N-terminal region of Helicobacter pylori CagA induces IL-8 production in gastric epithelial cells via the β1 integrin receptor

Abstract

Introduction. Helicobacter pylori is associated with gastrointestinal disease, most notably gastric cancer. Cytotoxin-associated antigen A (CagA), an important virulence factor for H. pylori pathogenicity, induces host cells to release inflammatory factors, especially interleukin-8 (IL-8). The mechanism by which C-terminal CagA induces IL-8 production has been studied extensively, but little is known about the role of the N-terminus.Aim. To investigate the effect of CagA_303-456aa (a peptide in the N-terminal CagA) on IL-8 production by gastric epithelial cells.Methodology. CagA_303-456aa was produced by a prokaryotic expression system and purified by Strep-tag affinity chromatography. An integrin β1 (ITGB1)-deficient AGS cell line was constructed using the CRISPR/Cas9 technique, and NCTC 11637 cagA and/or cagL knockout mutants were constructed via homologous recombination. The levels of IL-8 production were determined by enzyme-linked immunosorbent assay (ELISA), and p38 and ERK1/2 phosphorylation were examined by Western blot.Results. CagA_303-456aa induced IL-8 expression by AGS cells. IL-8 induction by CagA_303-456aawas specifically inhibited by ITGB1 deficiency. Notably, CagA_303-456aa activated the phosphorylation of both p38 and ERK1/2, and blocking p38 and ERK1/2 activity significantly reduced IL-8 induction by CagA_303-456aa. ITGB1 deficiency also inhibited the activation of p38 phosphorylation by CagA_303-456aa. Finally, experiments in CagA and/or CagL knockout bacterial lines demonstrated that extracellular CagA might induce IL-8 production by AGS cells.Conclusion. Residues 303-456 of the N-terminal region of CagA induce IL-8 production via a CagA_303-456-ITGB1-p38-IL-8 pathway, and ERK1/2 is also involved in the release of IL-8. Extracellular CagA might induce IL-8 production before translocation into AGS cells.

Keywords: CagA; ERK1/2; Helicobacter pylori; IL-8; ITGB1; p38.