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. 2011 Jul;79(7):2535–2543. doi: 10.1128/IAI.01370-10

Helicobacter pylori VacA Induces Programmed Necrosis in Gastric Epithelial Cells ▿

Jana N Radin 1, Christian González-Rivera 2, Susan E Ivie 1, Mark S McClain 1, Timothy L Cover 1,2,3,*
Editor: B A McCormick
PMCID: PMC3191986  PMID: 21482684

Abstract

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

INTRODUCTION

Helicobacter pylori is a Gram-negative bacterium that colonizes about half of the world's population. H. pylori colonization of the human stomach is consistently associated with gastric mucosal inflammation and is a risk factor for the development of peptic ulcer disease and distal gastric adenocarcinoma (14, 63). One of the major virulence factors of H. pylori is the vacuolating toxin VacA (13, 19, 26). VacA is expressed as a 140-kDa protoxin and undergoes proteolytic processing to yield an 88-kDa secreted toxin (13). VacA is secreted through an autotransporter (type V) pathway as a soluble protein into the extracellular space, and a proportion also remains attached to the bacterial cell surface (13). The secreted 88-kDa VacA protein forms anion-selective membrane channels in planar lipid bilayers (18, 50, 64), and consequently, VacA is classified as a pore-forming toxin. Multiple receptors for VacA have been identified, including sphingomyelin, receptor protein-tyrosine phosphatase alpha α (RPTPα), and RPTPβ on the surface of gastric epithelial cells and β2 integrin on the surface of T cells (28, 35, 53, 62, 73, 74). Upon internalization by cells, VacA localizes to endosomal compartments (31) as well as to mitochondria (3, 7, 21, 27, 30, 70).

VacA causes a wide array of alterations in target cells, including cell vacuolation, depolarization of the plasma membrane potential, permeabilization of epithelial monolayers, detachment of epithelial cells from the basement membrane, disruption of endosomal and lysosomal function, autophagy, interference with antigen presentation, and inhibition of T-cell activation and proliferation (13, 19, 26, 66). In addition, VacA can induce death of gastric epithelial cells. Thus far, most studies of VacA-induced cell death have been conducted using AGS or MKN28 gastric epithelial cell lines, as well as HeLa cells (5, 11, 16, 30, 44, 54, 70, 71). VacA-induced death of these cells is preceded by activation of Bax and Bak, induction of mitochondrial damage, reduction of the mitochondrial membrane potential, and cytochrome c release (30, 39, 70, 71, 75) and is accompanied by DNA fragmentation (16). On the basis of these observations, VacA-induced cell death has been classified as an apoptotic process (5, 11, 16, 44, 54).

Among several available gastric epithelial cell lines, the AZ-521 cell line is one of the most highly susceptible to VacA-induced cell death. AZ-521 cells have been used in previous studies for the identification of several VacA receptors and for studies of cellular alterations caused by VacA (20, 28, 53, 73–75). In the current study, we undertook an in-depth study of the process by which VacA causes death of these cells. We show that VacA-induced death of AZ-521 gastric epithelial cells occurs by a process consistent with programmed necrosis, resulting in extracellular release of cellular constituents. This leads to the hypothesis that VacA-induced programmed necrosis and the resulting release of proinflammatory cellular components augment H. pylori-induced gastric mucosal inflammation and thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

H. pylori wild-type (WT) strain 60190 (ATCC 49503) and isogenic mutants expressing VacA-G14A (50) or VacA Δ6-27 (68), mutant toxins that are defective in membrane channel formation, were grown on Trypticase soy agar plates containing 5% sheep blood at 37°C in ambient air containing 5% CO2. H. pylori liquid cultures were grown in brucella broth supplemented with 5% fetal bovine serum (FBS; Atlanta Biologicals) or 0.5% activated charcoal. WT VacA and VacA Δ6-27 were purified in oligomeric forms from H. pylori culture supernatants, as described previously (15). Before addition to cells, purified VacA was dialyzed into phosphate-buffered saline (PBS) and was then acid activated by the slow addition of 200 mM HCl until a pH of 3.0 was reached (15). For experiments using H. pylori broth culture supernatant (derived from cultures in brucella broth containing FBS), supernatants were concentrated 30-fold by ultrafiltration with a 30-kDa-cutoff membrane. The relative concentrations of VacA in broth culture supernatants from WT and mutant H. pylori strains were determined by Western blot analysis using rabbit anti-VacA antiserum (serum no. 958), and the concentrations of VacA in individual preparations were then normalized. Clostridium perfringens epsilon-toxin was expressed in Escherichia coli, purified, and trypsin activated as described previously (49, 56).

