Amino acid deletions at positions 893 and 894 of cytotoxinassociated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori(H.pylori)persistently colonizes the human gastric mucosa in more than 50%of the global population,leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma.Cytotoxin-associated gene A(CagA)protein,an important oncoprotein,has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus,which play crucial roles in pathogenesis.Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894.The cagA gene was amplified and mutated into cagA-NT and cagA-NE(sequence characteristics of strains from nongastric cancer patients),cloned and inserted into pAdtrack-CMV,and then transfected into AGS cells.The expression of cagA and its mutants was examined using realtime polymerase chain reaction and Western blotting,cell elongation via cell counting,F-actin cytoskeleton visualization using fluorescence staining,and interleukin-8(IL-8)secretion via enzyme-linked immunosorbent assay.RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE(40.88±3.10 vs 32.50±3.17,P<0.001 and 40.88±3.10 vs 32.17±3.00,P<0.001)at 12 hours after transfection.At 24 hours,pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT(46.02±2.12 vs 53.90±2.10,P<0.001 and 46.02±2.12 vs 51.15±3.74,P<0.001).The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE(27.54±17.37 vs 41.51±11.90,P<0.001 and 27.54±17.37 vs 41.39±14.22,P<0.001)at 12 hours after transfection.Additionally,pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection.CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity,which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H.pylori.