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The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity., identifier:Disease Models & Mechanisms, 5(4), pp.515-521; 2012

Objective: Prior reports suggested that infection with Helicobacter pylori was associated with respiratory diseases; pathogenetic mechanisms however, were not defined. We tested the hypothesis that VacA, an exotoxin of H. pylori, a gastric pathogen, was aspirated into the lung and could stimulate secretion of inflammatory cytokines by lung epithelial cells. Methods: The presence of VacA was determined by immunohistochemistry in surgical lung biopsy tissue samples from 72 patients with interstitial pneumonia. The effects of VacA on A549 human alveolar epithelial adenocarcinoma cells and normal human bronchial epithelial cells were determined. After incubation with VacA, the secretions of cytokines were measured by Multiplex LuminexR Assays. Results: VacA was detected with anti-VacA antibodies in bronchial epithelial cells and alveolar epithelial cells from 10 of 72 patients with interstitial pneumonia. VacA was more prevalent in lungs of patients with collagen vascular disease-associated interstitial pneumonia than in those of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and cryptogenic organizing pneumonia. Incubation of A549 cells and normal human bronchial epithelial cells with VacA for 24 h was cytotoxic, and resulted in vacuolation. VacA induced interleukin-8 production by A549 cells and normal human bronchial epithelial cells and interleukin-6 production by A549 cells. Based on multiplex screening, interleukin-8 and interleukin-6 were the primary secretory products induced by VacA. Conclusions: H. pylori VacA is present in human lung and can induce interleukin-8 and interleukin-6 production by human lung cells. VacA could have a role in the pathogenesis of respiratory diseases by its cytotoxic effects and by inducing the secretion of interleukin-8 and interleukin-6 by targeted airway epithelial cells., Biochemical and Biophysical Research Communications, 460(3), pp.721-726; 2015

Vacuolating cytotoxin A (VacA) is one of the important virulence factors produced by H. pylori. VacA induces apoptotic cell death, which is potentiated by ammonia. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. Our aim was to determine whether endoplasmic reticulum (ER) stress is associated with VacA-induced mitochondrial dysfunction and apoptosis. We found that C/EBP homologous protein (CHOP), a key signaling protein of ER stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with VacA. The effect of VacA on CHOP induction was significantly enhanced by co-incubation with ammonium chloride. Phosphorylation of eukaryotic initiation factor 2 (eIF2)-alpha, which is known to occur downstream of the ER stress sensor PKR-like ERlocalized eIF2-alpha kinase (PERK) and to regulate CHOP expression, was also observed following incubation with VacA in the presence of ammonium chloride. Knockdown of CHOP by siRNA resulted in inhibition of VacA-induced apoptosis. Further studies showed that silencing of the PERK gene with siRNA attenuated VacA-mediated phosphorylation of eIF2-alpha, CHOP induction, expression of BH3-only protein Bim and Bax activation, and cell death induced by VacA with ammonium chloride, indicating that ER stress may lead to mitochondrial dysfunction during VacA-induced toxicity. Activation of ER stress and up-regulation of BH3-only proteins were also observed in human H. pylori-infected gastric mucosa. Collectively, this study reveals a possible association between VacA-induced apoptosis in gastric epithelial cells, and activation of ER stress in H. pylori-positive gastric mucosa., PLoS ONE, 8(12), e82322; 2013

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that promotes the development of T-helper lymphocyte 1 (Th1). Chronic gastritis induced by Helicobacter pylori is considered a Th1-mediated process. IL-12 levels in gastric biopsy samples of H. pylori-infected patients are higher than in those of uninfected individuals, but the cellular source of IL-12 remains elusive. IL-12 staining was detected in mucosal epithelial cells, lymphocytes, and macrophages in specimens of patients with H. pylori-positive gastritis. Therefore, we investigated IL-12 p40 mRNA induction by H. pylori in gastric epithelial cells and T cells. Although cag pathogenicity island (PAI)-positive H. pylori induced IL-12 p40 mRNA expression, an isogenic mutant of the cag PAI failed to induce it in both cell types. Supernatants from H. pylori cultures and H. pylori VacA induced IL-12 p40 mRNA expression in T cells but not in epithelial cells. The activation of the IL-12 p40 promoter by H. pylori was mediated through NF-κB. The transfection of IκB kinase and NF-κB-inducing kinase dominant-negative mutants inhibited H. pylori-induced IL-12 p40 activation. Inhibitors of NF-κB, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and Hsp90 suppressed H. pylori- and VacA-induced IL-12 p40 mRNA expression. The results indicate that H. pylori induces IL-12 p40 expression by the activation of NF-κB, phosphatidylinositol 3-kinase, and p38 mitogen-activated protein kinase. Hsp90 is also a crucial regulator of H. pylori-induced IL-12 p40 expression. In addition to the cag PAI, VacA might be relevant in the induction of IL-12 expression and a Th1-polarized response only in T cells.

Udgivelsesdato: 2008-Apr-1, Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IkappaBalpha ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca(2+) entry (SKF96365), and intracellular Ca(2+) channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca(2+) chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca(2+) in mediating activation of MAPK and the canonical NF-kappaB pathway. VacA stimulated translocation of NF-kappaBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-kappaB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-kappaB site, suggesting direct involvement of the ATF-2/CREB binding region or NF-kappaB-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK via intracellular Ca(2+) release, leading to activation of the transcription factors, ATF-2, CREB, and NF-kappaB.

Udgivelsesdato: 2007-Sep, Treatment of AZ-521 cells with Helicobacter pylori VacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E(2) (PGE(2)) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE(2) production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-kappaB or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE(2) production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

Udgivelsesdato: 2006-Dec, 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.