Cell culture.

AZ-521 human gastric adenocarcinoma cells (Culture Collection of Health Science Resource Bank, Japan Health Science Foundation) were grown in minimal essential medium supplemented with 10% FBS (Atlanta Biologicals) and 1 mM nonessential amino acids. Initial experiments showed that acid-activated preparations of purified VacA induced significantly greater cell vacuolation and cell death than did nonactivated VacA preparations, and both VacA-induced cell vacuolation and cell death were potentiated by supplementation of the tissue culture medium with 5 mM ammonium chloride (data not shown). Therefore, in all subsequent experiments the tissue culture medium was supplemented with 5 mM ammonium chloride, and all preparations of purified VacA were acid activated prior to addition to cells (15, 16). Addition of 5 mM ammonium chloride did not alter the pH of the tissue culture medium. VacA concentrations of 10 μg/ml correspond to 114 nM. Madin-Darby canine kidney (MDCK) epithelial cells were grown in Leibovitz L-15 medium supplemented with 10% FBS.

Cell viability assay.

For cell viability assays, AZ-521 or AGS cells were seeded at 4 × 104 cells/well into 96-well plates and incubated overnight. Cells were then incubated with purified WT VacA or purified VacA Δ6-27 or with serial dilutions of H. pylori supernatant containing WT VacA or VacA-G14A. Cell viability was assessed using the CellTiterAQueous One Solution cell proliferation assay (Promega) according to the manufacturer's instructions. As a control, cells were treated with 1 μM staurosporine (Cell Signaling), an agent known to cause apoptosis, for various time intervals, and cell viability was assessed as described above.

Analysis of HMGB1 release by confocal microscopy and Western blotting.

For analysis of histone-binding protein high mobility group box 1 (HMGB1) by confocal microscopy, AZ-521 and MDCK cells were cultured on plastic chamber slides (5 × 104 cells/well in an 8-well chamber slide; LabTek) overnight. AZ-521 cells were then incubated with purified WT VacA or VacA Δ6-27 (40 μg/ml) for 12 h. MDCK cells were treated with 30 nM epsilon-toxin for 45 min. Cells were washed three times with PBS, fixed with 4% formaldehyde for 10 min at room temperature, and washed again with PBS. Cells were permeabilized with 0.25% Triton X-100 (in PBS) for 5 min at room temperature, followed by 3 washes with PBS, and then incubated with Image-iT signal enhancer (Invitrogen) for 30 min at room temperature. Cells were stained for HMGB1 using an anti-HMGB1 antibody (1:400 in 1% bovine serum albumin [BSA]–PBS; Abcam), followed by incubation with a secondary anti-goat Alexa Fluor 488-conjugated antibody (1:200 in PBS containing 1% BSA). The nuclei were stained by incubating the cells with 7-amino-actinomycin D (7-AAD; BD Biosciences). Samples were mounted with Fluor-Gel (Electron Microscopy Sciences) and examined at a ×65 magnification using an LSM 510 inverted confocal microscope (Carl Zeiss). Nonconfocal differential interference contrast (DIC) images were collected simultaneously with the confocal images.

For analysis of HMGB1 by Western blotting, AZ-521 cells were seeded at 7 × 105 cells/well into 6-well plates and incubated overnight. Following incubation with WT VacA or VacA Δ6-27, tissue culture supernatants were collected, proteins were precipitated from the supernatant using trichloroacetic acid (TCA), and cells were lysed in CelLyticM cell lysis reagent (Sigma). Samples were standardized on the basis of protein concentrations, which were determined using a bicinchoninic acid (BCA) assay kit (Pierce). HMGB1 in whole-cell lysates or preparations of TCA-precipitated supernatant proteins was detected by Western blot analysis using an anti-HMGB1 antibody (1:1,000; Abcam), followed by a horseradish peroxidase-conjugated secondary antibody (1:10,000; Promega). Proteins were visualized by incubation with a chemiluminescent substrate solution (Pierce) and exposure to X-ray film.

LDH release assay.

Cells (AZ-521, AGS, or MDCK) were seeded at 4 × 104 cells/well into 96-well plates. Following incubation with WT VacA, mutant VacA proteins, 1 μM staurosporine, or 30 nM epsilon-toxin, lactate dehydrogenase (LDH) release was measured using a CytoTox 96 nonradioactive cytotoxicity assay (Promega) according to the manufacturer's instructions. Maximum LDH release was determined following treatment of cells with lysis buffer.

