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.

CagA EPIYA polymorphisms in Colombian Helicobacter pylori strains and their influence on disease-associated cellular responses

AIM: To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori ) strains from Colombia on the host cell biology. METHODS: Eighty-four H. pylori-cagA positive strains with different Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs patterns, isolated from patients with gastritis (n=17), atrophic gastritis (n=17), duodenal ulcer (n=16), intestinal metaplasia (n=16) and gastric cancer (n=18), were included. To determine the integrity of the cag pathogenicity island (cag PAI) we evaluated the presence of cagA, cagT, cagE, and cag10 genes by polymerase chain reaction. AGS gastric epithelial cellswere infected with each strain and assayed for translo-cation and tyrosine phosphorylation of CagA by western blot, secretion of interleukin-8 (IL-8) by enzyme-linked immuno sorbent assay after taking supernatants from cocultures and cell elongation induction. For cell elongation quantification, coculture photographs were taken and the proportion of "hummingbird" cells (>15 μm) was determined. RESULTS: Overall 72% (60/84) of the strains were found to harbor a functional cag PAI. Levels of phos-phorylated CagA were significantly higher for isolates from duodenal ulcer than the ones in strains from gas-tritis, atrophic gastritis, intestinal metaplasia and gastric cancer (49.1% ± 23.1% vs 21.1% ± 19.5%, P < 0.02; 49.1% ± 23.1% vs 26.2%±14.8%, P<0.045; 49.1% ± 23.1% vs 21.5% ± 19.5%, P<0.043 and 49.1% ± 23.1% vs 29.5% ± 27.1%, P < 0.047 respectively). We observed variable IL-8 expression levels ranging from 0 to 810 pg/mL and from 8.8 to 1442 pg/mL at 6 h and 30 h post-infection, respectively. cagPAI-defective strains did not induce detectable levels of IL-8 at 6 h post-infection. At 30 h post-infection all strains induced IL-8 expression in AGS cells, although cagPAI-defective strains induced significantly lower levels of IL-8 than strains with a functional cagPAI (57.1 ± 56.6 pg/mL vs 513.6 ± 338.6 pg/mL,P < 0.0001). We did not observe differences in the extent of cell elongation induction between strains with a functional or a defective cagPAI in 6 h cocultures. At 24 h post infection strains with functionalcagPAI showed high diversity in the extent of hummingbird phenotype induction ranging from 7% to 34%. cag PAI defective strains induced significantly lower levels of elongation than strains with functional cag-PAI with one or more than one EPIYA-C motif (15.1% ± 5.2%vs 18.9% ± 4.7%,P < 0.03; and 15.1% ± 5.2% vs 20.0% ± 5.1%, P < 0.003 respectively). No differences were observed in cellular elongation inductionor IL-8 expression among H. pylori strains bearing one and more than one EPIYA-C motifs, neither at 6 h nor at 24 h of coculture. There were no associations between the levels of induction of cell elongation or IL-8 expression and number of EPIYA motifs or pathology. CONCLUSION: The present work describes a lack of association between H. pylori CagA protein EPIYA motifs variations from Colombian isolates and disease-associated cellular responses.

Discovery of unique African Helicobacter pylori CagAmultimerization motif in the Dominican Republic

BACKGROUND Helicobacter pylori(H.pylori)colonizes the human stomach and is a major cause of peptic ulcer disease and gastric cancer.However,although the prevalence of H.pylori is high in Africa,the incidence of gastric cancer is low,and this phenomenon is called to be African enigma.The CagA protein produced by H.pylori is the most studied virulence factor.The carcinogenic potential of CagA is associated with the Glu-Pro-Ile-Tyr-Ala(EPIYA)patterns and CagAmultimerization(CM)motifs.AIM To better understand the EPIYA patterns and CM motifs of the cagA gene.METHODS Gastric mucosal biopsy specimens were obtained from 258 patients with dyspepsia living in the Dominican Republic,from which 120 H.pylori strains were cultured.After the bacterial DNA extraction,the EPIYA pattern and CM motif genotypes were determined using a polymerase chain reaction-based sequencing.The population structure of the Dominican Republic strains was analyzed using multilocus sequence typing(MLST).Peptic ulcer disease and gastric cancer were identified via endoscopy,and gastric cancer was confirmed by histopathology.Histological scores of the gastric mucosa were evaluated using the updated Sydney system.RESULTS All CagA-positive strains carried the Western-type CagA according to the identified EPIYA patterns.Twenty-seven kinds of CM motifs were observed.Although the typical Western CM motif(FPLKRHDKVDDLSKVG)was observed most frequently,the typical East Asian CM motif(FPLRRSAAVNDLSKVG)was not observed.However,“FPLRRSAKVEDLSKVG”,similar to the typical East Asian CM motif,was found in 21 strains.Since this type was significantly more frequent in strains classified as hpAfrica1 using MLST analysis(P=0.034),we termed it Africa1-CM(Af1-CM).A few hpEurope strains carried the Af1-CM motif,but they had a significantly higher ancestral Africa1 component than that of those without the Af1-CM motif(P=0.030).In 30 cagA-positive strains,the"GKDKGPE"motif was observed immediately upstream of the EPIYA motif in the EPIYA-A segment,and there was a significant association between strains with the hpAfrica1 population and those containing the“GKDKGPE”motif(P=0.018).In contrast,there was no significant association between the CM motif patterns and histological scores and clinical outcomes.CONCLUSION We found the unique African CM motif in Western-type CagA and termed it Africa1-CM.The less toxicity of this motif could be one reason to explain the African enigma.