Study On The Pathogenesis Of Helicobacter Pylori IceA Gene

Helicobacter pylori are Gram-negative spiral shaped bacteria that colonizes in human gastric mucosa. H. pylori infect more than 50% of humans globally. Now it is generally accepted that Helicobacter pylori cause gastric and duodenal ulcer diseases. Additionally, H. pylori also play an important role in the pathogenesis of gastric carcinoma, primary B-cell gastric lymphoma and Sclerosing Cholangitis. There are many virulence factors related with the diseases caused by H. pylori.The iceA genetic locus that is one of virulence factors independent on cagA or vacA was suggested being induced to express following contact with epithelial cells.Two allelic variants, iceA1 and iceA2, have been identified. The iceA gene is thought to be involved in the pathogenesis to induce the expression of genes related to virulence which is also associated with H.pylori infection. Some researchers reported that iceA has relationship with the increased content of IL-8 in gastric mucous membranes and ulcer diseases. And IL-8 plays an important role in H. pylori-related inflammation and diseases. Several intriguing epidemiological studies have been reported about the Clinical relevance of the allelic iceA status of Helicobacter pylori isolated from different regions. While iceA alleles, iceAl or iceA2, show some specific relationship for gastroduodenal pathologies and is a virulence factor associated with inflammation and injury immunoreaction. However the function and the pathogenic role of iceA in Helicobacter pylori still is unclear.In the present study, iceA gene of H.pyloir MEL-Hp27 isolated from Chinese patients with chronic atrophy gastritis was cloned and its molecular character was analyzed. Then, insertional mutagenesis was carried out within the gene of H. pylori 27 by the method of molecule biology according to homologous recombination and the mutant strain was constructed. After that, difference of activities of urease between wild strain Hp27 and iceA isogenic mutant strain were assessed. Then we compared the mutant with wild strain as regards their characters, including the state, cell cycle, proliferation and apoptosis of SGC7901 cells, through co-culture of the bacteria and gastric epithelial cells. Finally, the effect of iceA gene on adherence of the bacteria to gastric SGC cells and on Helicobacter pylori associated IL-8 production from the epithelial cells was also assessed. This study provided the important experimental evidences for elucidating the function and the pathogenic role of the iceA gene in Helicobacter pylori.Methods1 Cluture of H.pylori and extraction of the genomic DNAH.pylori MEL-Hp27(Hp27) isolated from the gastric mucosa of patient with chronic atrophic gastritis was grown microarobically at 37℃for 3d in brucella broth plates.The H.pylori cells were harvested and the genomic DNA was extracted.2 Clone of iceA gene of Hp272.1 Primers designing and PCRReferring to the sequence of H.pylori strains published on GENBANK, primers were designed according to the nucleotide acid sequences of iceA gene and the sequences located in the upstream or downstream of the iceA gene. Then Hp27 iceA gene was amplified and cloned by using a polymerase chain reaction based approach.2.2 Cloning and sequencing the iceA gene of MEL-Hp27The purified product of PCR was subcloned into pMD19-T vector and transformed into DH5a.The positive clone was identified and the sequence was confirmed by DNA sequencing.2.3 Analysis of characteristics of iceA geneThe homology of iceA gene of MEL-Hp27 strain was compared with other Hp strains isolated from different regions published on GENBANK in nucleotide acid and the phylogenetic tree was constructed.3 Construction of targeting vector of iceA3.1 Recombinant plasmid pMD19-T-iceA and pBluescript SKⅡ(-) were digested with enzyme and connected into recombinant plasmid pBS-iceA.3.2 Recombinant plasmid pBS-iceA and the amplified kanamycin resistance gene were digested and connected into targeting vector of iceA mutant with kanamycin resistance gene pBS-iceA-km.4 Construction and identification of iceA mutant strain The recipient strain Hp27 was electrotransformed with the targeting vector pBS-iceA-km. The kanamycin resistance transformants were screened from brucella broth plates and identified by PCR and sequencing.5 Compare the characteristics between the wild and iceA mutant strains5.1 The activities of urease of wild strain Hp27 and iceA isogenic mutant were assayed by the urease-testing reagents5.2 Morphological changes of cells were observed and analyzed after a period of time through co-culture of the bacteria and gastric epithelial cells.5.3 The cellular proliferation was examined and compared by methyl thiazolyl tetrazolium(MTT) method through co-culture of the bacteria and gastric epithelial cells.5.4 Cell cycle were determined and analyzed by flow cytometry through co-culture of the bacteria and gastric epithelial cells for a period of time.5.5 Apoptosis of SGC7901 cells were determined and analyzed by flow cytometry through co-culture of the bacteria and gastric epithelial cells for a period of time.6 Study of the factors related with inflammation about iceA gene6.1 Analysis of the concentration of IL-8 induced by Hp27 from the SGC7901 cellsIL-8 was induced from the cells through co-culture of the wild or mutant Hp and gastric epithelial cells for a period of time. The concentration of IL-8 in the culture supernatants was determined and analyzed by enzyme-linked immunosorbent assay (ELISA)6.2 Analysis of the adherence of the wild or mutant strains to gastric SGC cells.After co-cultured the wild or mutant Hp and gastric epithelial cells for a period of time, they were treated using method of immunology. The adherence of bacteria to the cells was observed by fluorescent microscopy and adhesion to the cells was examined by flow cytometry.7 Statistical analysisThe experimental data were analyzed by the software of SPSS 11.0.The results of numerical data were expressed with (?)