PARP activation assay.

AZ-521 cells were seeded at 7 × 105 cells/well into 6-well plates and incubated overnight. Cells were then incubated for 8 h with H. pylori supernatant containing WT VacA or VacA-G14A or 1 μM staurosporine. Poly(ADP-ribose) polymerase (PARP) activity was measured using an HT Universal colorimetric PARP assay kit (Trevigen) according to the manufacturer's instructions.

Analysis of PARP cleavage by Western blotting.

Cells (AZ-521 or MDCK) were seeded at 7 × 105 cells/well into 6-well plates and incubated overnight. AZ-521 cells were then incubated with H. pylori supernatant containing WT VacA, or they were treated with 1 μM staurosporine. MDCK cells were treated with 30 nM epsilon-toxin. Cells were lysed in CelLyticM cell lysis reagent (Sigma). Cleavage of PARP was assessed by Western blot analysis using an anti-PARP antibody (1:1,000; Clontech), followed by a horseradish peroxidase-conjugated secondary antibody (1:10,000; Promega). Proteins were visualized by incubation with a chemiluminescent substrate solution (Pierce) and exposure to X-ray film.

Measurement of intracellular ATP levels.

AZ-521 cells (7 × 105 cells/well in 6-well plates) were incubated with H. pylori supernatant containing WT VacA or VacA-G14A, or they were treated with 1 μM staurosporine. MDCK cells (7 × 105 cells/well in 6-well plates) were treated with 30 nM epsilon-toxin. Cells were harvested by trypsinization and resuspended in 200 μl of PBS. Cell suspensions were mixed with 200 μl of a 10% TCA–4 mM EDTA solution and incubated on ice for 10 min. The extracts were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. ATP was measured using an Enliten ATP assay system bioluminescence detection kit (Promega) according to the manufacturer's instructions. Samples were standardized on the basis of protein concentration using a BCA assay kit (Pierce), and the ATP concentrations of samples were compared to ATP concentrations detected in extracts from untreated control cells. Fluorescence measurements and luminescence were measured using a BioTek FLx800 plate reader.

Caspase inhibition.

AZ-521 cells were seeded at 4 × 104 cells/well into 96-well plates and were incubated overnight. Cells were then preincubated with 100 μM Q-VD-OPh (a general caspase inhibitor; R & D Systems) for 3 h, followed by incubation with serial dilutions of H. pylori supernatant containing WT VacA or VacA-G14A for 24 h or incubation with staurosporine for the same length of time. Cell viability was then assessed using the One CellTiterAQueous Solution cell proliferation assay (Promega) according to the manufacturer's instructions.

Caspase-3 activation.

AZ-521 cells were seeded at 7 × 105 cells/well into 6-well plates and incubated overnight. Cells were then incubated with either WT VacA or 1 μM staurosporine for 4.5 h, and caspase-3 activation was measured using a caspase-3 colorimetric assay kit (Genscript) according to the manufacturer's instructions.

Select agent.

Plasmid DNA capable of expressing Clostridium perfringens epsilon-protoxin (or epsilon-toxin) is considered a select agent by the U.S. Department of Health and Human Services.

RESULTS

VacA causes death of AZ-521 gastric epithelial cells.

Most previous research on VacA-induced cell death was done with AGS or MKN28 human gastric epithelial cell lines or HeLa cells (16, 30, 44, 54, 70). Another human gastric epithelial cell line, AZ-521, has also been used extensively for studies of VacA, and several putative VacA receptors have been identified using this cell line (20, 28, 53, 73, 74). Previous studies have not investigated whether there are substantial differences in the susceptibility of different gastric cell lines to VacA-induced cell death. Therefore, we compared AZ-521 and AGS cells. Cells were incubated with serial dilutions of H. pylori broth culture supernatant containing WT VacA or an inactive mutant protein (VacA-G14A) (Fig. 1A) (50). Alternatively, cells were incubated with purified WT VacA or a purified inactive mutant protein (VacA Δ6-27) (Fig. 1B) (68). Cell viability was quantified by measuring cellular metabolic activity. WT VacA induced cell vacuolation within several hours after addition of the toxin, whereas the mutant toxins did not cause cell vacuolation (data not shown). Within 24 h, WT VacA induced death of AZ-521 cells, whereas the mutant toxins did not (Fig. 1A and B). WT VacA did not cause death of AGS cells after 24 h (Fig. 1C) and, consistent with previous results (11, 16, 44), caused death of AGS cells only after longer incubation periods (data not shown). These initial experiments showed that, in comparison to AGS cells, AZ-521 cells are much more susceptible to VacA-induced cell death. We conducted further experiments to investigate the process by which VacA causes death of AZ-521 cells.

