Host cell responses to genotypically similar Helicobacter pylori isolates from United States and Japan

Abstract

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA(+) vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA(+) vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA(+) vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.

FIG. 1.

IL-8 levels in culture supernatants from AGS cells coincubated with H. pylori cells. (A) Sixty-three cagA⁺ vacAs1/m1 and eight control cagA-negative vacAs2/m2 isolates were studied. IL-8 secretion by AGS cells was not significantly different between U.S. (n = 24) and Japanese (n = 39) cagA⁺ vacAs1/m1 isolates, but both were significantly (P < 0.001) greater than that for the cagA-negative vacAs2/m2 isolates. Horizontal lines indicate median values. (B) IL-8 levels according to disease outcome. Among the Japanese strains, those isolated from patients with DU induced the highest IL-8 values, but this trend was not present among U.S. strains. Horizontal lines indicate median values.

FIG. 2.

(A) Apoptosis of AGS cells following coculture with H. pylori cells, according to clinical diagnosis of patients from whom strains were isolated. Induction of DNA fragmentation was not significantly different between U.S. (n = 24) and Japanese (n = 39) cagA⁺ vacAs1/m1 isolates. Fragmentation was highest following coculture with Japanese isolates from GU patients (P < 0.005 for all comparisons with other isolates from Japanese patients). Horizontal lines indicate median values. For further analyses, the values for the eight isolates from GU patients that induced the highest apoptosis scores are boxed. (B) Comparison of histological findings present in the eight Japanese patients with GU from whom the isolates induced the highest apoptosis scores (columns A) and the other 31 Japanese patients (columns B), including nine other GU patients. ○, strain isolated from a patient with GU; •, strain isolated from a patient without GU. a and b, comparison of group A patients and group B patients (a, P < 0.05; b, P < 0.001). c, comparison of group A patients and group B GU patients (P < 0.01).

FIG. 2.

(A) Apoptosis of AGS cells following coculture with H. pylori cells, according to clinical diagnosis of patients from whom strains were isolated. Induction of DNA fragmentation was not significantly different between U.S. (n = 24) and Japanese (n = 39) cagA⁺ vacAs1/m1 isolates. Fragmentation was highest following coculture with Japanese isolates from GU patients (P < 0.005 for all comparisons with other isolates from Japanese patients). Horizontal lines indicate median values. For further analyses, the values for the eight isolates from GU patients that induced the highest apoptosis scores are boxed. (B) Comparison of histological findings present in the eight Japanese patients with GU from whom the isolates induced the highest apoptosis scores (columns A) and the other 31 Japanese patients (columns B), including nine other GU patients. ○, strain isolated from a patient with GU; •, strain isolated from a patient without GU. a and b, comparison of group A patients and group B patients (a, P < 0.05; b, P < 0.001). c, comparison of group A patients and group B GU patients (P < 0.01).

FIG. 3.

Effect of mutagenizing HP0638 and the cag island on AGS cell responses. (A) Confirmation of mutations interrupting HP0638 and/or deleting the cag island. PCR was performed with primers AN9260 and AN9261 (HP0638), OP1690 and OP1691 (cag site), and C3814 and C3815 (picB) to ascertain mutant construction. W, wild type; ΔC, cag island deleted; ΔO, HP0638 interrupted; ΔCO, HP0638 interrupted and cag island deleted. (B) IL-8 levels in culture supernatants from AGS cells coincubated with H. pylori wild-type and mutant strains. Data represent the means and standard deviations from three experiments. *, cagA⁺ control strain 88-23, **, cagA-negative control strain 88-22. (C) Induction of tyrosine-phosphorylated CagA protein by H. pylori wild-type and mutant strains in AGS cells.