cDNA array analysis of cag pathogenicity island-associated Helicobacter pylori epithelial cell response genes

Abstract

Helicobacter pylori strains containing the cag pathogenicity island (PAI) induce NF-kappaB activation and interleukin-8 secretion in gastric epithelial cells. The aim of this study was to investigate changes in epithelial gene expression induced by cag PAI-positive and -negative strains of H. pylori using high-density cDNA array hybridization technology. Radio-labeled cDNA prepared from H. pylori-infected Kato 3 gastric epithelial cells was hybridized to high-density cDNA arrays to identify changes in epithelial gene expression compared to noninfected controls. In vivo expression of selected, differentially expressed genes was examined by reverse transcription-PCR analysis of H. pylori-positive and -negative gastric mucosa. Screening of ca. 57,800 cDNAs identified 208 known genes and 48 novel genes and/or expressed sequence tags of unknown function to be differentially expressed in Kato 3 cells following H. pylori infection. Marked differences in gene expression profiles were observed following cag PAI-positive and cag PAI-negative infection with 15 novel cDNAs and 92 known genes being differentially expressed. H. pylori was found to change the expression of genes encoding growth factors and cytokine/chemokines and their receptors, apoptosis proteins, transcription factors and metalloprotease-disintegrin proteins (ADAMs), and tissue inhibitors of metalloproteinases. Gastric differential expression of selected known genes (amphiregulin and ADAM 10) and a novel gene (HPYR1) was confirmed in vivo in patients with H. pylori infection. Confirmation of the in vivo expression of selected genes demonstrates the usefulness of this approach for investigating pathogen-induced changes in host gene expression.

FIG. 1

Example of I.M.A.G.E. cDNA array hybridized with epithelial cell-derived probe from H. pylori-infected Kato 3 gastric epithelial cells (A) and a probe derived from uninfected Kato 3 cells (B). The circles highlight a differentially expressed gene.

FIG. 2

Representative RT-PCR with primers for ADAM 10 (A), amphiregulin (AR) (B), and HPYR1 (C) in gastric antral biopsy samples. G3PDH is the control gene GAPDH. Lane L, 100-bp ladder; lanes 1 to 3, H. pylori negative normal mucosa; lanes 4 to 6, mucosa from patients with cagA-negative H. pylori; lanes 7 to 9, mucosa from patients with cagA-positive H. pylori; lane 10, positive control (H. pylori-stimulated Kato 3 gastric epithelial cells); lane 11, negative control. Hp, H. pylori.

FIG. 2

Representative RT-PCR with primers for ADAM 10 (A), amphiregulin (AR) (B), and HPYR1 (C) in gastric antral biopsy samples. G3PDH is the control gene GAPDH. Lane L, 100-bp ladder; lanes 1 to 3, H. pylori negative normal mucosa; lanes 4 to 6, mucosa from patients with cagA-negative H. pylori; lanes 7 to 9, mucosa from patients with cagA-positive H. pylori; lane 10, positive control (H. pylori-stimulated Kato 3 gastric epithelial cells); lane 11, negative control. Hp, H. pylori.

FIG. 2

Representative RT-PCR with primers for ADAM 10 (A), amphiregulin (AR) (B), and HPYR1 (C) in gastric antral biopsy samples. G3PDH is the control gene GAPDH. Lane L, 100-bp ladder; lanes 1 to 3, H. pylori negative normal mucosa; lanes 4 to 6, mucosa from patients with cagA-negative H. pylori; lanes 7 to 9, mucosa from patients with cagA-positive H. pylori; lane 10, positive control (H. pylori-stimulated Kato 3 gastric epithelial cells); lane 11, negative control. Hp, H. pylori.

FIG. 3

Semiquantitative analysis of amphiregulin (AR) in antral and corpus mucosa of H. pylori-positive (Hp +ve) and -negative (Hp-ve) patients. Levels of amphiregulin relative to GAPDH (G3PDH) are indicated on the y axis. H. pylori-positive patients had significantly greater (P < 0.05) amphiregulin mRNA expression in the antrum than did H. pylori-negative patients with normal mucosa. NS, not statistically significant.

FIG. 4

Representative RT-PCR for HPYR1 and GAPDH (G3PDH) in Kato 3 gastric epithelial cells following coculture with cag PAI-positive strain G27 and an isogenic ΔcagM mutant. Cells were harvested for RT-PCR analysis at 45 min and 3, 6, and 24 h postinfection. Lane L, 100-bp ladder; lane -ve, negative.