Fig. 1.

Fig. 1.

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Effects of VacA on viability of AZ-521 cells. (A) AZ-521 cells were incubated with serial dilutions of H. pylori broth culture supernatant containing WT VacA or VacA-G14A. Concentrations of WT VacA and VacA-G14A were normalized by Western blot analysis, as described in Materials and Methods. After 24 h, cell viability was assessed using the CellTiterAQueous One Solution cell proliferation assay. (B) AZ-521 cells were incubated with the indicated concentrations of purified WT VacA or VacA Δ6-27, and cell viability was assessed after 24 h. Error bars represent means ± standard deviations based on triplicate determinations from a single representative experiment. The experiment was performed three times with similar results. (C) AGS cells were incubated with serial dilutions of H. pylori broth culture supernatant containing WT VacA or VacA-G14A. Cell viability was assessed after 24 h. Error bars represent means ± standard deviations based on combined results of two independent experiments, each performed in triplicate.

Incubation of AZ-521 cells with VacA leads to cell swelling.

To determine whether VacA-induced death of AZ-521 cells occurred by an apoptotic or a programmed necrosis pathway, we used several different experimental approaches. The first experiment examined cell size after incubation with VacA. Necrotic cells typically exhibit cell and organelle swelling (33), whereas apoptotic cells typically exhibit a reduction in cell size (29). To determine whether incubation of cells with VacA results in cell swelling or cell shrinkage, we incubated AZ-521 cells with WT VacA (10 μg/ml) or medium alone, and cell size was quantified by flow cytometric measurements of forward scatter (FSC-A). Treatment of cells with WT VacA resulted in a 1.6-fold increase in mean cell size compared to that of untreated cells (Fig. 2; P = 0.0001, Student's t test). The observed increase in cell size is atypical for an apoptotic process and suggested that VacA might cause death of AZ-521 cells through a necrotic pathway.

Fig. 2.

Fig. 2.

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Incubation of AZ-521 cells with VacA leads to cell swelling. AZ-521 cells were incubated with WT VacA (10 μg/ml) or medium alone for 6 h, and cell size was quantified by flow cytometric measurements of forward scatter (FSC-A). Treatment of cells with WT VacA resulted in a 1.6-fold increase in mean cell size compared to that of untreated cells (mean forward scatter fluorescence of 117,687 ± 10,133 compared to 71,943 ± 4,607) (P ≤ 0.0001, Student's t test). Mean fluorescence values reflect results of three independent experiments, and the images depict representative results. SSC-A, side scatter.

Cell death induced by VacA results in LDH release.

In addition to cell swelling, necrotic cells typically undergo organelle damage and plasma membrane rupture, leading to the release of intracellular components such as LDH (33, 52). In contrast, the cell membranes of apoptotic cells remain intact until late stages, when the cells undergo secondary necrosis (29, 59). We therefore assessed whether incubation of AZ-521 cells with VacA resulted in LDH release. Cells were incubated with either WT VacA or VacA Δ6-27 for various time intervals, and LDH release was analyzed at each time point. WT VacA induced the release of LDH beginning about 10 h after the addition of toxin, whereas VacA Δ6-27 did not induce LDH release (Fig. 3A). By 15 to 20 h after addition of WT VacA, maximum LDH release (corresponding to 100% cell killing) was detected.

Fig. 3.

Fig. 3.

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Cell death induced by VacA results in LDH release. (A) AZ-521 cells were incubated with purified WT VacA or VacA Δ6-27 (20 μg/ml; 228 nM), and LDH release was quantified at various time points. (B) AZ-521 cells were treated with 1 μM staurosporine. LDH release and cell viability (based on a metabolic assay) were assessed at various time points. (C) MDCK cells were treated with C. perfringens epsilon-toxin (30 nM), and LDH release was quantified at various time points. Error bars represent means ± standard deviations based on combined results of three independent experiments, each performed in triplicate. LDH release is expressed as a percentage of control, which is the maximal amount of LDH that can be released by the cells.

