| Helicobacter pylori(H.pylori)is accepted as the main pathogen of diseases such as gastritis,duodenal ulcer disease,gastric ulcer disease,gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue(MALT)lymphoma.Over 50%worldwide population are infected with H.pylori.Glycosylation of lipid or protein in gastric mucosa plays an important role in the host-pathogen interaction.The successful adhesion and colonization in gastric mucosa is the key step to establish infection of H.pylori.H.pylori adhesion to human gastric epithelial cells is linked with glycans,in particular,fucosylated glycans.and proteins as receptors of H.pylori adhesins can mediate the adhesion and colonization of H.pylori to gastric epithelial cells.The analysis of carbohydrate chains in the interaction of gastric epithelial cells with H.pylori is crucial to understand the mechanism of H.pylori adhesion to cells.Moreover,HpaA as an outer membrane protein of H.pylori located on the surface and flagellar sheath of H.pylori can bind to sialic acid and plays an important role in bacterial adhesion,but the HpaA-binding receptors on gastric epithelial cells has not been confirmed,and the function and mechanism of H.pylori adhesion and colonization mediated by HpaA are still not clear.Part 1:Analysis of carbohydrate chains in interaction of gastric epithelial cells with H.pyloriIn the study,we used lectin microarrays to detect the carbohydrate chains in gastric epithelial cells(GES-1 and AGS cells)infected by 10 H.pylori strains isolated from different diseases including chronic gastritis,duodenal ulcer and gastric cancer at 4h.Besides,we investigated the time-course expression of fucosyltransferases(FUTs)1-6 in GES-1 cells stimulated with H.pylori strains at 0.5-8h using qRT-PCR technology.The results showed that,in 20 H.pylori-infected cells samples,a total of 17 lectins spe-cific to glycans structure including RS-Fuc,Gal3C-S,Malectin,ACL,MOA,PALa,WFA,STL,PHA-P,PA-IL,PSA,LEA,BC2LCN,MAA,Calsepa,ACG and SBA had increased signals in more than half of samples compared to uninfected cells.Specially,the expression of poly LacNAc and aFuc recognized by Gal3C-S and RS-Fuc respectively was up-regulated in 14 samples compared to uninfected cells.Besides,the expression of Galα3Gal recognized by EEL was down-regulated in both cells infected by H,pylori strains compared to uninfected cells.The results indicated that Fuc,LacNAc,GalNAc,Man and Gal related glycans might be involved in the interaction between H.pylori and gastric epithelial cells.Besides,26 and 4 lectins specific to glycans structure had increased signals in more than half of AGS and GES-1 cell samples infected with H.pylori isolates,respectively,showing that there might be more glycans changed in gastric cancer cells than in normal gastric epithelial cells infected with H.pylori strains.The detection results of the expression levels of FUTs showed that the higher expression of FUT1 and FUT2 was detected in infected GES-1 cells by all H.pylori strains within 2 h.Specially,the expression of FUT2 was higher in H.py/ori-infected GES-1 cells with high fold change of BC2LCN and MOA lectin specific to α-1,2 linked fucose(Fuc)at 4 h.The results suggested that high level of al,2-linked Fuc synthesized by FUT1/2 might play roles in the preliminary stage of H.pylori adhesion,which provided pivotal information to understand the adhesion and colonization of H.pylori to human gastric epithelial cells.Part 2:Identification of functional interactome of gastric cancer cells with H.pylori outer membrane protein HpaA by HPLC-MS/MSTo screen the interaction between HpaA and proteins in gastric epithelial cells,the HpaA protein from H.pylori 26695 fused with a tag(6×His)was expressed and purified successfully,the secondary structure was estimated by the circular dichroism(CD)spectrum,and the purified recombinant protein was used to perform the pull-down assays with gastric cancer cell lines(AGS and SGC-7901)lysates respectively.The pull-down proteins were identified by high performance liquid chromatography tandem mass spectrometry system(HPLC-MS/MS).A total of 9 and 13 proteins related were analyzed from AGS and SGC-7901 cell lysates respectively.ANXA2 were considered as putative HpaA functional partners discovered from lysates of both cell lines with high score and coverage.It is hypothesized that HpaA may be involved in the biological process of regulation of transcription and nucleic acid metabolism during the adhesion of H.pylori to human gastric epithelial cells,and HpaA-binding proteins also be used as targets for the development of anti-adhesion drugs against H.pylori. |
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