Study On The Molecular Mechanisms Of H.pylori CagA Pathogenesis

Helicobacter pylori(H, pylori), a spiral-shaped bacterium that colonizes the human gastric mucosa, is estimated to inhabit at least half of the world's human population. Itis well known that H. pylori is recognized as the etiological agent of gastric diseases such as chronic atrophic gastritis and peptic ulcers, and the infection with H. pylori is associated with the development of gastric carcinoma. However, little is known about the molecular mechanisms of the pathogenesis induced by H.pylori and the fundamental reasons of diversity of infection outcomes. Cytotoxin-associated gene A (CagA) is one of the important virulent factors of H.pylori. Recent studies have indicated that the cagA gene product CagA is delivered from the bacterium into the cytoplasm of the bacterium-attached gastric epithelial cell via the type-Ⅳsecretion system. Upon membrane localization, translocated CagA interacts with a number of host proteins involved in cell signaling in both tyrosine phosphorylation-dependent and -independent manners. Epidemiological studies have shown that the cagA-positive H. pylori strains are associated with higher grades of gastric mucosal inflammation as well as severe atrophic gastritis and CagA has been suggested to play an important role in the development of gastric carcinoma. Thus, it is necessary that further works on biology functions of H. pylori CagA be done for shedding some light on the role of cagA in the pathogenesis of Chinese H.pylori strains, and on the molecular mechanisms of H.pyloricarcinogenesis.In the present study, firstly cagA gene in full length of H.pylori MEL-Hp27 isolated from the gastric mucosa of patient sufferred from chronic atrophic gastritis was cloned and its molecular characterization was analyzed. Then, referring to the sequence of cagA gene of H.pylori MEL-Hp27, the cagA gene knocked-out mutant strain Hp27△cagA was constructed via the method of homologous recombination. Thirdly, the biology functions and the virulent effects on host cell of CagA were preliminarily assessed via comparation of biological characteristics between wild-type strain Hp27 and isogenic mutant Hp27△cagA, and of the in vitro effects on the proliferation and apoptosis of BGC823 cells induced by two strains. In the end, proteomics was used to screen and identify the differential expression proteins between wild-type strains and isogenic mutants, and the expression patterns of proteins of the gastric epithelium cell Ges-1 co-cultured with the wild-type strains and the mutants were also investigated in the same way, respectively. This study provided the important experimental evidences for clarifying the roles that CagA plays in the pathogenesis induced by H. pylori and for elucidating the association between CagA and gastric cancer.1 Culture of H.pylori and extraction of the genomic DNAH.pylori MEL-Hp27(Hp27) isolated from the gastric mucosa of patient with chronic atrophic gastritis and international reference strain NCTC11637 were grown microarobically at 37℃for 3 days in brucella broth plates. The H.pylori cells were harvested and the genomic DNA was extracted.2 Clone of cagA gene of Hp272.1 Primers designing and PCRReferring to the sequence of H.pylori 26695 genome, two pairs of primers were designed according to the nucleotide acid sequence of conservative fragment in coding region of cagA gene and the sequences of genes located in the upper and downstream of cagA gene, respectively. Then Hp27 cagA gene in full length with its 5'AND 3'UTR was cloned by using a polymerase chain reaction (PCR)-based approach.2.2 Analysis of molecular characteristics of cagA geneThe complete sequence of cagA gene was published on GenBank. The characteristics of the regulatory sequence of Hp27 cagA gene were analyzed by being compared with those of other strains published on GenBank. The phylogenetic trees were constructed on the encoding sequences of cagA gene of H.pylori isolated from different regions and on the amino acid sequences of CagA of H.pylori isolated from East Asian which the clinical backgrounds of infection were known, respectively.3 Construction of targeting vector of cagA gene3.1 Two DNA fragments were amplified as homologous arms, according to the sequences of the upper and down stream of coding region of cagA gene, and one of homologous arms was consisted of the complete regulatory sequence of cagA gene. The