Analysis Of Virulence Factors And Function Of DupA Gene In H. Pylori Isolated From A Region At High Risk Of Gastroduodenal Diseases

ObjectivesHelicobacter pylori (H. pylori) colonize the surface area of the gastric mucosa in the human stomach, and are associated with an increased risk for various gastroduodenal diseases. In order to estimate the virulence of H. pylori isolated from the patients in a region at high risk of gastroduodenal diseases, we investigated the prevalence of H. pylori virulence markers, including cagPAI, vac A and dupA. The function of dupA of H. pylori was analyzed to further reveal the pathogenesis of H. pylori in the study.Methods1. H. pylori strains were isolated from gastric biopsies of patients in a region at high risk of gastroduodenal diseases. cagPAI genes (cagA, cagE, cagl, cagL, cagM, cagT and cagX), the cagA3’variable region, vac A, and dupA genotypes were determined by PCR. Some amplicons of the cagA3’variable region, cagPAI genes (cagl, cagL, cagM, cagT and cagX) and dupA were sequenced to compare with deposited sequences in NCBI (National Center of Biotechnology Information). The deduced amino acid sequence of proteins was obtained from the nucleotide sequence. The full-length amino acid sequences were aligned, and the phylo genetic trees were constructed by the program MEGA4.1.2. dupA genotype of H. pylori WH-21strain was analyzed, which was isolated from the patient with duodenal ulcer. The biological functions of the deduced DupA were analyzed using the structural bioinformatics analysis methods, including the primary structure, secondary structure, tertiary structure, the prediction of protein subcellular localization and the functional domains.3. After recombinant expression vector pET32a (+)-dupA was constructed and transformed into E. coli Rosetta (DE3), recombinant DupA fusion protein was induced by IPTG and affinity purified using Ni-nitrilotriacetic acid resin. ATPase activity of recombinant DupA fusion protein was tested using the assay based on the inorganic phosphate released by ATP hydrolysis. The polyclonal antibody to recombinant DupA fusion protein was produced by animal experiment.4. The subcellular localization of DupA (2499) was analyzed by Western blotting. The interaction proteins of DupA (2499) were investigated using Immunoprecipitation assay and MS. 5. After the pBluescript/△dupA::KMr was constructed and introduced into H. pylori WH21strain by electroporation, an isogenic mutant of H. pylori dupA was generated by allelic exchange mutagenesis.6. The CagA translocation activity of the dupA-deleted mutant was analyzed after GES-1cells infected with H. pylori by Western blotting. The adhesion activity of the dupA-deleted mutant was quantified by colony-forming units (CFU) assay and adhesion rate assay.7. Susceptibility of dupA-deleted mutant to low pH was measured by the bacterial growth in the different pH liquid media. The supernatants of the different pH liquid media were collected for analyzing urease levels using Western blotting. IL-8levels in the supernatants were determined after GES-1cells co-cultured with H. pylori strains to estimate the virulence of the dupA-deleted mutant.8. After MKN-45cells co-cultured with H. pylori strains, the death of MKN-45cells induced by H. pylori was studied in cytotoxicity assay by MTT assay, and Hoechst33258staining was performed for morphology analysis. Protein-involved mitochondria mediated apoptosis were analyzed by Western blotting, including Caspase-3, PARP, Bax and Bcl-2.Results1. After PCR amplification of the cagPAI genes, the results showed that100%(116/116) of the isolates were positive for cagA, cagL, cagM, cagT and cagX, whereas89.7%(104/116) and82.8%(96/116)strains had cagE and cagl, respectively. Isolates with partial cagPAI were found in25.9%(30/116) strains, which present at a higher frequency in chronic gastritis (44.4%,24/54) than that of gastric ulcer, duodenal ulcer and gastric cancer (9.7%,6/62)(p<0.001). None of the isolates completely lacked the cagPAI.cagA3’variable regions of24strains fell into two types:East Asian-type (91.7%) classified as EPIYA-A-B-D and Western-type (28.3%) classified as EPIYA-A-B-C. The proteins encoded by cagPAI genes (CagI、CagM、CagT、CagX) were conserved with high homology except CagL.The phylogenetic trees of the full-length CagPAI proteins (CagI, CagL, CagM, CagT and CagX) demonstrated that all CagPAI proteins in the isolates could be placed into two major groups (East Asian-group and Western-group), and most of them were