Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin-8

Abstract

SummaryTo determine the effect of Helicobacter pylori CagA expression on interleukin-8 (IL-8) induction in AGS cells, cagA and five of its fragments from strains 147A and 147C that vary in the 3 ′ ′ ′ ′ repeat region were cloned into the eukaryotic expression plasmid pSP65SR α α α α . IL-8, but not RANTES or IL-I β β β β , levels were increased in AGS cells transfected with 147A-cagA and to a greater extent with 147C-cagA , compared with negative controls. The 5 ′ ′ ′ ′ b fragment from the two strains had similar effects, but the 3 ′ ′ ′ ′ d and e fragments from 147C CagA had greater effects than those from 147A-CagA. When the Western CagA-specific sequence (WSS) of 147C-cagA was replaced with East Asian CagA-specific sequence (ESS) and cloned into pSP65SR α α α α as an East/West chimera, there was no significant effect on IL-8 production. Use of specific inhibitors indicates that Src kinase activation, and the mitogen-activated protein (MAP) kinase and NF-κ κ κ κ B pathways are the major intermediates for CagA effects on IL-8 induction, but the p38 MAP kinase pathway has little effect. These results indicate a direct CagA effect on IL-8 induction by gastric epithelial cells, and indicate signal pathway loci that can be targeted for amelioration.

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

“…1A). Differences in the mobility of the CagA protein reflect the previously observed microheterogeneity in the cagA open reading frame (31,32). These results confirm that the CagA molecules we study bind to SHP2, as determined by tyrosine phosphorylation status.…”

“…Bacteria, Cell Culture, and Co-incubation with H. pylori-A pair of naturally occurring isogenic cagA strains (147C, with a 3Ј tyrosine phosphorylation motif, and 147A, without the motif) isolated simultaneously from the same host have been described (31)(32)(33) …”

“…However, the molecular mechanisms involved are incompletely understood. In this study, we used a pair of isogenic strains (147A and 147C) with identical CagA proteins, differing only by the presence of a single C-EPIYA motif (31)(32)(33) to elucidate the mechanisms by which tyrosine-phosphorylated CagA affects signaling within gastric epithelial cells. Because the C-EPIYA ؉ (phosphorylated) CagA (from 147C) has greater binding affinity to SHP2, as shown previously (23,34,36) and confirmed in our co-immunoprecipitation experiments, the extent of this interaction could affect downstream signaling pathways.…”

Variations in the multimerization region of the Helicobacter pylori cytotoxin CagA affect virulence

“…The vector containing GFP without a CagA fragment was used as a control. Previous studies have demonstrated that the C-terminal CagA fragment exhibits CagA activity, including the induction of cell elongation and interleukin-8 secretion in AGS cells (14,18). This fragment, which contains the terminal third of the CagA protein, was observed to induce greater cell elongation than the complete CagA protein (18).…”

“…This CagA C-terminal fragment was amplified by polymerase chain reaction and cloned into the pAcGFP1-C3 vector (Clontech Laboratories, Inc., Mountainview, CA, USA) downstream to and in frame with green fluorescent protein (GFP). The primers used were as follows: CagA-DF, GGA TTG AGC TCA TGG GAA CAG GCG ATT TCA GTG GGG TAG and CagA-DR, CCG AGG TAC CTT AAG ATT TTT GGA AAC CAC CTT TTG TA (14). The CagA-DF and CagA-DR primers contained SacI and KpnI restriction sites (italicized above), respectively.…”