kanamycin resistance gene (km~R) was amplified as screening marker from pEGFP-N2 plasmid.3.2 The pBluescript SKⅡ(-) plasmid was digested and ligated with two homologous arms, between which the kanamycin resistance gene was inserted, and then the targeting vector(pBSK-cagA-mutant) was constructed.4 Construction and identification of a eagA knocked-out mutantHp27 served as a recipient strain was electrotransformed with the targeting vector of pBSK-cagA-mutant. The kanamycin resistance H.pylori transformants were screened from brucella broth plates (25μg/ml kanamycin) and identified by PCR and sequencing. The cagA knocked-out mutant was named as Hp27△cagA.5 Analysis of the biological characteristics of the cagA-knocked out mutant5.1 The activities of urease of NCTC11637,wild-type strain Hp27 and isogenic mutant Hp27△cagA were assayed by the urease-testing reagents.5.2 The stab agar test was used to observed the motility of wild-type strains and isogenic mutants. 5.3 The different adherent effects on human gastric cancer cell line BGC823 cells between wild-type strains and isogenic mutants were investigated by adherence experiments.5.4 The toxic effects of vacuolating toxin of wild-type strains and isogenic mutants on Hela cells were evaluated by both light microscopy and neutral red uptake (NRU) assay, and experiments on NCTC11637 strains were used as positive control.6 Effects of the cagA-knocked out mutant on the proliferation and apoptosis of BGC823 cells6.1 BGC823 cells were co-cultured with the wild-type strains and the isogenic mutants at a bacteria:cell concentration of 100:1, respectively.6.2 The cell phenotype was observed at 6,12,24 and 48h after co-culturing.6.3 The cellular proliferation was examined by methyl thiazolyl tetrazolium(MTT) method at time points of 5,10,20,25 and 30h after co-culturing.6.4 The induction of apoptosis of cells co-cultured with H.pylori for 48h was determined by flow cytometry.7 Methods of proteomics7.1 Protein isolation and concentration determinationThe wild-type strains of Hp27 and isogenic mutants of Hp27△cagA were harvested. The method of Sonication-Urea-CHAPS-DTT was employed to extract bacterial soluble proteins: Gastric epithelium cell line Ges-1 cells were co-cultured with wild-type strains and isogenic mutants at a bacteria:cell concentration of 100:1 for 10h, respectively. Total cellular proteins of two groups were extracted by the method of Sonication-Urea-CHAPS-DTT.The protein concentration was measured by using the Bradford's method.7.2 2-DE, Image analysis, MALDI-TOF-MS analysis and protein identification by protein database searchingTo obtain 2-DE maps, the soluble proteins were firstly separated by Isoelectric Focusing (IEF) electrophoresis using Bio-rad IPGphor and IPG strips with a linear pH range of 3～10, and secondly separated according to difference in molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Images of 2-DE gels were digitalized with ImageScanner (GS-800). Image analyses were conducted with PDQuest gel image analysis software 7.1(Bio-rad). Only those spots that have statistical significance in differential expression were selected for further investigation. Spots of interest were cut out with a clean scalpel and subjected to in-gel digestion with trypsin, then gained the peptide mass finger-printer (PMF) by matrixes assisted laser adsorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Then the biology information was used to search protein database of these PMF and identify the proteins, and analyze the biological significance and function of these proteins.7.3 Quality guaranteeThe normalized volumes of protein spots was used to analyze the differential level of protein expression. The differential spots were selected for subsequent MS analysis only if statistical significance generated. The 2-DE maps of each sample were run at least three times to ensure the reproducibility and reliability.8 Statistical analysisThe experimental data were analyzed by using the soft-ware of SPSS11.0. The results of numerical data were expressed with x±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=0.05.Results1. Cloning and analyzing the molecular characteristics of cagA gene in full length of H.pylori isolated from ChinaCagA gene was successfully cloned in full length from MEL- Hp27 genomic DNA and published on GenBank(Accession No.:DQ306710). The coding region of cagA gene is consisted of 3,510 bp, which encodes 1169 amino