The Research Of Cagi Gene From Cag Pathogenicity Island In Helicobacter Pylori

Helicobacter pylori (H. pylori), one of the common pathogens, is a spiral-shaped bacterium that colonizes the human gastric mucosa. It is estimated to inhabit at least half of the world's human population. H. pylori has been identified as the major causative factor of chronic gastric and peptic ulcer disease and closely related to the occurrence and development of gastric carcinoma, mucosa-associated lymphoid tissue (MALT) lymphoma. Cytotoxin-associated gene A (CagA) is one of the most 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 IV secretion system that encoded by the Cag pathogenicity island (Cag-PAI). However, the function of genes and the mechanism of pathogenicity in Cag-PAI are not clear. Therefore, we focused on the identification and characterization of the cagI gene from Cag-PAI in this study. We explored the funtion of cagl gene through molecular biology, immunology and microbiology techniques. All of these results will shed light on the mechanism of pathogenicity in Cag-PAI.Methods:1. Oligonucleotide primers were designed according the sequence of H. pylori 22695 in GenBank, using Primier 5.0 software. H. pylori cagI gene was amplified from the genome DNA of H. pylori NCTC 11637 by PCR. The plasmid pMD18-T-cagI was constructed via T-A clone method, and sequenced by Sangong (shanghai, China). The sequence of the gene was analyzed through bioinformatics approach by DNA Star,Clustal 1.8 and Merga4.0. The phylogenetic trees were constructed on the encoding sequences of cagI gene.2. The expression vector pET-28a-cagI was constructed and transformed into E. coli BL21 (DE3) by molecular cloning method. The protein was expressed and characterized via SDS-PAGE and Western Blot methods after induced by Isopropylthio-β-D-Galacgoside (IPTG). The target protein was purified and collected by Ni2+-NTA column. Prepared antibody of the CagI protein by immunizing rabbit, and detected the titer by using enzyme-linked immunosorbent assay (ELISA).3. H. pylori was harvested and resuspended. The lysate was fractionated into periplasmic fractions,cytosolic fractions and membrane fractions (including inner membrane and outer membrane) by ultracentrifuge. The subcellular localization of CagI was detected by Western Blot.4. Human gastric epithelial (GES-1) cells infected by H. pylori were harvested and resuspended. The lysate was fractioned into p fractions(a pellet containing bacteria) and S (supernatant containing cell cytosol) fractions. The ability of CagI's translocation was dertermined by Western Blot method.5. The bacteria membrane(including inner and outer membrane) fraction were immunoprecipitated with CagI polyclonal antibody and Protein G. Then proteins were analyzed by SDS-PAGE analysis and immunoblotted with anti-CagI and anti-CagA, respectively. The lysate of His-CagI E. coli were incubated with the bacteria membrane. Using His pull-down, co-immunoprecipitation and peptide mass fingerprinting with MALDI-TOF, it was found that CagI interacted with CagA.6. Two DNA fragments were amplified as homologous arms, according to the sequences of th upper and down stream of coding region of cagl gene, and the targeting vector(pBluescript/△cagI::KMr) was constructed. Then we introduced the suicide plasmid pBluescript/△cagI::KMr into H. pylori NCTC 11637 by electroporation and screened the mutant based on antibiotic selection. The mutant was checked by PCR and gene sequencing. The cagL knocked-out mutant was constructed as same as cagI knocked-out mutant(positive control).7. The cagl gene mutant and wild type were co-cultured with GES-1 cells. The ability of CagA's translocation and H. pylori strain adherence were dertermined.8. The experimental data were analyzed by using the software 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. Results:1. We cloned cagl gene of H. pylori NCTC 11637 genomic DNA. It is 1086 base pairs long, which encodes a product of 361 amino acids. GenBank accession number is HM126476. The sequence of the nucleotide analysis for cagl showed that it shares 98.3% and 98.1% homology with H. pylori 26695 and J99 in Genbank, respectively. The sequence of the amino acids for CagI showed the highest homology(99.5%) with H. pylori 26695. DNA Star software predict that the CagI's molecular weight is 39.37kDa.2. The prokaryotic expression vector pET-28a-cagI was efficiently transformed into E. coli BL21 (DE3). The protein was expressed in E. coli BL21 (DE3)at 30℃after 0.8mmol/L IPTG induction for 8h. After expressed by Isopropylthio-β-D-Galacgoside (IPTG) induction, the result of Western Blot indicates that the recombinant protein could be recognized by the antibody of anti-His. The expressed product reached a purity of 98 % after Ni2+-NTA column chromatography. Using recombinant CagI to immunize rabbit, polyclonal antibody titer achieved 1:1.6×105.3. The result of Western Blot suggested that CagI was localized to the bacterial membraner(including inner and outer membrane). CagI remained to be associated with the bacterial pellet and did not appear to be translocated into host cells. Similarly, we detected CagA delivery which is translocated into host cells as a control. Then the result of immunoprecipitation and pull-down showed that CagI interacted with CagA. Moreover, MALDI-TOF detected CagI co-immunoprecipitated with CagA.4. Two homologous-arm fragments (612bp and 796bp) and (651bp and 666bp) were PCR-amplified, respectively. These DNA fragments were engineered into the pBluescript SKⅡ(-) plasmid to construct the targeting vector pBluescript/△cagI::KMr and pBluescript/△cagL::KMr, respectively. Restriction endonuclease analyses showed that pBluescript/△cagI::KMr and pBluescript/△cagL::KMr had been correctly recombined. H. pylori NCTC11637 strains were electrotransformated with the targeting vector of pBluescript/△cagI::KMr and pBluescript/△cagL::KMr, and the kanamycin resistance H. pylori transformants were screened in the Colombia plates containing kanamycin. The cagI and cagL deleted status was identified by the PCR.5. The cagI knocked-out mutant and wild type were co-cultured with GES-1 cells. CagI could attenuate the CagA's translocation in cagI knocked-out mutant. CagL could interrupt the CagA's tranlocation in the cagL knocked-out mutant. Moreover, the co-cultured assay indicated that the ability of H. pylori strain adherence was lower in cagI knocked-out mutant than H. pylori. Conclusion:1. We have cloned the cagI gene successfully, the cagI was the structure genes of T4SS encoded by Cag-PAI in H. pylori. The homogenicity of nucleic acid and that of amino acid was high.2. Constructed the prokaryotic expression vector pET-28a-cagI, obtained CagI recombinant proteinsand prepared polyclonal antibody.3. CagI was a bacterial membrane-associated protein and localized in the bacterial membrane(including inner and outer membrane). Moreover, CagI was not translocated into host cells and co-immunoprecipitated with CagA.4. pBluescript/△cagI::KMr and pBluescript/△cagL::KMr were constructed and the cagI and cagL knocked-out mutant were constructed.5. CagI could attenuate the CagA's translocation in cagI knocked-out mutant and the ability of H. pylori strain adherence was lower in cagI knocked-out mutant than H. pylori.