Helicobacter pylori vacA transcription is genetically-determined and stratifies the level of human gastric inflammation and atrophy

Abstract

Aims: Helicobacter pylori infection is the major cause of peptic ulceration and gastric cancer, and an important virulence determinant is its vacuolating cytotoxin vacA. Previously, we have described allelic variation in vacA which determines toxin activity and disease risk. Here we aimed to quantify vacA mRNA expression in the human stomach, define its genetic determinants and assess how well it predicts gastric pathology.

Methods: Gastric biopsies were donated by 39 patients with H. pylori infection attending for endoscopy at Queen's Medical Centre, Nottingham, UK. Total RNA was extracted, and vacA mRNA quantified by reverse transcriptase quantitative PCR. Separate biopsies were histologically scored for inflammation and atrophy using the updated Sydney system. H. pylori strains were isolated from further biopsies, and the nucleotide sequence upstream of vacA determined.

Results: vacA mRNA levels in human stomachs varied by two orders of magnitude independently of vacA allelic type. Among vacA i1-type (toxic) strains, increased vacA expression was strongly associated with higher grade gastric inflammation (p<0.02), neutrophil infiltration (p<0.005) and the presence of atrophy (p<0.01). A polymorphism at nucleotide +28 near the base of a potential stem-loop structure within the 5' untranslated region was significantly associated with vacA transcript level and inflammation.

Conclusions: Increased gastric vacA expression during H. pylori infection is associated with inflammation and premalignant pathology. The +28 nucleotide within the vacA 5' stem-loop stratifies disease risk among toxic vacA i1-type strains.

Keywords: GASTRIC PATHOLOGY; HELICOBACTER PYLORI; INFLAMMATION; MICROBIAL PATHOGENIC; TOXIN.

Figure 1

vacA mRNA level in the human stomach infected with Helicobacter pylori is associated with inflammation and atrophy. Gastric biopsies were scored for inflammation (A and B), neutrophil infiltration (C and D) and atrophy (E and F) using the updated Sydney system by a qualified histopathologist blind to all data. Relative vacA mRNA levels in separate gastric biopsies were quantified by reverse transcriptase quantitative PCRs using the Pfaffl method with 16S rRNA as a reference gene (arbitrary units). Data were stratified by vacA i1-type (A, C and E; n=25) or vacA s1i1m1, cagA⁺ genotype (B, D and F; n=20). Multiple comparisons for inflammation and neutrophil infiltration grades (A and C) were assessed by Kruskal–Wallis analysis of variance (ANOVA) and multiplicity adjusted pairwise p values calculated by Dunn's test are shown. Pairwise comparisons (B and D–F) were assessed by Mann–Whitney U tests for statistical significance.

Figure 2

Peptic ulcer disease (PUD) does not correlate with in vivo vacA mRNA level. Pathological findings were recorded by a qualified gastroenterologist during upper gastrointestinal endoscopy for dyspeptic symptoms. Patients with past or present gastric or duodenal ulcers or erosions were defined as a PUD group. Patients showing no pathology were defined as normal. Patients taking proton pump inhibitors or antibiotics in the 2 weeks preceding endoscopy were excluded from the study. Relative vacA mRNA levels in gastric biopsies were quantified by reverse transcriptase quantitative PCRs with 16S rRNA as a reference gene (arbitrary units). Data were assessed by Mann–Whitney U tests for statistical significance.

Figure 3

A predicted stem-loop sequence is present near the start of the vacA 5′ untranslated region (UTR). (A) The intergenic region between the 3′ end of the upstream gene cysS (diagonal hatched box) and the start of the vacA open reading frame (white box) is shown with the −35 and −10 consensus promoter sequence positions, the transcriptional start site (+1) and the 5′ UTR (thick line) indicated. (B) The predicted mRNA fold of a potential stem-loop sequence located at +4 to +30 in the vacA 5′ UTR, with a G/A polymorphism at position +28 shown.

Figure 4

A natural G/A polymorphism within a potential stem-loop in the vacA 5′ untranslated region (UTR) is associated with in vivo vacA mRNA level, inflammation score and neutrophil infiltration. The nucleotide at position +28 in the vacA 5′ UTR (A or G) was determined by sequencing for the infecting strain from 27 patients. Gastric biopsies were used to quantify the relative in vivo vacA mRNA level by reverse transcriptase quantitative PCRs for all 27 patients (A), and scored for inflammation (B) and neutrophil infiltration (C) using the Sydney system for 19 patients infected with i1-type strains. Data were assessed by Mann–Whitney U tests for statistical significance.