Identification And Characterization Of Hp0523 Gene From Cag Pathogenicity Island In Helicobacter Pylori

Cytotoxin-associated gene A(CagA) is the most important virulence factor in Helicobacter pylori(H.pylori),which involved in the development of gastric carcinoma in human.It was translocated into the human gastric cell via a typeâ…£-like secretion apparatus that encoded by the Cag pathogenicity island(Cag-PAI) in H. pylori.However,the function of genes and the mechanism of pathogenicity in Cag-PAI are not dear.Therefore,we focused on the identification and characterization of the hp0523 gene from Cag-PAI in this study.Moreover,we explored the role of hp0523 in the process of CagA's translocation.All of these results will shed light on the mechanism of pathogenicity in Cag-PAI.Methods:1.The hp0523 gene was amplified from the H.pylori genomic DNA via a PCR method.The plasmid pGEM-T-hp0523 was constructed via T-A clone method,and sequenced by Sangong(shanghai,China).The sequence of hp0523 was analyzed through bioinformatics approach.2.The expression vector pET-28a-hp0523 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 IPTG.The target protein HP0523 was purified and collected by Ni²⁺-NTA column.3.The fragment F1 and F2,which from the upstream and downstream of hp0523 gene, were amplified by PCR method for the construction of suicide plasmid pBlueKM40/AhpO523::Km^r.The suicide plasmid was introduced into H.pylori via electroporation.The mutant was screened via antibiotic selection medium and identified by PCR method.4.The hp0523 gene mutant and wild type were co-cultured with human gastric epithelial cell BGC-823.The ability of CagA's translocation was dertermined by Western blot method. Results:1.We cloned hp0523 gene from the H.pylori genomic DNA.DNA sequence analysis showed that hp0523 is composed of 510 bp,which encoded a polypeptide containing 169 amino acids with a predicted molecular mass(Mr) of 19.7 kDa and a theoretical iso-electric point(pI) at 9.53.The deduced amino acid sequence of hp0523 in NCTC11637 is shares 92～95%identity to hp0523 from 26695 and J99 from GenBank. The secondary structure analysis indicated that hp0523 might contain a conserved SLT domain between residues 33 to 165.The alignment indicated that the GLU56 site in hp0523 amino acid sequence that may be the central site for the catalytic activity and the "AVGAY" in C terminal is a motif for the peptidoglycan binding.Combining these data,we predict the hp0523 gene might act as a peptidoglycan hydrolase.2.We constructed the expression vector pET-28a-hp0523 and transformed into E.coli BL21(DE3).The result of SDS-PAGE and Western blot suggested that HP0523 protein expressed after induced by IPTG.The target protein HP0523 was purified and collected.3.The spot assay indicated that the purified protein HP0523 could exhibit the lytic activity after renatured.The zymogram gel analysis suggested that HP0523 has peptidoglycan hydrolase activity against the bacterial peptidoglycan,which separated from S.aureus by SDS boiling method.The biochemical characterization analysis suggested that HP0523 has broad-spectrum lytic activity,which much lower lytic activity than lysyzome and the optimum activity occurred at approximately pH 6.0.4.We amplified the fragment F1 and F2,which from the upstream and downstream of hp0523 gene,and constructed the suicide plasmid pBlueKM40/ΔhpO523::Km^r.The PCR result suggested that hp0523 gene mutant that selected from antibiotic medium after electroporation.The co-cultured assay indicated that HP0523 could interrupt the CagA's tranlocation.Conclusion:In this study,we identified and characterized hp0523 gene as a peptidoglycan hydrolase gene in Cag-PAI.It also suggests that hp0523 gene may be a component of Cag Type IV like secretion apparatus that involved in CagA's translocation.