For comparison, we analyzed the effects of staurosporine, an agent that is known to induce apoptosis. On the basis of the results of a cell viability assay, staurosporine-treated cells underwent cell death beginning several hours after addition of the drug, but the cells did not release any substantial amounts of LDH until much later time points (Fig. 3B). As a control for an agent expected to induce necrosis, we analyzed effects of another bacterial pore-forming toxin, C. perfringens epsilon-toxin, on a susceptible cell line (MDCK cells). Incubation of MDCK cells with 30 nM epsilon-toxin resulted in near-maximal LDH release within 6 h after addition of the toxin (Fig. 3C).

To evaluate whether the observed necrosis of AZ-521 cells in response to VacA was due to primary necrosis or secondary necrosis (i.e., subsequent to apoptosis), we assessed cell viability as well as LDH release at early time points. We incubated AZ-521 cells with WT VacA and VacA-G14A for 4, 6, and 24 h and quantified both LDH release and cell viability. These experiments showed that there was relatively little loss of cell viability at early time points (≤6 h), and at these early time points, the level of LDH release was comparable to the proportion of dead cells (Fig. 4). When they are combined with the results shown in Fig. 3, these data suggest that the observed cell death is due to primary necrosis rather than secondary necrosis.

Fig. 4.

Fig. 4.

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Incubation of AZ-521 cells with VacA results in primary necrosis. AZ-521 cells were incubated with H. pylori broth culture supernatants containing normalized concentrations of WT VacA or VacA-G14A. (A) LDH release was quantified 4, 6, and 24 h after addition of VacA. (B) Cell viability was assessed 4, 6, and 24 h after addition of VacA. Error bars represent means ± standard deviations based on combined results of three independent experiments, each performed in six replicates. LDH release is expressed as a percentage of control, which is the maximal amount of LDH that can be released by the cells.

We then compared the ability of VacA to cause LDH release from AZ-521 cells with its effect on AGS cells. Similar to what we observed in the cell viability assays (Fig. 1), WT VacA caused substantial LDH release from AZ-521 cells but minimal LDH release from AGS cells after 24 h of incubation (Fig. 5A and B). After 48 h of incubation, VacA caused LDH release from AGS cells corresponding to about 30 to 40% of maximal lysis (Fig. 5D). These experiments confirmed that AZ-521 cells are much more susceptible to VacA-induced cell death than AGS cells and revealed that VacA induces LDH release, a characteristic of necrotic cell death, in both cell lines.

Fig. 5.

Fig. 5.

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Cell death induced by VacA results in LDH release in both AZ-521 and AGS cells. AZ-521 and AGS cells were incubated with serial dilutions of H. pylori broth culture supernatants containing normalized concentrations of WT VacA or VacA-G14A. LDH release was quantified at 24 h (A and B) and 48 h (C and D) after addition of VacA. Error bars represent means ± standard deviations based on combined results of two independent experiments, each performed in triplicate. LDH release is expressed as a percentage of control, which is the maximal amount of LDH that can be released by the cells.

Cell death induced by VacA results in PARP activation.

Poly(ADP-ribose) polymerase is a nuclear enzyme that catalyzes the transfer of ADP-ribose moieties from NAD+ to itself and other acceptor proteins in response to DNA damage (36, 52). PARP initially functions to alter chromatin structure and facilitate DNA repair following low levels of DNA damage. However, following severe damage, sustained PARP activity leads to depletion of NAD+ and ATP, resulting in irreversible energy failure and eventually necrotic cell death (8). PARP activation is commonly observed in necrotic cells but not in apoptotic cells. PARP is inactivated by caspase cleavage in apoptosis, an ATP-dependent process (46, 61), to prevent PARP-induced ATP depletion.

To determine whether VacA treatment induced PARP activation, we treated AZ-521 cells with WT VacA or VacA-G14A for 8 h and then analyzed activation of PARP. Incubation of AZ-521 cells with WT VacA resulted in increased PARP activity, while incubation with VacA-G14A or staurosporine did not (Fig. 6A). We also examined PARP cleavage by Western blot analysis. As expected, PARP cleavage occurred in staurosporine-treated cells, but PARP cleavage was not detected in VacA-treated cells or in MDCK cells treated with epsilon-toxin (Fig. 6B). Since WT VacA did not induce PARP cleavage, the responses to WT VacA and VacA-G14A were indistinguishable (Fig. 6B). Activation of PARP and absence of detectable PARP cleavage in response to VacA or epsilon-toxin are consistent with necrotic cell death.

