Clustering of Helicobacter pylori VacA in lipid rafts, mediated by its receptor, receptor-like protein tyrosine phosphatase beta, is required for intoxication in AZ-521 Cells

Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflammatory responses and cell detachment. Our recent data demonstrated that VacA uses receptor-like protein tyrosine phosphatase beta (RPTPbeta) as a receptor, of which five residues (QTTQP) at positions 747 to 751 are involved in binding. In AZ-521 cells, which mainly express RPTPbeta, VacA, after binding to RPTPbeta in non-lipid raft microdomains on the cell surface, is localized with RPTPbeta in lipid rafts in a temperature- and VacA concentration-dependent process. Methyl-beta-cyclodextrin (MCD) did not block binding to RPTPbeta but inhibited translocation of VacA with RPTPbeta to lipid rafts and all subsequent events. On the other hand, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), which disrupts anion channels, did not inhibit translocation of VacA to lipid rafts or VacA-induced activation of p38 mitogen-activated protein (MAP) kinase, but inhibited VacA internalization followed by vacuolation. Thus, p38 MAP kinase activation did not appear to be required for internalization. In contrast, phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited translocation, as well as p38 MAP kinase/ATF-2 activation, internalization, and VacA-induced vacuolation. Neither NPPB nor PI-PLC affected VacA binding to cells and to its receptor, RPTPbeta. Thus, receptor-dependent translocation of VacA to lipid rafts is critical for signaling pathways leading to p38 MAP kinase/ATF-2 activation and vacuolation.     

Resistance of primary murine CD4+ T cells to Helicobacter pylori vacuolating cytotoxin

Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of gastric cancer and peptic ulcer disease. H. pylori secretes a toxin, VacA, that targets human gastric epithelial cells and T lymphocytes and enhances the ability of H. pylori to colonize the stomach in a mouse model. To examine how VacA contributes to H. pylori colonization of the mouse stomach, we investigated whether murine T lymphocytes were susceptible to VacA activity. VacA inhibited interleukin-2 (IL-2) production by a murine T-cell line (LBRM-33), similar to its effects on a human T-cell line (Jurkat), but did not inhibit IL-2 production by primary murine splenocytes or CD4+ T cells. VacA inhibited activation-induced proliferation of primary human CD4+ T cells but did not inhibit the proliferation of primary murine CD4+ T cells. Flow cytometry studies indicated that the levels of VacA binding to primary murine CD4+ T cells were significantly lower than levels of VacA binding to human CD4+ T cells. This suggests that the resistance of primary murine CD4+ T cells to VacA is attributable, at least in part, to impaired VacA binding to these cells.     

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.     

The intermediate region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay

Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.     

Vacuolating cytotoxin purified fromHelicobacter pyloricauses mitochondrial damage in human gastric cells

We investigated the effects of vacuolating cytotoxin (VacA) prepared fromHelicobacter pylorion 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 PO₂in 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/G₁phase 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.     

Kinetics and Mechanisms of Extracellular Protein Release by Helicobacter pylori

To investigate the kinetics and mechanisms of extracellular protein release by Helicobacter pylori, we analyzed the entry of metabolically radiolabeled bacterial proteins into broth culture supernatant. At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein. Subsequently, culture supernatants accumulated many proteins that were components of intact bacterial cells. This nonselective release of proteins was associated with a decreasing turbidity of cultures and loss of bacterial viability, indicative of an autolytic process. The rates of VacA secretion and autolysis were each influenced by medium composition, and therefore these may be regulated phenomena. Extracellular release of proteins by H. pylori may be an important adaptation that facilitates the persistence of H. pylori in the human gastric mucus layer. Moreover, entry of proinflammatory proteins into the gastric mucosa may contribute to the induction of a mucosal inflammatory response.     

Vacuolating Cytotoxin of Helicobacter pylori Induces Apoptosis in the Human Gastric Epithelial Cell Line AGS

Helicobacter pylori induces cell death by apoptosis. However, the apoptosis-inducing factor is still unknown. The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS. The supernatant from the vacA wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not. That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA. Furthermore, a recombinant VacA produced in Escherichia coli was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation. These findings demonstrate that the vacuolating cytototoxin of H. pylori is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.     

Novel activities of the Helicobacter pylori vacuolating cytotoxin: from epithelial cells towards the immune system

H. pylori has developed a unique set of virulence factors, which allow its survival in a unique ecological niche, the human stomach. The vacuolating cytotoxin (VacA) and the cytotoxin-associated antigen (CagA) are major bacterial factors involved in modulating the host. VacA, so far mainly regarded as a cytotoxin for the gastric epithelial cell layer, apparently has profound effects in modulating the immune response. In this review we discuss some of the classical effects of VacA, such as cell vacuolation, and compare them with more recently identified mechanisms of VacA on immune cells.     