acids. And the 5'UTR and 3'UTR of cagA gene are consisted of 649 bp and 476 bp, respectively. The sequences of-10 box and -35 box were found in the upper stream of coding region, and the distance from -10 box and -35 box to start codon "ATG" was 89bp and 154bp, respectively. The analysis of phylogenetic tree constructed on the encoding sequences of cagA gene showed that there was significant difference between the strains isolated from West countries and from East Asia. The regulatory sequences of cagA gene was also found the regional differences mentioned above. The analysis of phylogenetic tree constructed on the the amino acid sequences of CagA showed that H. pylori strains could be classified two groups: one was only consisted of strains isolated from patients with chronic gastritis and duodenal ulcer; the other was consisted of strains isolated from patients with gastric cancer and others gastric diseases. And the cagA gene of Hp27 belonged to the latter.2. Construction of a cagA knocked-out mutant strain of Chinese H.pyloriTwo homologous-arm fragments (710bp and 970bp) and the kanamycin resistance gene (800bp)were PCR-amplified, respectively. These three DNA fragments were engineered into the pBluescript SKⅡ(-) plasmid to construct the targeting vector (pBSK-cagA-mutant). Restriction endonuclease analyses showed that pBSK-cagA-mutant vector had been correctly recombined. Hp27 strains were electrotransformated with the targeting vector of pBSK-cagA-mutant, and the kanamycin resistance H. pylori transformants were screened in the brucella broth plates containing kanamycin (25μg/ml) and named as Hp27△cagA. The cagA deleted status of Hp27△cagA was identified by their PCR products with primers specific for the cagA and the kanamycin resistance gene. PCR results showed that DNA fragments of cagA gene could be amplified from genomic DNA of wild-type strains Hp27, while could not be amplified from that of mutants Hp27△cagA; the kanamycin resistance gene could be amplified from genomic DNA of mutants, but could not be amplified from that of wild-type strains. Furthermore, the long fragments were amplified by using a pairs of primers designed according to the flanking sequences of cagA gene, which indicated that a DNA fragment about 5100bp could be obtained from genomic DNA of wild-type strains, while only a DNA fragment about 2500bp could be amplified from that of mutants. The sequencing result was in coincidence with PCR results that the coding region(3510bp) of cagA gene was knocked-out, which was substituted by the kanamycin resistance gene(800bp). And the sequence was published on GenBank(Accession No.: DQ789393).3. Analysis of the biological characteristics of the cagA-knocked out mutant of Chinese H. pyloriIt was showed that NCTC 11637 and wild-type strains Hp27 could lead to remark-ably color changes of the urease-testing reagents in a very short time, while the isogenic mutants Hp27△cagA needed to spend much more time on causing the same changes. The OD492nm values of urease reagents treated by H. pylori investigated that OD values of isogenic mutants at different time points from 5 to 80 minutes were significantly lower than those of wild-type strains and NCTC11637, which indicated that the activity of urease of isogenic mutants was decreased. In the stab agar test, isogenic mutants only formed a smaller swarming zone than that of wild-type strains after at least 5 days of incubation in 0.3% motility agar, which indicated that isogenic mutants were less motile than wild-type strains and cagA gene may have some relationship with the motility of H.pylori. And it was not found that ablation of cagA gene had any apparent effect on vacuolating cytotoxin activity and adherence of H. pylori.4. The effects of the cagA-knocked out mutant on the proliferation and apoptosis of BGC823 cellsThe changes of cell morphology were observed when BGC823 cells were co-cultured with H. pylori for 6 hours, and cell scatterring and hummingbird morphology (cell elongation) were presented after co-cultured 48 hours in both groups. The MTT assay showed that the viability f BGC823 cells co-cultured with the wild-type strains was inhibited significantly compared with the isogenic-mutants group after co-cultured 20, 25 and 30 hours, respectively, while there was no remarkably difference in cellular proliferation between the isogenic mutants group and control group. As demonstrated by flow cytometry, the apoptotic