Dataset Information

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

ABSTRACT: Helicobacter pylori VacA is a secreted pore-forming toxin that induces cell vacuolation in vitro and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NH4Cl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the hypothesis that NH4Cl augments VacA toxicity by altering the intracellular trafficking of VacA or inhibiting intracellular VacA degradation. We observed VacA colocalization with LAMP1- and LC3-positive vesicles in both the presence and absence of NH4Cl, indicating that NH4Cl does not alter VacA trafficking to lysosomes or autophagosomes. Conversely, we found that supplemental NH4Cl significantly increases the intracellular stability of VacA. By conducting experiments using chemical inhibitors, stable ATG5 knockdown cell lines, and ATG16L1 knockout cells (generated using CRISPR/Cas9), we show that VacA degradation is independent of autophagy and proteasome activity but dependent on lysosomal acidification. We conclude that weak bases like ammonia, potentially generated during H. pylori infection by urease and other enzymes, enhance VacA toxicity by inhibiting toxin degradation.

SUBMITTER: Foegeding NJ

PROVIDER: S-EPMC6434121 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

Foegeding Nora J NJ Raghunathan Krishnan K Campbell Anne M AM Kim Sun Wook SW Lau Ken S KS Kenworthy Anne K AK Cover Timothy L TL Ohi Melanie D MD

Infection and immunity 20190325 4

Helicobacter pylori VacA is a secreted pore-forming toxin that induces cell vacuolation in vitro and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NH4Cl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the h ...[more]

PMID: 30692181

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Project description:Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA(+)/VacA(+)H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.

Project description:Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of ?-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and ?ggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than ?ggt. By using semi-quantitative neutral red uptake assay, we next showed that ?ggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of ?ggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of ?ggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.

Dataset Information

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

Publications

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

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