Background
Tyrosine phosphorylation of Helicobacter pylori cytotoxin-associated protein (CagA) of in gastric epithelial cells is reported. Phosphorylated CagA trigger downstream cellular signaling that alters cell morphology and proliferation and induce pro-inflammatory process. Translocated CagA involves epithelial-to-mesenchymal transition and disruption of cellular apical-junction complex in MDCK cells. These events may play important role in the development of malignancy. To clarify these issues, our aims in this study are: 1) to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from differnet patients with gastric adenocarcinoma and gastritis, 2) to analyze the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation, 3) to establish primary human gastric epithelial cell for better understanding effects of CagA protein in non-cancer epithelial cells and 4) to observe cellular junction change in primary human gastric epithelial cell but not non-gastric epithelial cells .

Methods

Fifty-eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. To elucidate whether events of CagA translocation and phosphorylation take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. Tight junction proteins: ZO-1 and occludin changes were observed by confocal microscopy.

Results

Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients, (p<0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Our results also demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeleton rearrangement and the disruption of tight junctions in primary gastric epithelial cells.

Conclusions

CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation. The establishment of primary culture gastric epithelial cells also provides direct evidence of the modulation of gastric epithelial cells by CagA translocation, and advances our understanding of the carcinogenesis of H. pylori infection.