Fig. 6.

Fig. 6.

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Cell death induced by VacA results in PARP activation. (A) AZ-521 cells were treated for 8 h with H. pylori broth culture supernatants containing normalized concentrations of WT VacA or VacA-G14A or with 1 μM staurosporine. The background level of PARP activity detected in untreated cells was subtracted from the values determined for treated cells. Error bars represent means ± standard deviations from combined results of two independent experiments, each performed in triplicate. (B) Western blot analysis of PARP in AZ-521 cells treated for 8 h with H. pylori supernatant containing WT VacA or VacA-G14A or with 1 μM staurosporine (ST) and MDCK cells treated with C. perfringens epsilon-toxin (30 nM) for 30 min.

Cell death induced by VacA leads to reduction of intracellular ATP levels.

Sustained PARP activity can lead to depletion of NAD+ and ATP, and a reduction in intracellular ATP levels is considered to be a hallmark of necrosis (23). In contrast, apoptosis is an ATP-dependent process and intracellular ATP levels typically remain relatively unchanged in apoptotic cells until the late stages of apoptosis (34). We therefore assessed whether incubation of AZ-521 cells with VacA resulted in decreased intracellular ATP levels. Cells treated with WT VacA showed progressively decreasing ATP levels over time (Fig. 7A), whereas this phenomenon was not observed when cells were treated with VacA-G14A. Similarly, when MDCK cells were treated with 30 nM epsilon-toxin, a toxin that is known to induce necrosis, intracellular ATP was rapidly depleted (Fig. 7B). Cells treated with staurosporine exhibited only a small reduction in ATP levels (Fig. 7A). The observed depletion of intracellular ATP induced by VacA is consistent with induction of cell death by a necrotic pathway.

Fig. 7.

Fig. 7.

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Cell death induced by VacA leads to reduction of intracellular ATP levels. (A) AZ-521 cells were treated for the indicated times with H. pylori broth culture supernatants containing normalized concentrations of WT VacA or VacA-G14A, or cells were treated with 1 μM staurosporine. Cellular ATP concentrations were measured at various time points and are reported as a percentage of the ATP concentrations of untreated control cells. (B) MDCK cells were treated for the indicated times with C. perfringens epsilon-toxin (30 nM), and cellular ATP concentrations were determined. Error bars represent means ± standard deviations from combined results of three independent experiments, each performed in triplicate. Statistical significance was assessed using analysis of variance and Dunnett's post hoc test. *, P < 0.05 compared to time zero.

VacA-induced cell death does not depend on caspase activation.

Since caspases are important mediators of apoptosis (42, 67), we next investigated whether inhibition of caspase activation would prevent VacA-induced cell death. AZ-521 cells were preincubated with the general caspase inhibitor Q-VD-OPh, which can prevent apoptosis by the three major apoptotic pathways (caspase 9/3, caspase 8/10, and caspase 12) (9), and we then treated the cells with either WT VacA or staurosporine. While inhibition of caspase activation blocked apoptosis induced by staurosporine (Fig. 8A), it had no effect on VacA-induced cell death (Fig. 8B).

Fig. 8.

Fig. 8.

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VacA-induced cell death does not depend on caspase activation. AZ-521 cells were pretreated with the general caspase inhibitor Q-VD-OPh (100 μM) or dimethyl sulfoxide (DMSO) alone for 3 h and then treated with 2 μM staurosporine for 24 h (A) or incubated for 24 h with serial dilutions of H. pylori broth culture supernatant containing WT VacA (B). The inhibitor remained present throughout the experiment. Cell viability was assessed using the CellTiterAQueous One Solution cell proliferation assay. Error bars represent means ± standard deviations of measurements from combined results of two independent experiments, each performed in triplicate. (C) AZ-521 cells were incubated for 4.5 h with H. pylori broth culture supernatant containing WT VacA or with 1 μM staurosporine, and caspase-3 activation was determined using a caspase-3 colorimetric assay kit. OD415, optical density at 415 nm.

To further analyze the role that caspases play in VacA-induced cell death, we assessed whether incubation of cells with VacA resulted in activation of caspase-3. While incubation of AZ-521 cells with staurosporine resulted in activation of caspase-3, incubation of cells with VacA had no effect on caspase-3 (Fig. 8C). These results provide evidence that VacA-induced cell death occurs through a caspase-independent process.

