To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori) strains from Colombia on the host cell biology.
METHODS: Eighty-four H. pylori-cagA positive strains with different Glu-
Pro-Ile-Tyr-Ala (EPIYA) motifs patterns, isolated from patients with
gastritis (n = 17),
atrophic gastritis (n = 17),
duodenal ulcer (n = 16), intestinal
metaplasia (n = 16) and
gastric cancer (n = 18), were included. To determine the integrity of the cag pathogenicity island (cagPAI) we evaluated the presence of cagA, cagT, cagE, and cag10 genes by polymerase chain reaction. AGS gastric epithelial cells were infected with each strain and assayed for translocation and
tyrosine phosphorylation of CagA by western blot, secretion of
interleukin-8 (IL-8) by
enzyme-linked immuno sorbent assay after taking supernatants from cocultures and cell elongation induction. For cell elongation quantification, coculture photographs were taken and the proportion of "hummingbird" cells (> 15 μm) was determined.
RESULTS: Overall 72% (60/84) of the strains were found to harbor a functional cagPAI. Levels of phosphorylated CagA were significantly higher for isolates from
duodenal ulcer than the ones in strains from
gastritis, atrophic gastritis, intestinal
metaplasia and
gastric cancer (49.1% ± 23.1% vs 21.1% ± 19.5%, P < 0.02; 49.1% ± 23.1% vs 26.2% ± 14.8%, P < 0.045; 49.1% ± 23.1% vs 21.5% ± 19.5%, P < 0.043 and 49.1% ± 23.1% vs 29.5% ± 27.1%, P < 0.047 respectively). We observed variable
IL-8 expression levels ranging from 0 to 810 pg/mL and from 8.8 to 1442 pg/mL at 6 h and 30 h post-
infection, respectively. cagPAI-defective strains did not induce detectable levels of
IL-8 at 6 h post-
infection. At 30 h post-
infection all strains induced
IL-8 expression in AGS cells, although cagPAI-defective strains induced significantly lower levels of
IL-8 than strains with a functional cagPAI (57.1 ± 56.6 pg/mL vs 513.6 ± 338.6 pg/mL, P < 0.0001). We did not observe differences in the extent of cell elongation induction between strains with a functional or a defective cagPAI in 6 h cocultures. At 24 h post
infection strains with functional cagPAI showed high diversity in the extent of hummingbird phenotype induction ranging from 7% to 34%. cagPAI defective strains induced significantly lower levels of elongation than strains with functional cagPAI with one or more than one EPIYA-C motif (15.1% ± 5.2% vs 18.9% ± 4.7%, P < 0.03; and 15.1% ± 5.2% vs 20.0% ± 5.1%, P < 0.003 respectively). No differences were observed in cellular elongation induction or
IL-8 expression among H. pylori strains bearing one and more than one EPIYA-C motifs, neither at 6 h nor at 24 h of coculture. There were no associations between the levels of induction of cell elongation or
IL-8 expression and number of EPIYA motifs or pathology.
CONCLUSION: The present work describes a lack of association between H. pylori CagA
protein EPIYA motifs variations from Colombian isolates and disease-associated cellular responses.