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. 2014 Feb 28;20(1):57-71.
doi: 10.2119/molmed.2013.00076.

Differential Proteomics of Helicobacter Pylori Associated With Autoimmune Atrophic Gastritis

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Free PMC article

Differential Proteomics of Helicobacter Pylori Associated With Autoimmune Atrophic Gastritis

Ombretta Repetto et al. Mol Med. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Representative analytical DIGE proteome map of HP from AAG (A) and micro-preparative two-dimensional protein map of HP from AAG (B). (A) Proteins were resolved by IEF over the pI 3–10, followed by 8–16% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and overlaid by DeCyder. After extraction, proteins were labeled with Cy3 and Cy5. In particular, this gel (number 19) refers to samples P102cy3 (antrum) and P103cy5 (corpus) extracted from AAG-associated HP strains. An internal standard comprised of equal amounts of proteins from all samples (AAG, DU and GC) was labeled with Cy2 and included in all gels. (B) Gel number 18 is shown as representative of DIGE maps with proteins extracted from gastric cancer (cy5) comigrated with those extracted from H. pylori associated with DU (cy3). (C) Numbered spots indicate the differentially expressed proteins in AAG-associated HP strains in regards to either GC or DU. Identified spots are listed in Tables 2 and 3. Around 300 μg AAG-associated HP unlabeled protein pooled from amounts of samples was resolved by IEF over the pI range 3–10 NL, followed by 8–16% gradient SDS-PAGE and stained with CBB G-250. MW, Bio-Rad two-dimensional molecular weight standards.
Figure 2
Figure 2
Principal component analysis of HP proteins isolated from AAG, DU and GC patients. (A) Score plot showing an overview of the proteins. Each circle represents a protein. The ellipse represents a 95% significance level. Proteins outliers can either be very strongly differentially expressed proteins or mismatched spots. (B) Loading plot showing an overview of the spot maps from the three groups AAG, DU and GC. Each circle represents a spot map. The ellipse groups the five spot maps from AAG, which can be separated from DU and GC ones and only one DU sample (4Cgel8). AAG-associated HP spot maps are displayed in black; those from GC and DU are displayed in red and blue, respectively. For each spot map, abbreviations indicate the identifier of the patients reported in Table 3 and include the diagnosis (AAG, GC, DU), the stomach location of HP isolation (a, antrum; c, corpus), and a superscript gel number in the Deyder work flow.
Figure 3
Figure 3
Protein contents of selected HP spots and HP gene characterization in the analyzed atrophic autoimmune gastritis-affected patients. (A) Protein content, expressed as log standard volume, as calculated by the DeCyder software, is represented for spots 168, 166 (peroxiredoxin), 57 (HSP70), 40 (F-ATPase subunit) and 42 (flagellin A) in the four patients of Table 3, with “4a” and “4c” above being for antrum and corpus, respectively. (B) The presence (+) or absence (−) of the CagA, CagE and VirB11 genes, together with the polymorphisms for VacA and Hom genes are shown for each patient. *Patient at the first visit without atrophy, but with increasing anti-PC antibody (levels increasing form 1:160 to 1:1,280 after 1 month) and still under follow-up.

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