Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells

Institute of Human Physiology, University of Pavia and Istituto Ricovero e Cura a Carrattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy.
The American journal of physiology (Impact Factor: 3.28). 11/1998; 275(4 Pt 1):G681-8.
Source: PubMed


The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.

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    • "Late trafficking of VacA inside intoxicated cells is not very well-researched. VacA is taken up into endocytic compartments that were shown to be GEECs (GIP-AP-enriched early endosomal compartments (Gauthier et al., 2005)), and is then routed through early and late endosomes, finally reaching compartments carrying both endosomal and lysosomal markers (Li et al., 2004;Molinari et al., 1997), but avoiding degradation (Sommi et al., 1998). After four hours, VacA is found in vesicles scattered throughout the cytosol (Fig. 4A, first column), which become enlarged VCVs in the presence of weak bases (Morbiato et al., 2001). "
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    ABSTRACT: The human pathogen Helicobacter pylori colonises half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion-selective channels in the endosome membrane that cause the compartment to swell, but the composition and purpose of the resulting VacA-containing vacuoles (VCVs) is still unknown. VacA exerts influence on the host immune response in various ways, including inhibition of T-cell activation and proliferation and suppression of the host immune response. In this study, for the first time the composition of VCVs from T-cells was comprehensively analysed to investigate VCV function. VCVs were successfully isolated via immunomagnetic separation and the purified vacuoles were analysed by mass spectrometry. We detected a set of 122 VCV-specific proteins, implicated amongst others in immune response, cell death and cellular signalling processes, all of which VacA is known to influence. One of the individual proteins studied further was stromal interaction molecule (STIM1), a calcium sensor residing in the endoplasmic reticulum (ER) important in store-operated calcium entry (SOCE). Live cell imaging microscopy data demonstrated co-localisation of VacA with STIM1 in the ER and indicated that VacA may interfere with the movement of STIM1 towards the plasma membrane-localised calcium release activated calcium (CRAC) channel protein ORAI1 in response to Ca(2+) store depletion. Furthermore, VacA inhibited the increase of cytosolic free Ca(2+) in the Jurkat E6-1 T-cell line and human CD4(+) T cells. The presence of VacA in the ER and its trafficking to the Golgi apparatus was confirmed in HeLa cells, identifying these two cellular compartments as novel VacA target structures. This article is protected by copyright. All rights reserved.
    Cellular Microbiology 06/2015; 17(12). DOI:10.1111/cmi.12474 · 4.92 Impact Factor
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    • "In this regard, VacA-induced vacuolation is inhibited by the V-ATPase activity inhibitor, Bafliomycin A1 [17]. In contrast, weak bases including NH4Cl that can be produced by the high urease activity of H. pylori significantly potentiates VacA-mediated vacuole formation in cultured cells [5], [18], [19]. "
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    ABSTRACT: 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 ER-localized 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 12/2013; 8(12):e82322. DOI:10.1371/journal.pone.0082322 · 3.23 Impact Factor
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    • "Vacuolating toxin, VacA, has been detected closely associated with the membrane of H. pylori vesicles (Keenan et al. 2000). Vesicular VacA is stable in the eukaryotic cell, and its toxicity, which is urease dependent, can be induced days following internalization (Sommi et al. 1998). Since urease is secreted by H. pylori into the gastric mucosa but vesicles do not carry urease (Keenan et al. 2000), this may be part of an elaborately regulated virulence mechanism that is not yet fully understood. "
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    ABSTRACT: Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.
    Genes & Development 12/2005; 19(22):2645-55. DOI:10.1101/gad.1299905 · 10.80 Impact Factor
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