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Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

ABSTRACT: Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

SUBMITTER: Asahi M

PROVIDER: S-EPMC2195829 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Asahi M M Azuma T T Ito S S Ito Y Y Suto H H Nagai Y Y Tsubokawa M M Tohyama Y Y Maeda S S Omata M M Suzuki T T Sasakawa C C

The Journal of experimental medicine 20000201 4

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced ...[more]

PMID: 10684851

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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:BackgroundHelicobacter pylori colonises the stomach of approximately 50% of the global population. Cytotoxin-associated gene A protein (CagA) is one of the important virulent factors responsible for the increased inflammation and increases the risk of developing peptic ulcers and gastric carcinoma. The cytokine interleukin-6 (IL-6) has particularly important roles in the malignant transformation of gastric and intestinal epithelial cells as it is upregulated in H. pylori-infected gastric mucosa. In this study, we investigated the underlying mechanisms of CagA-induced IL-6 up-regulation during H. pylori infection. AGS cells, a human gastric adenocarcinoma cell line, lacking eEF1A1 were infected with CagA+ H. pylori (NCTC11637), CagA- H. pylori (NCTC11637ΔcagA), or transduced by Ad-cagA/Ad-GFP. The expression and production of IL-6 were measured by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The interactions among CagA, eukaryotic translation elongation factor 1-alpha 1 (eEF1A1), protein kinase Cδ (PKCδ), and signal transducer and activator of transcription 3 (STAT3) were determined by western blot or co-immunoprecipitation.ResultsDuring H. pylori infection, CagA-M (residues 256‒871aa) was found to interact with eEF1A1-I (residues 1‒240aa). NCTC11637 increased the expression of IL-6 in AGS cells compared with NCTC11637ΔcagA whereas knockdown of eEF1A1 in AGS cells completely abrogated these effects. Moreover, the CagA-eEF1A1 complex promoted the expression of IL-6 in AGS cells. CagA and eEF1A1 cooperated to mediate the expression of IL-6 by affecting the activity of p-STATS727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3S727 and induction of IL-6 by CagA.ConclusionsCagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.

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Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Publications

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

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