Duodenal ulcer promoting gene of Helicobacter pylori

Abstract

Background & aims: Identification of a disease-specific H pylori virulence factors predictive of the outcome of infection remains unachieved.

Methods: We used the polymerase chain reaction and Southern blot to compare the presence of 14 vir homologue genes with clinical presentation of H pylori infection, mucosal histology, and mucosal interleukin (IL)-8 levels.

Results: We examined 500 H pylori strains from East Asia and South America, including 120 with gastritis, 140 with duodenal ulcer (DU), 110 with gastric ulcer (GU), and 130 with gastric cancer. Only 1 gene that encompassed both jhp0917 and jhp0918 called dupA (duodenal ulcer promoting gene) was associated with a specific clinical outcome. dupA was present in 42% of DU vs. 21% of gastritis (adjusted odds ratio [OR] = 3.1, 95% confidence interval [CI]: 1.7-5.7). Its presence was also associated with more intense antral neutrophil infiltration and IL-8 levels and was a marker for protection against gastric atrophy, intestinal metaplasia, and gastric cancer (OR for gastric cancer = 0.42, 95% CI: 0.2-0.9 compared with gastritis). In vitro studies in gastric epithelial cells using dupA -deleted and -complemented mutants showed that the dupA plays roles in IL-8 production, in activation of transcription factors responsible for IL-8 promoter activity, and in increased survivability at low pH.

Conclusions: dupA is a novel marker associated with an increased risk for DU and reduced risk for gastric atrophy and cancer. Its association with DU-promoting and -protective effects against atrophy/cancer was evident in both Asian and Western countries.

Figure 1

Univariate analysis showing the relationship between the jhp0917-0918 gene (dupA) and the gastric mucosal histology. The scores (0: absent/normal to 5: maximal intensity) for neutrophil infiltration, intestinal metaplasia, and atrophy scores are presented separately in relation to clinical presentation in 1 East Asian and 1 Western country (Japan and Colombia, respectively). The strains from Japanese patients included 34 dupA-positive strains (7 from gastritis, 11 from DU, 13 from GU, and 3 from gastric cancer) and 124 dupA-negative strains (42 from gastritis, 19 from DU, 37 from GU, and 26 from gastric cancer). Strains from Colombia included 50 dupA-positive strains (14 from gastritis, 21 from DU, 9 from GU, and 6 from gastric cancer) and 87 dupA-negative strains (22 from gastritis, 17 from DU, 15 from GU, and 33 from gastric cancer). The data are expressed as mean ± standard error.

Figure 2

In vivo gastric mucosal IL-8 levels and the jhp0917-0918 status. Mucosal IL-8 levels from biopsy specimens measured by ELISA are presented on the left panel, and adjusted IL-8 levels for H pylori density (IL-8 per H pylori) are presented on the right panel. IL-8 in the supernatant was assayed in duplicate. Data are expressed as mean ± standard error.

Figure 3

Schematic structure of the jhp0917 gene and jhp0918 gene in strain J99 and that of the dupA gene in the clinical isolates. All 24 clinical isolates examined contains “C or T” insertion after position 1385 to produce 1 continuous open-reading frame homologue to the virB4 gene. We denote the gene containing the sequences of the jhp0917 gene and jhp0918 as the duodenal ulcer-promoting (dupA) gene.

Figure 4

In vitro IL-8 production from MKN45 cells cocultured with wild-type strain C142 and its dupA-deleted mutant and dupA-complemented mutant and clinical isolates is shown. H pylori (MOI of 100) was cocultured with MKN45 cells for 20 hours. Experiments were performed at least 4 times for the wild type and its isogenic mutants and were performed in duplicate for clinical samples. IL-8 in the supernatant was assayed by an ELISA in duplicate. Data are expressed as mean ± standard error.

Figure 5

Shows the luciferase reporter activity of ISRE, NF-κB, AP-1, and CRE associated with H pylori infection. Luciferase reporter gene assay was performed as previously described at least 4 times for (A) wild type and its isogenic mutant and was performed in duplicate for (B) clinical samples. Luciferase activity was normalized by renilla luciferase vector DNA (Promega), and normalized luciferase activity (fold induction) is presented. Data are expressed as mean ± standard error of normalized luciferase activity.

Figure 6

Susceptibility of H pylori to exposure to low pH. Log10 kill represents the difference in viable H pylori before and after exposure to low pH. Assays were performed at least 4 times. Data are expressed as mean ± standard error.