Objective To investigate the effects of cyto toxin associated gene A protein(cagA)gene on protein expression in outer membrane vesicles(OMVs)by isolating,identifying and comparing differentially expressed proteins(DEPs)in cagA,positive Helicobacter pylori(H.pylori),East Asian strain GZ7/cagA+and cagA knockout strain GZ7/△cagA-derived OMVs.Methods The OMVs of GZ7/△cagA and GZ7/cagA+were extracted by ultracentrifugation,and their morphology and particle size were identified by transmission electron microscopy and nanoparticle tracking technology.The expression of cagA protein in the two groups of OMVs was verified by Western blot,and the proteomics of OMVs was analyzed.After quality control analysis and principal component analysis of proteomic data,DEPs were screened under the screening conditions of up-regulated protein fold change(FC)＞2.0,down-regulated protein FC＜0.5,FDR ≤ 0.05,and bioinformatics analysis of DEPs was performed using OmicsBean online tool,Gene Ontology and KOBAS.The localization of OMVs cells in cells was identified by immunofluorescence,and the cell activity was detected by real-time cell analyzer(RTCA).Results The successful separation and purification of OMVs were confirmed by electron microscopy and particle size.Proteome analysis showed that there were a total of 79 DEPs in the GZ7/cagA+-OMVs group compared with the GZ7/△cagA-OMVs group,of which 38 proteins were down-regulated and 41 proteins were up-regulated.Bioinformatics analysis showed that DEPs were mainly related to metabolic pathways such as pyruvate metabolism,propionic acid metabolism,glycolysis/gluconeogenesis and citric acid cycle.Immunofluorescence and RTCA confirmed that H.pylori-derived OMVs could enter cells and locate in mitochondria and inhibit cell proliferation.Conclusion The present study demonstrates that cagA can affect the protein composition of OMVs secreted by H.Pylori and DEPs may promote the colonization and pathogenicity of cagA*H.pylori on gastric mucosa.
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