Cell death induced by VacA results in the release of HMGB1.

The results obtained so far suggested that VacA-induced cell death occurs primarily by a necrotic pathway rather than an apoptotic pathway. Previous studies have reported that Clostridium septicum alpha-toxin- and Staphylococcus aureus alpha-toxin-induced necrosis of cells results in the release of HMGB1 (a proinflammatory protein) into the cytoplasm and then into the culture supernatant (24, 38). Release of HMGB1 is a hallmark of necrotic cells but not apoptotic cells (60). To determine whether VacA-induced cell death resulted in release of HMGB1, we incubated AZ-521 cells with purified WT VacA or VacA Δ6-27 and assessed localization of HMGB1 by confocal microscopy (Fig. 9A). In untreated cells and cells that were treated with VacA Δ6-27, HMGB1 remained in the nucleus. In contrast, in cells treated with WT VacA, HMGB1 localized in the cytoplasm and not the nucleus. Immunoblot analysis confirmed that, in response to treatment with WT VacA, there was a progressive reduction of cellular HMGB1 accompanied by release of HMGB1 into the culture supernatant (Fig. 9B). As expected, HMGB1 colocalized with the nucleus of untreated MDCK cells, but as was observed with VacA-treated cells, HMGB1 was released into the cytoplasm of epsilon-toxin-treated cells (Fig. 9C) and was subsequently released into the culture supernatant (data not shown). These results indicate that VacA induces cell death by a necrotic pathway, resulting in the release of HMGB1.

Fig. 9.

Fig. 9.

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Cell death induced by VacA results in release of HMGB1. (A) AZ-521 cells were treated with purified WT VacA or VacA Δ6-27 (40 μg/ml; 456 nM) for 12 h. (B) AZ-521 cells were treated with 10 μg/ml of WT VacA or VacA Δ6-27 for various time intervals. (C) MDCK cells were treated with C. perfringens epsilon-toxin (30 nM) for 45 min. Localization of HMGB1 was assessed by confocal microscopy (A and C). Cell-associated HMGB1 and release of HMGB1 into the culture supernatant were detected by Western blot analysis (B).

DISCUSSION

Bacteria and bacterial protein toxins are capable of causing cell death by multiple pathways, including apoptosis, necrosis or lysis, programmed necrosis, pyroptosis, and autophagic cell death (45, 58). In contrast to the cellular necrosis that occurs in response to nonspecific cellular insults (such as treatment with detergent, high concentrations of peroxide, or freeze-thaw cycles), programmed necrosis (also known as pyronecrosis, oncosis, necroptosis, or PARP-induced cell death) is initiated through activation of specific signaling pathways (29, 33). These include PARP-1 hyperactivation (77), tumor necrosis factor alpha receptor signaling (10), and RIP1 kinase signaling (12). Programmed necrosis can be induced by extracellular signals or can be the result of intracellular perturbations (78).

Apoptotic cells and necrotic cells share several common features but can be differentiated by a variety of morphological and biochemical criteria. Typical morphological features of cells undergoing apoptosis include cell rounding, cell shrinkage, and plasma membrane blebbing (29, 33). Other features of apoptotic cells include caspase activation, PARP cleavage, retention of intracellular ATP levels, and absence of plasma membrane rupture (29). Cell swelling, vacuolation of the cytoplasm, PARP activation, ATP depletion, early plasma membrane rupture, and the release of proinflammatory proteins are characteristics of cells undergoing programmed necrosis, but these features are not characteristics of apoptotic cells (40, 47). Mitochondrial alterations and DNA damage can occur in both cells undergoing apoptosis and necrotic cells (6, 32, 33, 40, 43, 72).

In the current study, we observed that AZ-521 gastric epithelial cells were much more susceptible to VacA-induced cell death than were AGS gastric epithelial cells. Therefore, we investigated the process by which VacA causes death of AZ-521 cells. As controls, we analyzed the effects of staurosporine (an agent known to cause apoptosis) and C. perfringens epsilon-toxin (a pore-forming toxin that causes necrosis). Our results showed that the effects of VacA were different from those of staurosporine and resembled the effects of epsilon-toxin. Therefore, we conclude that VacA induces death of AZ-521 cells by a programmed necrosis pathway.

