Differential Proteomics of Helicobacter pylori Associated with Autoimmune Atrophic Gastritis

Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which development can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, such as duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluorescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG, to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to compare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC, whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-assisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common between DU- and GC-HP (peroxiredoxin, heat shock protein 70 [HSP70], adenosine 5'-triphosphate [ATP] synthase subunit α, flagellin A). Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common protein expression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposed as putative markers of AAG-associated HP. Other comparative studies by two-dimensional maps integrated with functional genomics of candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains.

Description of interactions involving known pathogenic factors such as: (i) urease with the heat shock protein GroEL or the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island (PAI) CagA protein with the DNA gyrase GyrA, as well as some partners of TsaA, and a peroxide reductase/stress-dependent molecular chaperone. 4 cag PAI proteins; numerous OMPs; the vacuolating cytotoxin VacA; other potential virulence factors, and few ribosomal proteins were detected in the structurebound fraction. In contrast; catalase KatA; γ-glutamyltranspeptidase Ggt, and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. Among the secreted proteins were: several redox-active enzymes; various components of the flagellar apparatus; 6 putative oxidoreductase; 3 fragments of the vacuolating toxin VacA; the serine protease and chaperone HtrA, and 8 previously uncharacterized proteins.
9 proteins previously shown to be surfaceexposed; 7 proteins virulence-associated, and 11 highly immunogenic in infected patients.
60 proteins, such as urease-subunit; 60 kDa chaperonin, and thioredoxin. New proteins identified included type I restriction enzyme R protein; type IIS enzyme R; M protein, and DNA polymerase III-subunit.

Strain17875
To systematically analyze HP proteome. Up to 1800 proteins separated, of which of 152 proteins identified and organized in a 2-DE database accessible via the Internet. To identify and characterize the cellsurface proteins expressed by HP in order to further develop vaccines. 40 proteins from a detergent-solubilized HP preparation were identified, and over one-third were membrane or membrane-associated. Pfr), memberof the tricarbon acid cycle (isocitrate dehydrogenase Idh; fumarate reductases FrdA, FrdB and FldA; aconitate hydratase 2 AcnB) and heat shock proteins (chaperone and heat shock protein GroEL and heat shock protein ClpB).
18 GC biomarkers were selected and 3 of them were purified and identified as: a neutrophilactivating protein NapA, a RNA-binding protein, and a DNA-binding histone-like protein HU.
4 proteins were present only on the 2D map of the strain isolated from DU patients (6phosphogluconolactonase; S-ribosylhomocysteine lyase; aliphatic amidase; and hypothetical protein HP0697), while 5 were specific for that one isolated from GC patients (hypothetical protein HP0958; transcription elongation factor greA; quinone reactive Ni/Fe hydrogenase large subunit; and NADPH-flavin oxidoreductase RecA protein).
OipA positive status was significantly associated with the presence of DU and GC, high HP density, and severe neutrophil infiltration. While, SabA positive status was associated with GC, intestinal metaplasia, and corpus atrophy, and negatively associated with DU and neutrophil infiltration.
8 proteins were identified but not specific of any among the analyzed disease (alkyl hydroperoxide reductase TsaA, inorganic pyrophosphatase PpaA, unknown function, two 3dehydroquinase type II AroD, 3-ketoacid-coenzyme A transferase subunit BScoB, and elongation factor P). the number indicates the gel in the Decyder workflow; b) the number indicates the AAG, GC or DU patient; c) AAG, autoimmune atrophic gastritis; GC, gastric cancer; DU, duodenal ulcer; d) A, antrum; C, corpus; e) Cye-Dye, cyanine dye.