Characterization Of0520Gene In The Cag Pathogenicity Island Of Helicobacter Pylori

The Helicobacter pylori (H. pylori) is one of the human gastric pathogens which infects half of the world’s population. Type I H. pylori strains harboring the cag pathogenicity island (cag PAI) are highly relevant with several gastric diseases. The cag PAI encoded Type IV Secretion System (TFSS) is able to synthesize and deliver its major effector protein CagA into host cells for cell damage. So far, studies have shown that cag PAI encodes27proteins and the function of most genes is still unclear. We concentrated on hp0520, the No.l gene of cag PAI. Microbiology, molecular biology, immunology and bioinformatics methods were used for exploring the function of hp0520. All of these results will shed light on the function and pathogenic mechanism of cag PAI.Methods:1. A primer pair of hp0520gene was designed by Primer Premier6.0according to the complete gene sequence of H. pylori26695from Genebank. The target genome was obtained through T-A cloning method and studied using bioinformatics methods.2. The expression vector pET-32a-hp0520was constructed and transformed into E. coli Rosetta (DE3). After induced by IPTG, the target protein was expressed and purified by Ni2+-NTA via QIAGEN system. The purified protein was identified via Western blot analysis and mass spectrum methods.3. The purified HP0520protein was used to immunize BALB/c mice. Then the spleen cells were fused with myeloma SP2/0cells via cell fusion methods. The positive hybridoma cell lines, which were able to secret the target monoclonal antibody (mAb) were screened by means of several rounds of subclone and indirect ELISA methods. The positive hybridoma cell lines were intraperitoneal inoculated BALB/c mice to produce ascetic-type mAb. Immunoglobin isotype of the mAb was identified by ELISA methods. Indirect ELISA methods were used to test the titer of the mAb in cell culture supertanant and ascites. Indirect ELISA methods and Western blot analysis were used to indentify the specific reaction between the mAb and its antigen.4. The suicide vector pBlueKM40-△hp0520::Kmr was constructed on the basis of an allelic exchange mutagenesis strategy and introduced into H. pylori NCTC11637by electroporation. Positive clones were selected based on antibiotic selection and identified by PCR and western blot analysis and an hp0520-deficient mutant was obtained.5. The H. pylori NCTC11637bacteria cells were collected and separated into three individual fractions including periplasm, cytoplasm and membrane (including inner and outer membrane). Then the subcellular fractionation of the HP0520protein was determined by western blot analysis. Meanwhile, The H. pylori NCTC11637strains were co-cultured with human cell GES-1to determine whether the HP0520protein was secreted into the GES-1cell.6. To explore the interaction of HP0520and other cag PAI proteins, the bacterial two-hybrid system was used. Then the hp0520gene mutant and wild type NCTC11637were collected and prepared for whole-cell lysates. Western blot analysis was used to detect the expression of CagX, CagM, CagL and CagA after hp0520gene mutation.7. The hp0520gene mutant and wild type NCTC11637were co-cultured with GES-1cells respectively. Cell morphology, cytokines secretion and the ability of CagA translocation were detected.Results:1. H. pylori NCTC11637hp0520gene is348bp long and its encoded protein is115amino acids long. The relative molecular mass of HP0520is12.42kDa and the isoelectric point is8.58. There are multiple hydrophilic regions on the both sides of the protein, whereas hydrophobic transmembrane regions exist on the middle area. It is predicted that HP0520owns one signal peptide which is constituted with first21bases with a cleavage between the twenty-first and twenty-second base on the C-terminal.2. The expression vector pET-32a-hp0520was constructed and the target protein was obtained by IPTG induction and purified via QIAGEN system.3. The purified protein was used to immunize mice to obtain the HP20-1cell line, which was able to secret anti-HP0520monoclonal antibody. The titer of the antibody is1:3.2叁105.4. The suicide vector pBlueKM40-A hp0520::kan was constructed and introduced into H. pylori NCTC11637.The H. pylori Ahp0520mutant was screened via kanamycin resistance, PCR and Western blotting.5. The monoclonal antibody, HP20-1, was used for Western blot analysis. The results confirmed that H. pylori was able to synthesize HP0520under natural conditions. Moreover, HP0520was localized in the bacterial membrane and not secreted into host cells.6. The bacterial two-hybrid system has screened that HP0520interacted with HP0521, HP0525, HP0526and HP0538, among which HP0525, HP0526and HP0538were located in the bacterial inner membrane.7. The expression of4critical outer-membrane structural and effector proteins in the Cag-T4SS were compared between the hp0520gene mutant and wild type NCTC11637. The result suggested that deletion of hp0520had no significant influence on the expression of CagX, CagM, CagL and CagA.0. The hp0520gene mutant and wild type NCTC11637were co-cultured with GES-1cells. Then Cell morphology, CagA translocation and cytokines secretion were detected. The data showed that deletion of hp0520gene decreased CagA translocation into host cells and cytokines secretion.Conclusion:1. The full-length hp0520gene was successfully cloned. Bioinformatics methods were used to understand the physico-chemical properties of the hp0520encoded protein and predict signal peptides and transmembrane regions.2. The hp0520gene encoded protein was localized in the bacterial membrane and not able to be secreted into the host cell.3. HP0520protein interacted with HP0525, HP0526and HP0538, which were chaperone-like proteins in the TFSS localized in the inner membrane. Moreover, deficiency of HP0520did not influence the stablization of CagX, CagM, CagL (inner membrane proteins) and CagA (effector protein). This result suggested that HP0520was located in the membrane of H. pylori.4. Deletion of hp0520abolished the host cell ability of cytokines secretion and partial function of CagA translocation, which means the function of T4SS was partly weakened. These results suggested that HP0520was a structure protein in the TFSS localized in the inner membrane.