Protein signature characterizing Helicobacter pylori strains of patients with autoimmune atrophic gastritis, duodenal ulcer and gastric cancer

Abstract

Background: Helicobacter pylori (H. pylori) represents a key factor in the etiology of autoimmune atrophic gastritis (AAG), duodenal ulcer (DU) and gastric cancer (GC). The aim of this study was to characterize the differential protein expression of H. pylori isolated from gastric biopsies of patients affected by either AAG, DU or GC.

Methods: The H. pylori strains were isolated from endoscopic biopsies from the stomach of patients with gastric disease. Protein profiles of H. pylori were compared by two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) for the identification of significantly different spots (Student t-test, p < 0.05).

Results: A total of 47 differentially expressed spots were found between H. pylori isolated from patients with either DU or AAG diseases and those isolated from patients with GC (Anova < 0.05, log fold change >1.5). These spots corresponded to 35 unique proteins. The identity of 7 protein spots was validated after one-dimensional electrophoresis and MS/MS analyses of excised gel portions. In H. pylori isolated from DU-patients a significant increase in proteins with antioxidant activity emerged (AroQ, AspA, FldA, Icd, OorA and ScoB), together with a higher content of proteins counteracting the high acid environment (KatA and NapA). In H. pylori isolated from AAG-patients proteins neutralizing hydrogen concentrations through organic substance metabolic processes decreased (GroL, TrxB and Tuf). In addition, a reduction of bacterial motility (FlhA) was found to be associated with AAG-H. pylori isolates. In GC-H. pylori strains it was found an increase in nucleic acid-binding proteins (e.g. DnaG, Tuf, RpoA, RplU) which may be involved in a higher demand of DNA- and protein-related processes.

Conclusion: Our data suggest the presence of specific protein signatures discriminating among H. pylori isolated from either AAG, DU or GC. Changes in protein expression profiles evaluated by DIGE succeeded in deciphering part of the molecular scenarios associated with the different H. pylori-related gastric diseases.

Keywords: Adenocarcinoma; Autoimmune atrophic gastritis; Comparative proteomics; DIGE; Duodenal ulcer; Gastric cancer; Helicobacter pylori.

Fig. 1

Representative micropreparative two-dimensional (2-D) protein map of Helicobacter pylori associated with duodenal ulcer (B). (A) Around 300 μg of DU-associated H. pylori unlabelled proteins were resolved by IEF over the pI range NL 3–10, followed by 8–16% gradient SDS-PAGE and stained. Numbered spots indicate the H. pylori differentially expressed proteins, which are listed in Table 2

Fig. 2

Principal component analysis of proteome maps of Helicobacter pylori isolates related to autoimmune atrophic gastritis (AAG), duodenal ulcer (DU) and gastric cancer (GC). The loading plots show an overview of the H. pylori spot maps of GC versus DU (a) and GC versus AAG (b). Each circle represents a spot map. AAG, DU and GC associated H. pylori spot maps are displayed in grey, white and black, respectively

Fig. 3

Protein separation by one-dimensional electrophoresis (1-PAGE) of Helicobacter pylori proteins extracted from duodenal ulcer (DU) or gastric cancer (GC) bioptic samples, and validation of the presence of some proteins. H. pylori protein extracts of either DU or GC biopsies were mixed and then aliquoted. After protein separation and gel staining, the 8 gel portions indicated by rectangles and numbers (nr.) were cut and processed for identification by mass spectrometry (LC-MS/MS)

Fig. 4

Protein-protein interaction maps of Helicobacter pylori proteins associated with gastric diseases. a The STRING tool (http://string-db.org) was used for making the networks with all the proteins listed in Table 2. Confidence views are shown, where the thickness of the connecting lines indicates the level of confidence. Stronger associations are represented by thicker lines. Different colors indicate different biological processes: (i) blue (organic substance metabolic process); (ii) green (defense against extreme environment conditions); (iii) yellow (oxidation reduction); (iv) brown (chemical reactions involving various nitrogenous compounds), and (v) red (bacteria motility). b The STRING tool was used for making the networks with the H. pylori proteins up-regulated in gastric cancer versus duodenal ulcer, which were mainly involved in nucleic acid binding (red color)