Characterization of CagA variable region of Helicobacter pylori isolates from Chinese patients.

AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients. METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20 isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively. RESULTS: About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA(+) strains contained 2-4 tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro. CONCLUSION: CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.

A new subtype of 3' region of cagA gene in Helicobacter pylori strains isolated from Zhejiang Province in China.

AIM: To isolate the subtypes of 3' region of cagA gene in Helicobacter pylori (H pylori) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3' region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3' region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pylori isolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes I, II, and III according to their sizes. The sizes of subtypes I, II, and III were 648-650 bp, 705-707 bp, and 815 bp, respectively. Among the 132 cagA-positive H pylori strains, 123 (93.2%) belonged to the group of subtype I, 6 (4.5%) presented subtype II, 1 (0.8%) was subtype III, and 2 (1.5%) presented subtypes I and III both. The primary structure of subtype I was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype II possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3' region of cagA gene which had not been reported before. The primary structure of subtype III consisted of 4 repeats of R1, 1 repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype I strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype III strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype II strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%), respectively. There were no statistical differences in the distribution of subtypes of 3' region of cagA gene among different H pylori-associated gastroduodenal diseases (chi(2) = 11.544, P>0.05). CONCLUSION: There are three subtypes (I, II, and III) of 3' region of cagA gene in H pylori strains isolated from Zhejiang Province, and subtypeIis predominant. Subtype II is a newly found subtype of 3' region of cagA gene. The result of this study does not support the view that the subtypes of 3' region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.

Biological activity of the virulence factor cagA of Helicobacter pylori.

BACKGROUND: China is one of the countries with the highest incidence of H. pylori and more than 9090 isolates possessed the cagA gene. This study was to evaluate the biological activity of the H. pylori virulence factor cagA isolated from Chinese patients. METHODS: cagA DNA fragments were amplified from the genomic DNA and subsequently cloned into the mammalian expression vector for cell transfection and DNA sequencing. cagA protein, phosphorylated-tyrosine cagA and the complex of cagA precipitated with SHP-2 were identified respectively by western blot in the crude cell lysate from conditionally immortalized gastric epithelial cells at 48 hours after transfection with cagA DNA. In addition, the ability of induction of scattering phenotype was examined after transient expression of cagA in AGS cells. RESULTS: The C-terminal half of cagA contained only one repeated sequence and three tandem five-amino-acid motifs glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA). Moreover, the amino acid sequence of D2 region in repeated sequence was aspartic acid-phenylanaline-aspartic acid (D-F-D) which was significantly distinguished from the three repeated sequences and aspartic acid-aspartic adid-leucine (D-D-L) in the western standard strain NCTC11637. Western blot revealed that cagA became phosphorylated in tyrosine site and bound with SHP-2 after transient expression of cagA DNA in gastric epithelial cells. Transient expression of cagA in AGS cells showed that cagA was able to induce the elongation phenotype although to a lesser extent than western strains. CONCLUSIONS: cagA perturbs cell signaling pathways by binding with SHP-2. However, significant difference exists in amino acid sequence and biological function of cagA in Chinese compared with those of western countries.

Gene distribution of cagII in Helicobacter pylori-infected patients of Zhejiang Province.

