Study On Genetic Diversity Of 3' Region Of CagA Gene In Helicobacter Pylori Strains Isolated From Zhejiang Population

Background and Aim Helicobacter pylori (H. pylori) is a spiral Gram-negative microaerophilic bacterium that chronically colonizes the gastric epitheliums of more than half of all people worldwide. It causes chronic inflammation that is frequently associated with chronic atrophic gastritis and peptic ulcer disease. Epidemiological and animal studies have suggested that the chronic infection of H. pylori is an important risk factor for the development of gastric adenocarcinoma. Accordingly, the World Health Organization has classified H. pylori as a group I carcinogen in 1994. Chronic infection with H. pylori has also been suggested to be an important risk factor for the development of gastric mucosa-associated lymphoid tissue lymphoma. Despite of high percentage of H. pylori infection, more than 80% of the carriers present asymptomatic gastritis. Only 10-20% of the H. pylori carriers develop chronic gastritis and peptic ulcer disease, and a smaller proportion (0.1 ~4%) develop gastric carcinoma. Variation in virulence of the infecting strains is thought to be an important reason for different outcomes of H. pylori infection. CagA, encoded by cytotoxin-associated gene A (cagA), is an important virulence factor of H. pylori. The CagA-positive H. pylori strains are more virulent than the CagA-negative strains and are associated with higher grades of gastric inflammation. CagA is injected intogastric epithelial cells by the bacterial type IV secretion system encoded by the cag pathogenicity island (cag PAI) and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds to a cytoplasmic Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase called SHP-2, activates the phosphatase activity, and thereby induces morphologic transformation and abnormal proliferation of gastric epithelial cells. The structure of cagA gene contains a 5' highly conserved region and a variable 3' region, in which the presence of a variable number of repeat sequences results in CagA with a molecular mass of 120 to 145 kDa. Tyrosine phosphorylation of CagA occurs at the EPIYA motif -a 5-amino-acid sequence (Glu-Pro-Ile-Tyr-Alu) that is present in the carboxy-terminal variable region of the protein. The potential of CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. Therefore, variation of the EPIYA motif caused by genetic diversity of 3' region of cagA gene is supposed to be responsible for the degree of virulence of CagA~+ H. pylori and the different clinical outcomes of H. pylori infection. In this study, the aim is to investigate the genetic diversity of 3' region of cagA gene in H. pylori strains isolated from Zhejiang population and its relations to H. pylori-associated gastroduodenal diseases.Materials and Methods A total of 182 H. pylori isolates were obtained from H. pylori-infected adults living in Zhejiang Province. Eighty-one isolates were from the patients in Hangzhou City and the others were from the patients in Daishan County. The patients, consisting of 128 men and 54 women with a mean age of 41.1 years (ranging from 16 to 71 years), were classified into 3 groups including chronic gastritis (n=101), peptic ulcer (n=79) and gastric adenocarcinoma (n=2), according to the results of endoscopic and histological examination. H. pylori isolates from biopsy specimens were cultured on ECY selective solid medium at 37℃ for 5 days, under 100% humidity and microaerophilic conditions (5%O2, 10%CO2, and 85% N2). H. pylori was identified by the following criteria: characteristic of colony, rapid urease test, catalase test and morphology on Gram staining. H. pylori isolates were harvested from the agar plates and genomic DNA was extracted by phenol/chloroform method.The primers for 3' region of cagA gene were synthesized according to the references and used to amplify the gene by PCR. PCR amplification was performed as following: an initial denaturation at 95 ℃ for 3 min, followe...