Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.
ABSTRACT 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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ABSTRACT: Outer membrane vesicles (OMVs) are constitutively produced by all Gram-negative bacteria. OMVs form when buds from the outer membrane (OM) of cells encapsulate periplasmic material and pinch off from the OM to form spheroid particles approximately 10 to 300nm in diameter. OMVs accomplish a diversity of functional roles yet the OMV's utility is ultimately determined by its unique composition. Inclusion into OMVs may impart a variety of benefits to the protein cargo, including: protection from proteolytic degradation, enhancement of long-distance delivery, specificity in host-cell targeting, modulation of the immune response, coordinated secretion with other bacterial effectors, and/or exposure to a unique function-promoting environment. Many enriched OMV-associated components are virulence factors, aiding in host cell destruction, immune system evasion, host cell invasion, or antibiotic resistance. Although the mechanistic details of how proteins become enriched as OMV cargo remain elusive, recent data on OM biogenesis and relationships between LPS structure and OMV-cargo inclusion rates shed light on potential models for OM organization and consequent OMV budding. In this review, mechanisms based on pre-existing OM microdomains are proposed to explain how cargo may experience differing levels of enrichment in OMVs and degrees of association with OMVs during extracellular export. This article is part of a Special Issue entitled: Protein Trafficking & Secretion.Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor
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ABSTRACT: Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH (‘activation’) results in structural changes in the protein and a marked enhancement of its cell-vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]-VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]-VacA to cells. Binding of acid-activated [125I]-VacA to cells at 4°C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non-specifically or to an abundant, low-affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS–PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time-dependent process at 37°C but not at 4°C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]-VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2-deoxy-d-glucose. These results indicate that acid or alkaline pH-induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.Molecular Microbiology 01/2002; 37(2):433 - 442. · 5.03 Impact Factor
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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 01/2013; 8(12):e82322. · 3.53 Impact Factor