps, and bafilomycin A1 is reported to inhibit VacA-induced vacuolation (33, 38, 39). AZ-521 cells were incubated with bafilomycin A1 before exposure to VacA, and Bax activation was assessed by flow cytometric analysis (Fig. 8). As expected, no vacuoles were detected in bafilomycin A1-treated AZ-521 cells, even after 24 h of incubation with VacA (Fig. 8, A, G, and H, phase contrast images); Bax, however, was activated to the same extent in cells with and without bafilomycin A1 treatment (Fig. 8, C-H). Similarly, VacA-induced AZ-521cell death was not inhibited by bafilomycin A1 treatment (Fig. 8B), so that both VacA-induced Bax activation and subsequent cell death were independent of VacA-induced vacuolation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is increasing evidence that apoptosis plays an important role in the pathogenesis of a variety of infectious diseases (40). Although apoptosis may be a natural physiological occurrence, excessive apoptosis results in tissue damage. Several H. pylori virulence factors induce apoptosis of different types of cells, including epithelial cells (41), polymorphonuclear leukocytes (42), T lymphocytes (43), and macrophages (44), suggesting that apoptosis is important in the pathogenesis of H. pylori infection. Recent reports indicate that VacA is one of the major virulence factors of H. pylori and induces apoptosis in various types of cells via a mitochondria-dependent pathway (14). We have now demonstrated the involvement of Bax activation with release of cytochrome c from mitochondria in VacA-induced apoptosis of cultured AZ-521 gastric epithelial cells, leading to PARP cleavage and cell death. In agreement, we also have observed VacA-induced Bax activation in HeLa cells (data not shown).

H. pylori infection was associated with elevated levels of Bax protein expression in chronic gastritis and premalignant lesions (45). The apoptotic index in Bax-positive cells in intestinal metaplasia, gastric dysplasia, and gastric carcinoma was higher than in Bax-negative cells. In the present study, we found that VacA-induced Bax activation in AZ-521 cells was correlated with cell damage and apoptosis, without changes in the Bax protein level.

The process of apoptosis involves a wide variety of effector molecules, and the Bcl-2 family proteins constitute one of the most biologically relevant classes of apoptosis regulatory molecules. The Bcl-2 family comprises two subfamilies: one of anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Bfl-1, Brag-1, and Mcl-1) and the other of opposing pro-apoptotic proteins (Bax, Bak, Bcl-XS, Bad, Bid, Bik, and Hrk) (46). Some of the Bcl-2 family proteins localize to mitochondria, forming homo- and hetero-oligomers to regulate efflux of apoptosis-executioner proteins, including cytochrome c and Smac/DIABLO. Here, we demonstrate that Bax was activated and localized to mitochondria in gastric cells exposed to VacA. It was reported that some bacteria, in addition to H. pylori, can induce Bax activation in response to infection (47, 48), suggesting that Bax activation plays important role in the pathogenesis of a variety of infectious diseases. Recently, it was reported that infection with a cag-positive H. pylori strain (s1a/m2/cagA+) resulted in Bax translocation to mitochondria, caspase-3 activation, and cell death (49). However, the presence of additional H. pylori cytotoxic factors including VacA, lipopolysaccharide, and/or the pathogenicity island may also be important in causing tissue destruction during H. pylori infection.

The proapoptotic Bcl-2 homologs, Bax and Bak, are both critical regulators of mitochondrial membrane permeabilization (23, 36), with partially redundant functions. Bax is a cytosolic, monomeric protein in nonapoptotic cells; during apoptosis, it undergoes conformational changes near the amino and carboxyl termini to expose a functionally crucial Bcl-2 homology domain 3 and translocates to the mitochondrial outer membrane. Bak is largely associated with the mitochondrial outer membrane and endoplasmic reticulum, even in healthy cells; it, too, changes conformation in response to apoptotic stimuli. Activated Bax and Bak undergo homo-oligomerization and may participate in formation of a large mitochondrial transition pore complex that facilitates cytochrome c release. In addition to Bax activation in VacA-treated AZ-521 cells, we observed Bak activation by confocal microscopy using conformation-specific anti-Bak antibodies (Fig. 6). Consistent with the involvement of Bax and Bak in the VacA mediated process, we observed a small amount of cytochrome c release even in cells in which the Bax level was decreased (Fig. 5). These results indicate that VacA may utilize both Bax and Bak to induce apoptosis.