rates in both groups treated by wild-type strains (8.4%) and mutants(9.1%) were significantly higher than that of control group(2.7%), but there was no difference between these two experimental groups5. Study on differential expression proteins of the wild-type strains and isogenic cagA knocked-out mutants of Chinese H. pyloriAnalysis of differential expression proteins between the wild-type strains and isogenic cagA knocked-out mutants by using PDQuest gel imagination analysis software showed that there were 22 differential protein spots appeared. Compared to the total bacteria proteins of wild-type strains, there were 8 spots down-regulated, 4 spots up-regulated and 10 spots absent expression in the bacteria proteins of mutants. 5 protein spots which were high abundance in bacteria proteins of wild-type strains while were down-regulated or absent-expressed in bacteria proteins of mutants were selected to be identified and analyzed. The identified proteins were all important antioxidant proteins of H. pylori including Alkyl hydroperoxide reductase(Ahp), Superoxide dismutase (Sod) and Modulator of drug activity(Mda66). This study indicated that cagA gene may have some influence on the expressions of antioxidant proteins of H. pylori in the direct or indirect way, which was extremely important to maintain normal activity of antioxidative stress and ensure H.pylori persistent colonization in the host stomach.6. Comparative proteomic analysis of gastric epithelium cell line Ges-1 treated by the wild-type strains and isogenic cagA knocked-out mutants of Chinese H.pylori2-DE map showed that there were 20 differential protein spots appeared between two groups of total proteins of gastric epithelium cell line Ges-1 cells treated by wild-type strains of Hp27(wild-type strain group) and isogenic mutants of Hp27△cagA(mutant group), respectively. Compared to the wild-type strain group, there were 11 spots down-regulated. 1 spots up-regulated and 8 spots absent expression in the mutant group. 5 protein spots which were high abundance in the cellular proteins of wild-type strain group while were down-regulated or absent expressed in the proteins of mutant group were selected to be identified and analyzed. The identified proteins could be classified as two groups: one was antiinflammatory proteins consistinging of Annexin Al and Platelet-activating factor acetylhydrolase IB subunitγ(PAF-AH IBγ); the other was cancer-related proteins including Annexin Al, Triosephosphate isomera (TIM) andα-Enolase, respectively. This study may provide the direct evidences to prove that cagA is involved in the process of inflammation and carcinogenesis of H. pylori. 1 CagA gene was successfully cloned in full length. It was firstly indicated that there was regional difference in the regulatory sequences of cagA gene between the isolates of West countries and East Asia countries. Analysis of phylogenetic tree constructed on the the amino acid sequences of CagA showed that H. pylori strains could be classified as two groups of including or not including the strains isolated from patients with gastric cancer.2 A cagA knocked-out mutant of Chinese H. pylori was firstly constructed, which is the essential way for studying the biological functions of CagA.3 It was firstly observed that cagA gene knocked-out could inhibit the activity of urease and decrease the motility of H. pylori, which may be one of pathogenesis of H. pylori cagA4 The cagA gene may play the crucial role in inhibiting the cellular proliferation.5 It was firstly showed that cagA gene may have some influence on the expressions of antioxidant proteins(Alkyl hydroperoxide reductase, Superoxide dismutase and modulator of drug activity) of H. pylori in the direct or indirect way, which was extremely important to maintain normal activity of antioxidative stress and ensure H. pylori persistent colonization in the host stomach.6 The cagA related proteins were firstly found in the total cellular proteins of Ges-1 treated by H. pylori, which could be classified as two groups: one was antiinflammatory proteins consistinging of Annexin Al and Platelet-activating factor acetylhydrolase IB subunitγ(PAF-AH IBγ); the other was cancer-related proteins including Annexin Al, Triosephosphate isomerase (TIM) andα-Enolase, respectively. This study may provide the direct evidences to prove that cagA is involved in the process of inflammation and the carcinogenesis of H. pylori.