±S. T test was used to compare the statistical difference between two groups. According to the features of data, one-way analysis of variance or repeated measures analysis of variance were applied to test the statistical difference among three or more groups. The significance level was set at a=0.05Results1 Cloning and analysis of gene iceA of Helicobacter pylori isolatedThe iceA gene of Hp27 strain was successfully cloned. The PCR product was about 790bp. The recombinant plasmid pMD 19-T-iceA was digested by one restriction enzyme to produce a 3 820bp DNA fragment and digested by two enzymes to produce 3 OOObp and 820bp DNA fragments. Sequence analysis showed that the iceA gene of MEL-Hp27 have more than 85% homology with H.pylori American strains and less than 70% of homology with H.pylori Japanese or Indian strains. The-10 box was located in the up-stream of iceA gene and the distance from ATG was 17bp. The SD sequence is located in the up-stream of iceA gene and the distance from ATG was 7bp.There are two avariable numbers of tandem repeats between ATG and the up-stream gene cysE. There are differences about the characters of the gene between Hp27 strains and other Helicobacter pylori strains from other regions.2 Construction of MEL-Hp27 iceA mutant strainIn this study, Kanamycin resistance gene was inserted into the target gene by the method of gene recombination to construct the targeting vector pBS-iceA-km, with which the recipient strain Hp27 was electrotransformed. IceA mutant strain was screened on brucella broth plates with kanamycin and identified.DNA fragments, which were amplified from genomic DNA of mutant or wild strains separately by primers designed according to the flanking sequences of the iceA gene, from mutant strains are longer 800bp than the wild. Kanamycin resistance gene can be amplified from the mutant strains but not wild. The sequencing result of amplified product also showed that Kanamycin resistance gene had been inserted into the target gene 3 Comparison the activities of urease of wild strains with iceA gene mutantsRemarkable color change can be seen immediately when wild strain and constructed isogenic mutant were assayed by the urease-testing reagents. After being treated for 25 minutes, the OD values of wild strains reached a climax when urease had been decomposed completely. Before that, all its OD values at different time point were higher than mutant strains. The OD values of mutant strains reached a climax until 50 minutes later. There are some differences between two groups but not significant.4 The effect of iceA gene on growth state of SGC7901Cell elongation and hummingbird morphology were showed through co-culture of the bacteria and gastric epithelial cells for 48h and similar changes of cell morphology were observed in wild and mutant strains groups. The MTT assay showed that the vigor of cells increased slightly after co-cultured for 12h or 18h with Hp compared with contral group, but began to descend after co-cultured for 24h. There was no remarkable difference in cellular proliferation among all groups when co-cultured for 12h,18h or 24h, but there were distinct differences when co-cultured for 30h or 36h (P<0.05).Especially cellular proliferation of mutant group was significantly higher than wild group when co-cultured for 30h (P<0.05).The primary influence of wild strain on the cell cycle was that it arrested the cell cycle of SGC at G2 phase compared with contral group. But for mutant group, it arrested at S phase. SGC7901 cells infected with wild or mutant strains,24h or 48h, all displayed a significant increase in apoptosis, with statistically significant levels of apoptosis compared to uninfected cells (P<0.05). However there were no significant differences (P>0.05) between mutant group and wild group.5 Study of the factors related with inflammation about iceA geneAfter SGC7901 cells were co-cultured with Helicobacter for 8h or 24h, the concentration of IL-8 in the culture supernatants was determined and analyzed. Significant differences were observed between SGC cells interacting with the Hp strains (wild and mutant) and the uninfected cells (P<0.05). There were significant differences for the concentration of IL-8 between wild and mutant groups if co-cultured for 24h (P<0.05), but not for 8h ((P>0.05).When SGC7901 cells were co-cultured with Helicobacter, both of wild and mutant strains were bounded to cells rapidly which was confirmed by fluorescent microscopy. Adhesion to cells was examined by flow cytometry. The mean numbers of wild or mutant strains adhering to cells were 32.49±2.96% and 25.03±2.74%, respectively. The difference was not statistically significant (P>0.05).Conclusions1 There are some differences for iceA gene among Helicobacter pylori strains from different regions.2 The mutant of iceA gene had significantly effect on the activities of proliferation and the cell cycle of SGC7901 cells. However, it did not change adhesion and apoptosis of Hp to cells significantly.3 IL-8 was induced by Hp27 from the SGC7901 cells. The mutant of iceA gene decreased the concentration of IL-8 distinctly.