Figure 8
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  		FIGURE 8.
Inhibition of VacA-induced cellular vacuolation but not Bax activation by bafilomycin A1. A and B, effect of bafilomycin A1 on VacA-induced cellular vacuolation and cell death. AZ-521 cells were grown overnight as described under "Materials and Methods" and incubated with or without bafilomycin A1 (5 nM) for 30 min and then with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with NH4Cl (5 mM). Vacuolation (A) or cell viability (B) was assessed by neutral red uptake assay or MTS assay. The data are the means ± S.D. of values from three separate experiments with assays in triplicate. The S.D. value of the data without error bars is <0.01. C-F, effect of bafilomycin A1 on VacA-induced Bax activation. AZ-521 cells were grown overnight, and incubated without (C and D) or with (E and F) bafilomycin A1 (5 nM) for 30 min and then without (C and E) or with (D and F) 120 nM acid-activated VacA for 24 h in EMEM supplemented with NH4Cl (5 mM) before assessment of Bax activation as described in the legend to Fig. 4. The data are representative of at least three experiments. In parentheses are percentages ± S.D. of values of cells that are Baxact from two separate experiments. G and H, Bax activation in AZ-521 cells in which VacA-induced cytochrome c release was observed but VacA-induced cellular vacuolation was inhibited. AZ-521 cells were grown overnight on 8-well chamber microscope slide as described under "Materials and Methods" and incubated without (G) or with (H) bafilomycin A1 (5 nM) for 30 min and then with or without 120 nM acid-activated VacA for 24 h in EMEM supplemented with NH4Cl (5 mM) before immunocytochemistry and confocal analysis were performed. The cells were incubated with anti-cytochrome c antibody (red) and anti-Bax-NT antibodies (green). Left column, phase-contrast images are shown; VacA-induced vacuoles were indicated by arrows. The data are representative of at least two experiments. DMSO, dimethyl sulfoxide.

 
One of the most extensively studied actions of VacA is its induction of large cytoplasmic vacuoles in cultured cells. VacA-induced vacuolar formation is reported to require vacuolar-type ATPase, and its inhibitor, bafilomycin A1, inhibits VacA-induced vacuolation. It was hypothesized that VacA inserts into endosomal membranes to form anion-selective channels with the resulting influx of anions into endosomes, enhancing vacuolar-type ATPase proton pumping activity (7). Membrane-permeant protonated weak bases, such as NH4Cl, then accumulate in the endosomes, and the resulting osmotic swelling results in formation of large vacuoles. In the present study, bafilomycin A1 inhibited vacuole formation but did not inhibit VacA-induced Bax activation and cell death. These data indicate that vacuole formation and Bax activation were independent effects of VacA. NH4Cl enhanced VacA-induced Bax activation, indicating that NH4Cl participated not only in vacuole formation but also in VacA-induced cell death. The unusually high urease activity of H. pylori increases the ammonium concentration of gastric fluid, which was reported to be 1.0-13.0 mM in infected people versus 0.02-1.4 mM in those without infection (50). In our study, the addition of 5 mM NH4Cl to incubation medium, a concentration found in gastric juice of H. pylori-infected patients, amplified the effects of VacA; NH4Cl alone did not affect cytochrome c release and Bax activation. This observation is consistent with enhancement of gastric mucosal damage by ammonium ions, which alone do not promote tissue injury.

Although it was reported that, under certain conditions, VacA localized to the mitochondria (17, 21), it had not been clear whether cytochrome c release resulted from a direct action of VacA on mitochondria. We found that most of the VacA was associated with vacuoles even in AZ-521 cells in which cytochrome c release had been induced, and VacA did not induce cytochrome c release from isolated mitochondria. These data suggested that VacA-induced cytochrome c release did not necessarily require direct interaction of VacA with mitochondria; rather, an alternative signaling pathway resulting in Bax and Bak activation appears to be important in VacA action. The relevance of Bax to the action of VacA was supported by the siRNA knock-down experiments where a reduction in Bax reduced the extent of cytochrome c release.

In summary, we show that VacA does not itself directly induce cytochrome c release from mitochondria, but rather Bax activation is involved in VacA-induced cytochrome c release and cell death. VacA-induced Bax activation was enhanced by NH4Cl, but was independent of vacuole formation, indicating that VacA has biological activity in addition to vacuole formation, and these activities might be functionally independent. Interestingly, we and other groups reported that VacA-induced derangements in phosphorylation of intracellular proteins, including mitogen-activated protein kinase, p38 (19, 24, 51), which was not associated with vacuolation (19). Further, SB203580, which inhibits p38, did not block VacA-induced cytochrome c release (19), indicating that p38 does not participate in VacA-induced cytochrome c release and Bax activation. Thus, VacA affects multiple different signaling pathways in gastric cells; some of these effects are independent of vacuolation.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and from Institute of Tropical Medicine, Nagasaki University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental material. Back

2 Supported by the Intramural Research Program, NHLBI, National Institutes of Health. Back

1 To whom correspondence should be addressed: Dept. of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan. Tel.: 81-95-849-7833; Fax: 81-95-849-7805; E-mail: a-wada{at}net.nagasaki-u.ac.jp.

3 The abbreviations used are: GFP, green fluorescent protein; EMEM, Eagle's minimal essential medium; PBS, phosphate-buffered saline; PARP, poly(ADP-ribose)polymerase; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; siRNA, small interfering RNA; MOPS, 4-morpholinepropanesulfonic acid; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-13-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt. Back


   ACKNOWLEDGMENTS
 
We thank K. Maeda and K. Tamura for skillful assistance and S. Goto (Nagasaki University), K. Rokutan (Tokushima University), T. Niidome (Kyushu University), and I. Kato (Medical School of Chiba University) for helpful discussions. We thank M. Vaughan (Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, MD) for helpful discussion and critical review of the manuscript.



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