Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells.

We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.

Kimura M ，Goto S ，Wada A ，Yahiro K ，Niidome T ，Hatakeyama T ，Aoyagi H ，Hirayama T ，Kondo T ... -  《MICROBIAL PATHOGENESIS》

Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin.

Chronic gastritis induced by Helicobacter pylori is a strong risk factor for the development of distal gastric adenocarcinoma. A specific host response to H. pylori that may contribute to gastric carcinogenesis is epithelial cell apoptosis. The aim of this study was to investigate the capacity of H. pylori vacuolating toxin (VacA) to induce gastric epithelial cell apoptosis. When cocultured with AGS gastric epithelial cells, H. pylori strain 60190, which expresses a type s1/m1 VacA toxin, induced significantly higher levels of apoptosis than did an isogenic vacA null mutant strain. VacA purified from strain 60190 induced apoptosis in a dose-dependent manner, which required acid activation of the purified toxin and the presence of ammonium chloride. In contrast, apoptosis was not induced after incubation with a chimeric s2/m1 toxin (in which the s1 sequence at the NH(2) terminus of VacA from strain 60190 was replaced with the s2 sequence from the nontoxigenic strain Tx30a) or a VacA mutant protein (VacA Delta 6-27) that lacks a unique strongly hydrophobic region near the VacA NH(2) terminus. Moreover, when an equimolar mixture of purified VacA Delta 6-27 and purified wild-type VacA were added simultaneously to AGS cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the apoptosis-inducing activity of wild-type VacA. These results indicate that VacA induces gastric epithelial cell apoptosis and suggest that differences in levels of gastric mucosal epithelial apoptosis among H. pylori-infected persons may result from strain-dependent variations in VacA structure.

Cover TL ，Krishna US ，Israel DA ，Peek RM Jr ... -  《CANCER RESEARCH》

Impairment of glutathione metabolism in human gastric epithelial cells treated with vacuolating cytotoxin from Helicobacter pylori.

Helicobacter pylori vacuolating cytotoxin (VacA) is believed to be one of the factors that induces gastric disease. Our previous study indicated that VacA causes a decrease in the intracellular ATP level in human gastric epithelial cells, suggesting to impair mitochondrial membrane potential followed by a decrease in energy metabolism (Kimura et al., Microb. Pathog., 1999, 26: 45--52). In the present study, we investigated whether the decrease in ATP level affects glutathione metabolism, in which its synthesis and efflux are ATP-dependent. Treatment of AZ-521 human gastric epithelial cells with 120 nM VacA for 6 h suppressed the efflux of oxidized glutathione (GSSG) in a dose- and time-dependent manner. The efflux of GSSG from the cells and glutathione (GSH) synthesis of cells treated with VacA were approximately 50 and 70% of those of the control, respectively. The turnover rate of intracellular GSH was also suppressed by VacA. Viability of the cells pretreated with VacA, then further incubated with H(2)O(2), was decreased by 50% at 6 h and 70% at 12 h. These results suggested that VacA impairs GSH metabolism in the gastric epithelial cells, which weakens the resistance of the cells against oxidative stress or cellular redox regulation by GSH.

Kimura M ，Goto S ，Ihara Y ，Wada A ，Yahiro K ，Niidome T ，Aoyagi H ，Hirayama T ，Kondo T ... -  《MICROBIAL PATHOGENESIS》

Helicobacter pylori vacuolating cytotoxin binds to the 140-kDa protein in human gastric cancer cell lines, AZ-521 and AGS.

To investigatie a potential mechanism of how Helicobacter pylori establishes infection, we purified a lot of vacuolating toxin (VacA) from supernatant of H. pylori ATCC49503 (tox+ strain 60190). We used an antibody which was prepared by immunizing rabbits with a synthetic peptide consisting of 16 amino acids reflecting a portion (Glu69-Arg83) of amino acid sequence of Vac A. VacA caused vacuoles in human gastric cancer cell lines AZ-521 AGS, and monkey kidney cell line COS-7, but not human promyeloblastic cell line HL-60. By immunoprecipitation analysis using anti VacA antibody, a biotinylated cell surface protein of 140kDa (p140) was precipitated only when the lysates of VacA-susceptible cells were incubated with VacA but not with inactivated VacA, indicating the association of p140 with VacA.

Yahiro K ，Niidome T ，Hatakeyama T ，Aoyagi H ，Kurazono H ，Padilla PI ，Wada A ，Hirayama T ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Relationship between Vac A toxin and ammonia in Helicobacter pylori-induced apoptosis in human gastric epithelial cells.

VacA toxin is one of the most important virulence factors produced by H. pylori even though neither its role nor its action mechanisms are completely understood. First considered as a toxin inducing only cell vacuolation, VacA causes apoptosis of gastric epithelial cells by targeting mitochondria. A hotly debated question about VacA action is its relationship with ammonia, which is produced in vivo by H. pylori urease. While ammonia is strictly required for VacA-dependent vacuolation, its role in VacA-induced apoptosis is much less defined. This study was thus aimed to investigate the relationship between VacA toxin and ammonia in H. pylori-induced mitochondrial damage and apoptosis of human gastric epithelial cells in culture by means of flow cytometry. Our results show that, unlike cell vacuolation, in MKN 28 cells neither apoptosis nor dissipation of mitochondrial transmembrane potential induced by VacA require ammonia. Nevertheless, ammonia significantly potentiates both these VacA-induced effects, but independently of the swelling of VacA-containing endosomes (i.e., vacuolation). Our findings make unlikely the hypothesis that ammonia-dependent swelling and rupture of endosomal vesicles in which VacA is sequestered after cell internalization may allow the toxin to reach mitochondria and trigger apoptosis.

Chiozzi V ，Mazzini G ，Oldani A ，Sciullo A ，Ventura U ，Romano M ，Boquet P ，Ricci V ... -  《-》