Previous studies have reported that VacA is capable of causing death of AGS gastric epithelial cells and several other cell lines (5, 7, 16, 39, 44). On the basis of the observations that VacA-induced cell death is preceded by the activation of Bax and Bak (75), that VacA can localize to mitochondria (7, 21, 27, 30, 70), and that incubation of cells with VacA results in reduction of the mitochondrial transmembrane potential and release of cytochrome c (30, 39, 70), VacA-induced cell death of these cell lines was classified as an apoptotic process (5, 16, 44, 54, 71). However, these phenomena can occur in cells undergoing either apoptotic or nonapoptotic cell death (33, 40, 41). Several findings in a previous study (70) suggested that there might be differences between the process by which VacA induces death of HeLa cells compared to the process by which known apoptosis-inducing agents cause cell death. (i) Inhibition of cellular caspases had no effect on VacA-induced reduction of the mitochondrial transmembrane potential or cytochrome c release (70). (ii) VacA-induced reduction of the mitochondrial transmembrane potential occurred prior to cytochrome c release (70), whereas the apoptosis-inducing agent actinomycin D induced reduction in the mitochondrial transmembrane potential only after the release of cytochrome c (69). (iii) A lower VacA concentration was needed to induce the reduction of the mitochondrial transmembrane potential than was needed to induce cytochrome c release (70). In the current study, we did not conduct detailed studies of the process by which VacA causes death of AGS or HeLa cells. However, we observed that VacA-induced death of AGS cells is associated with LDH release. This suggests that, similar to VacA-induced death of AZ-521 cells, VacA-induced death of AGS cells may occur at least in part through programmed necrosis.

An important difference between apoptosis and programmed necrosis is that the plasma membrane remains intact in apoptotic cells but not in necrotic cells. Apoptotic cells are engulfed in vivo by resident phagocytes, thereby preventing cells from undergoing secondary necrosis and releasing intracellular contents. As a consequence, apoptosis usually elicits a very limited inflammatory response (59). In contrast, necrotic cells undergo early plasma membrane rupture, resulting in release of intracellular contents that can promote an inflammatory response (33). One of the proinflammatory proteins released by necrotic cells is HMGB1. HMGB1 is expressed by almost all cells and is one of the most abundant proteins in the nucleus. In healthy cells, it is involved in facilitating transcription factor binding, nucleosome remodeling, and DNA repair (4). The release of HMGB1 from necrotic cells occurs by a process that requires PARP activation, and the release of HMGB1 results in a proinflammatory response (57, 60, 76). When it is released from cells, HMGB1 can bind to several receptors on immune cells, including receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4 (55). This results in maturation of dendritic cells, activation of T cells, and the release of proinflammatory cytokines by monocytes, T cells, and endothelial cells (22, 51). Additionally, HMGB1 has been shown to promote colon carcinogenesis in an inflammation-based model (48). Our results indicate that VacA treatment of cells results in the release of HMGB1, which would be capable of stimulating a proinflammatory response.

Colonization of the stomach with H. pylori results in chronic gastric inflammation, and inflammation contributes to the pathogenesis of peptic ulcer disease and distal gastric adenocarcinoma (14, 63). Data from numerous studies suggest that VacA contributes to gastric mucosal inflammation and development of peptic ulceration or gastric cancer, in a manner that is independent of the effects of the cag pathogenicity island (1, 2, 25, 65). The data reported in the current study suggest that VacA may contribute to gastric inflammation by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins.

In summary, these results indicate that VacA-induced cell death of AZ-521 gastric epithelial cells occurs by a programmed necrosis pathway. Several other bacterial pore-forming toxins, including Staphylococcus aureus alpha-toxin, Escherichia coli hemolysin, Clostridium septicum alpha-toxin, and Clostridium perfringens epsilon-toxin, are also known to induce cell death by programmed necrosis (17, 24, 37, 38). VacA can therefore be included among the growing number of bacterial pore-forming toxins that induce cell death by programmed necrosis and thereby trigger host inflammatory responses. We propose that VacA augments mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

ACKNOWLEDGMENTS

This work was funded by NIH grants AI39657, AI068009, CA116087, and AI079123, the Molecular Microbial Pathogenesis Training Program (T32 AI007281-21), and the U.S. Department of Veterans Affairs. The VMC Flow Cytometry Shared Resource and Cell Imaging Shared Resource are supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, or the U.S. Department of Veterans Affairs.

We thank Beverly Hosse for assistance with VacA purification, Holly M. Algood for helpful discussions, and David K. Flaherty and Brittany Matlock for assistance with flow cytometry.

Footnotes

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Published ahead of print on 11 April 2011.

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