AIM: To determine the prevalence of genotypes of cagII in Helicobacter pylori (H pylori)-infected patients in Zhejiang Province and investigate the relationship between these genotypes and the types of gastroduodenal diseases. METHODS: One hundred and seventy one clinical isolates were collected from 70 chronic superficial gastritis, 31 chronic atrophic gastritis, 41 gastric ulcer, 21 duodenal ulcer, 3 gastric and duodenal ulcer, and 5 gastric adenocarcinoma patients. Polymerase chain reaction assays were performed for analysis of cagT, ORF13 and ORF10 genes in the cagII region. RESULTS: Of 171 H pylori isolates from Zhejiang patients, 159(93.0%) were positive for all the three loci. One isolate (0.6%) was negative for all the three loci, and 11(6.4%) were partially deleted in cagII. The positive rates of cagT, ORF13 and ORF10 genes were 97.1%, 94.7% and 99.4%, respectively. In the strains isolated from the patients with diseases including chronic superficial gastritis, chronic atrophic gastritis, gastric ulcer and duodenal ulcer, the sitive rates of cagT were 95.7%, 100.0%, 95.1% and 100.0%, respectively. The positive rates of ORF13 were 94.3%, 93.5%, 95.1% and 100.0%, respectively. The sitive rates of ORF10 were 98.6%, 100.0%, 100.0% and 100.0%, respectively. The three genes were all positive in the three H pylori strains isolated from the patients with both gastric and duodenal ulcer. In the five strains isolated from the patients with gastric adenocarcinoma, only one isolate was negative for ORF13. There were no significant differences of the cagT, ORF13 and ORF10 genes among the different gastroduodenal diseases including chronic superficial gastritis, chronic atrophic gastritis, gastric ulcer, duodenal ulcer, both gastric and duodenal ulcer and gastric adenocarcinoma (chi(2)=3.098, P>0.05 for cagT; chi(2)=3.935, P>0.05 for ORF13 and chi(2)=6.328, P>0.05 for ORF10). CONCLUSION: The cagII is not a uniform and conserved entity. Although the genes in cagII are highly associated with the gastroduodenal diseases, the clinical outcome of H pylori infection is not reliably predicted by the three genes in cagII in patients from Zhejiang Province.

Purification and relationship with gastric disease of a 130 kDa (CagA) protein of Helicobacter pylori.

OBJECTIVE: The aims of this research were to purify and identify the 130 kDa (CagA) protein of H. pylori clinical isolate HP97002 and evaluate the relationships between the purified 130 kDa (CagA) protein and gastric diseases. METHODS: The procedure for isolating the protein included 6 mol/L guanidine extract, size exclusion chromatography and elusion from gel. Sera of 68 patients with gastric diseases (44 with chronic gastritis, 15 with atrophic gastritis, 7 with peptic ulcer disease, 2 with gastric cancer) were obtained, and the serological response to CagA was studied by Western-blot using the purified protein. RESULTS: The purified protein was 130 kDa and preserved good antigenicity and revealed basic isoelectric point about of 8.1. Among 68 sera, 43 sera could recognize the purified protein associated with chronic gastritis 47.7% (21/44), atrophic gastritis 86.7% (13/15), peptic ulcer disease 100% (77), gastric cancer 100% (2/2). Compared with each other, the difference was significant (chi2 = 13.327, P = 0.004), and 130 kDa (CagA) protein was associated with severe gastric diseases ( r(S) = 0.442, P = 0.001). CONCLUSION: The 130 kDa (CagA) protein was associated with severe gastric diseases.

[Analysis of babA2 cagA and vacA genotypes of Helicobacter pylori in chronic gastritis and peptic ulcer].

OBJECTIVE: To investigate the distribution of babA2 cagA and vacA genotypes of Helicobacter pylori (Hp) in chronic gastritis and peptic ulcer and to discuss the relationship between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer. METHODS: babA2, cagA genotypes and vacA subtype of 58 Hp strains isolated from patients of Zhejiang province with chronic gastritis or peptic ulcer were tested by polymerase chain reaction. RESULTS: The positive rates of babA2, cagA, vacAs1a, vacA m1 and vacA m2 of 58 Hp strains were 87.9%, 100%, 93.1%, 1.7% and 65.5%, respectively. There were no significant differences between the positive rates of babA2, vacA s1a and vacA m2 genes of Hp strains isolated from patients with chronic gastritis and peptic ulcer. CONCLUSION: The genotypes of Hp isolated from patients of Zhejiang province were predominatly babA2 positive, cagA positive and vacA s1a/m2. The relationships between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer can not be identified.