Virulence factors of Helicobacter pylori

To date a number of virulence factors have been identified and characterised from the gastric pathogen Helicobacter pylori. The vacuolating toxin (VacA) is a major determinant of H. pylori-associated gastric disease. In non-polarised cells, VacA alters the endocytic pathway, resulting in the release of acid hydrolases and the reduction of both extracellular ligand degradation and antigen processing. The toxin forms trans-membrane anion-specific channels and reduces the transepithelial electrical resistance of polarized monolayers. Localization of the VacA channels in acidic intracellular compartments causes osmotic swelling which, together with membrane fusion, leads to vacuole formation. The neutrophil-activating protein of H. pylori (HP-NAP) induces the production of oxygen radicals in human neutrophils via a cascade of intracellular activation events which may contribute to the damage of the stomach mucosa. This protein has recently been shown to be an important antigen in the human immune response to H. pylori infection. In addition, mice vaccinated with recombinant HP-NAP were protected against H. pylori challenge. H. pylori strains that are associated with severe tissue damage and inflammation possess the cag pathogenicity island that contains several genes encoding factors involved in the induction of proinflammatory cytokines/chemokines and of a type IV secretion system involved in the delivery of a highly immunogenic protein, CagA, into eukaryotic cells. Recent advances in our understanding of the involvement of VacA, HP-NAP and the CagA/Type IV secretion system in the H. pylori-associated disease process are discussed in this review.     

Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions

The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.     

Host cell signaling in Helicobacter pylori infection

Helicobacter pylori represents a highly successful human microbial pathogen that has infected approximately half of the world's population. This gram-negative microorganism colonizes the human epithelial layer in the stomach and induces a state of chronic inflammation that does not resolve the underlying infection and often leads to gastric or duodenal ulcers, or more rarely to gastric cancer. Among the reactions in H. pylori-infected epithelial cells the induction of proinflammatory cytokines, cell spreading and movement, as well as a scattered phenotype appear strictly dependent on the expression of pathogenicity island-encoded proteins in H. pylori. This review will discuss the features of the H. pylori-induced signal transduction leading to changes in host cellular function. Topics discussed comprise the signaling and the phenotypes associated with the type IV secretion system, the activation of target genes involved in gastric physiology, and putative mechanisms leading to the development of gastric cancer.     

Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection

B7‐H1 [programmed death‐ligand‐1 (PD‐L1)] is a B7‐family member that binds to programmed death‐1 (PD‐1). Recently, deficiency of PD‐L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD‐L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD‐L1 expression, regulation and function during Helicobacter pylori infection. PD‐L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co‐culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD‐L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon‐γ or tumour necrosis factor‐α. Moreover, PD‐L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD‐L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach.     

Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA.

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.     

Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.     

Complex cellular responses of Helicobacter pylori-colonized gastric adenocarcinoma cells

Helicobacter pylori is an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer. H. pylori pathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms of H. pylori were performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative analysis of 14 different human gastric epithelial cell lines after colonization with H. pylori. We found remarkable differences in host cell morphology, extent of CagA tyrosine phosphorylation, adhesion to host cells, vacuolization, and interleukin-8 (IL-8) secretion. These data might help in the selection of suitable cell lines to study host cell responses to H. pylori in vitro, and they imply that different host cell factors are involved in the determination of H. pylori pathogenesis. A better understanding of H. pylori-directed cellular responses can provide novel and more balanced insights into the molecular mechanisms of H. pylori-dependent pathogenesis in vivo and may lead to new therapeutic approaches.     

Pathogenicity island-dependent effects of Helicobacter pylori on intracellular signal transduction in epithelial cells

Helicobacter pylori is a microbial pathogen that colonises the stomach of more than half of the world's human population. H. pylori infection can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. A prominent feature of H. pylori is the presence of a pathogenicity island (PAI) which encodes a type IV secretion system (T4SS). This review focuses on the processes of how H. pylori induces PAI-dependent epithelial cell signalling that might be of importance in gastric inflammation and the pathogenesis of neoplasia.     

Helicobacter pylori -induced apoptosis in gastric epithelial cells is blocked by protein kinase C activation

Apoptosis plays a major role in gastrointestinal epithelial cell turnover. We have examined induction of apoptosis by Helicobacter pylori in gastric AGS cells and the role of protein kinase C (PKC) which has been shown to modulate programmed cell death. Incubation of AGS cells with H. pylori resulted in an activation of caspases 3 and 9 and induced programmed cell death. The PKC activator 12- O -tetradecanoylphorbol-13-acetate (TPA) caused translocation of PKC gamma, delta and var epsilon, prevented H. pylori -induced caspase activation and programmed cell death. Cocultivation of AGS cells with H. pylori resulted in a translocation of the atypical PKC isoform PKC lambda. We suggest that inhibition of H. pylori induced apoptosis by PKC activation can play a role in the process of neoplastic transformation.