Effect Of Variable Region Polymorphism Of CagA Gene Of Helicobacter Pylori On Its Function

Helicobacter pylori(H.pylori) is a kind of gram-negative(GN) microaerophilic bacterium in a spiral rodlike form[1, 2]. The infection rate of H.pylori exceed 50% across the world, and it is even higher in Asia, Africa, South America and other less developed areas[3]. H.pylori may colonize in human gastric mucosa for a long term and thus becomes an important pathogen that causes gastritis, peptic ulcer, gastric mucosa associated lymphoid tissue lymphoma and gastric adenocarcinoma[4-7]. It has been listed as a type I carcinogenic factor by the World Health Organization(WHO) since 1994. It is the only type I carcinogenic factor from bacteria so far.H.pylori is closely associated with gastroduodenal diseases. Its cytotoxin associated gene A(Cag A) is one of the most important virulence factors[8]. Colonizing on the gastric epithelium surface, H.pylori can interfere the functions of the host cells and induce cytotoxicity[9] by injecting Cag A protein into the host cells via the unique type IV secretion system(TFSS).Utilizing different artificially optimized and humanized cagA genotypes, we built six eukaryotic expression vectors of cag A genotypes before gastric epithelium AGS cells were transfected with these vectors respectively. The effects of H.pylori Cag A EPIYA polymorphism on cellular morphology and IL-8 were observed. Meanwhile, we collected H.pylori-positive specimens from 131 cases with gastric diseases, detected Cag A EPIYA motif infected with H.pylori, and analyzed the influences of the characteristics of EPIYA motif types on the varieties and severities of gastric diseases among patients with gastric diseases.The experiment can be divided into the following three parts:I. The optimization and artificial synthesis of cag A genotypes and the construction of their eukaryotic expression vectorsThrough NCBI Blast H.pylori cag A sequence, we selected a total of 6 sequences representing different cag A genotype: cag A 98-10, cag A Ca52, cag A NCTC11637, cag A F75, cag A PNGhigh85 and cag A Shi470. After analyzing the above genes by utilizing condon preference, gene GC content, dinucletide sequence and so on, 4 brand new C-terminal sequences(NCTC11637, F75, PNGhigh85 and Shi470 cag AHS) were obtained through optimized synthesis(cag A 98-10 HS gene and cag A Ca52 HS gene were preserved in the laboratory in advance).The pcDNA3.1 and N terminals of cagA 98-10 N were connected to the Cterminals of NCTC11637 HS, cag A SHI470 HS, cag A PNGhigh85 HS and cag A F75 HS with recombinant clone kit method to build eukaryotic expression vectors pc DNA3.1-cag A NCTC11637 HS, pc DNA3.1-cag A F75 HS, pc DNA3.1-cag A PNGhigh85 HS and pc DNA3.1-cag A Shi470 HS. That is to say, plus the established cag A 98-10 HS and cag A Ca52 HS, a total of six eukaryotic expression vectors were obtained.II. The influences of different CagA genotypes on the morphology of AGS cells and IL-8 expressionAGS were recovered, cultured and passaged, and the expressions of CagA protein after the transient transfection of AGS cells with different types of cag AHS plasmids were detected by Western Blotting. GAPDH was used as the internal reference protein, while the cells transfected with mock-vehicle pc DNA3.1 were taken as negative controls, suggesting that different types of cag A were successfully expressed in AGS cells and that there was no statistically significant difference in transfection efficiency. Cellular morphological changes and IL-8 expression were observed within 48 hours after the transient transfection of AGS cells with different types of cag AHS plasmids, with the cells transfected with mock-vehicle pc DNA3.1 as negative controls. Cellular morphological observation was conducted every 6h after the transfection, and the numbers of hummingbird-like cells were counted at 24 h and 36 h respectively. The statistical results showed that all Cag A genotypes could increase the number of cells with hummingbird-like change. At 24 h and 36 h after transfection, compared with the mock-vehicle controls, all the six Cag A subtypes showed a remarkably significant differences(P<0.001), Compare with Cag A ABCCC, the five Cag A subtypes left showed a remarkably significant differences(P<0.001), Compare with Cag A ABDD, Cag A ABC and Cag A J-Western showed a remarkably significant differences(P<0.001); Cag A ABDD and Cag A Amerindian showed a remarkably significant differences 24 h after transfection(P<0.001); Compare with Cag A ABD, Cag A ABC and Cag A J-Western showed a remarkably significant differences 36 h after transfection(P<0.01).In addition, IL-8 secreted by these cells 12 h and 36 h after transfection was detected respectively. The statistical analysis revealed that, at 12 h after transfection, Compare with the mock-vehicle controls, Cag A ABDD showed a remarkably significant differences(P<0.01); at 36 h after transfection, compare with the mock-vehicle controls, Cag A ABD and Cag A J-Western showed a remarkably significant differences(P<0.01), Cag A ABDD and Cag A ABCCC showed aremarkably significant differences(P<0.001); Compare with Cag A Amerindian, Cag A ABD and Cag A J-Western showed a remarkably significant differences(P<0.01), Cag A ABDD and Cag A ABCCC showed a remarkably significant differences(P<0.001); Although both Cag A ABD and Cag A ABDD belonged to East Asian type, they had significantly different effects on the IL-8 secretion levels of AGS cells(P<0.001). Meanwhile, both Cag A ABC and Cag A ABCCC which belong to Western type had significantly different effects on the IL-8 secretion levels too(P<0.001), and compare with Cag A J-Western, Cag A ABDD and Cag A ABCCC showed a remarkably significant differences(P<0.001).Research on the Relationship between Cag A EPIYA Polymorphism and Gastric DiseasesThe incidence rates of H.pylori infection and gastric carcinoma are higher in China. However, there were few reports about the correlation of H.pylori cytotoxin-associated gene A(Cag A) with the types and severities of gastric diseases, not mention to reports about the correlation of Cag A EPIYA motif with gastric diseases and gastric carcinoma. In response, the present study preliminarily explored the association of H.pylori Cag A(+) EPIYA motif with the severities of gastric diseases.H.pylori-positive specimens from 131 cases with gastric diseases were collected in this study. Gastroscopic observation and pathological diagnosis revealed 37 cases with chronic gastritis, 42 cases with gastric ulcer, 35 cases with duodenal ulcer and 17 cases with gastric adenocarcinoma. 117 Cag A-positive cases were affirmed by PCR amplification, accounting for 89.3% of the total cases. And among these positive cases, there were 92 cases(78.6%) with East Asian type EPIYA-D motif, most of which were Cag A ABD(64.1%); there were 19 cases with EPIYA-C motif, accounting for 16.2% of the total cases. There was a statistically significant difference in the distribution of Cag A ABD patients among gastric adenocarcinoma and patients with no gastric adenocarcinoma(P<0.001). Cag A ABD was the only subtype among gastric adenocarcinoma patients infected with H. pylori. It was estimated that the infection of H.pylori containing Cag A ABD might increase the risk of gastric adenocarcinoma among patients with gastric diseases.