included in the East Asian-group.It showed that sla/m2(48.3%) and s1c/m2(13.8%) strains were the most prevalent H. pylori strains in this region. Thirty-one percent of strains (31.0%) possessed dupA, and the prevalence of dupA was significantly higher in strains from patients with gastric ulcer, duodenal ulcer and gastric cancer (40.3%) than that from chronic gastritis (20.4%, p=0.02). Sequencing of dupA revealed an ORF of2,449-bp, which was called as dupA (2499).2. Sequencing of dupA in H. pylori WH-21revealed an ORF of2,449-bp, which was dupA (2499). There was homology among dupA sequences of the nine strains from Weihai, approximately99.3%. The homology among strains from Weihai and two strains from Japan was99.25%, while it was91.65%among fourteen strians isolated from different regions. Using the bioinformatics software, DupA (2499) was classified as stable one with high hydrophilicity and antigenicity. There was transmembrane helices but no signal peptide in DupA (2499), which located in membrane. The major structural element of polypeptide chain was α-helix, and one coiled coil was predicted in DupA (2499). It contained combined sites of ADP、ATP and Mg2+in the tertiary structure. The protein was predicted to have high homology with the CagE_TrbE_VirB superfamily with ATP binding site.3. The expressed recombination DupA fusion protein was obtained, and the protein exhibited a clear ATPase activity (129.5±17.8U/mgproft. The polyclonal antibody to recombinant DupA fusion protein was produced in the study, and the antibody titer was1:6.4×104.4. DupA (2499) was located in bacterial membrane using Western blotting, which interacted urease and HSP60by IP and MS.5. The isogenic dupA mutant of H. pylori was obtained and identified by PCR using one pair of primers (P1and P4) and Western blotting using anti-DupA antibody.6. After used H. pylori to infect GES-1cells, the quantities of CagA protein translocated into the cells were similar between the wild type strain and the dupA-deleted mutant. No Significantly decreased numbers of the dupA-deleted mutant bound to GES-1cells were compared with the wild type strain both in CFU assay [(27.2±7.1)×106and (28.5±6.9)×106, p=0.772] and adhesion rate assay (81.0%and78.8%,p=0.724).7. The result revealed that the wild type strain had a stronger growth than the dupA-deleted mutant in low pH (p<0.05). Reduced urease secretion levels were found in dup A-deleted mutant especially in low pH comparing to the wild type strain.After the two strains infected GES-1cells, IL-8levels were significantly higher in supernatants co-cultured with the wild type strain than in those with its dupA-deleted mutant (p<0.001)8. The amounts of vital cells were decreased which were infected with wild type strain, compared to those for the mutant-infected cells after12h (p<0.05). After12h, apoptosis was observed in MKN-45cells treated both with the wild type strain and the deleted-dupA mutant. The number of apoptotic cells resulting from treatment with the wild type strain was significantly higher than in cells treated with the deleted-dupA mutant (p<0.05). The increase of cleaved Caspase-3, cleaved PARP and Bax in MKN-45cells exposed to the wild type strain was significantly higher than that exposed to the deleted-dupA mutant, while the decrease of Bcl-2in the wild type strain was more obvious from6h.Conclusions1. H. pylori isolated from patients are highly virulent strains, which associates with the high risk of gastroduodenal diseases in the region at high risk of gastroduodenal diseases. The virulent characteristics of H. pylori involve the more active East Asian-type cagA, intact cagPAI, East Asian-type cagPAI, highly virulent vacA genotypes, and dupA (2499).2. dupA (2499) is conserved and the homology is high, which is suitable for cloning and expression in vitro. DupA (2499) is classified as stability and has strong antigenicity, which is suitable for inducing antibody.3. DupA (2499) locates in membrane independently of CagPAI to involved the urease secretion as ATPase, which is not involved the adhesion and CagA delivery to host cells4. DupA interacted and involved the urease secretion in H. pylori cells, and the characteristic makes dupA-positive strains resistant to high acid conditions. dupA-positive strain is resistant to high acid conditions and induces IL-8secretion, and the characteristics associated with duodenal ulcer development. DupA inhibits tumor cell growth, and leads to mitochondria mediated apoptosis to reduce the risk for gastric cancer.