Study On The Function And Pathogenicity Of A New Serine Protease Autotransporter In Neisseria Gonorrhoeae

Objective:In the early stage of our research group,bioinformatics analysis suggested that NGO2105 protein may be a new serine protease autotransporter in N.gonorrhoeae.In this study,we intend to prove that whether the NGO2105 protein has serine protease autotransport characteristics and its role in the the pathogenic process,to provide important experimental evidence for further understanding the pathogenic mechanism of N.gonorrhoeae and exploring new protein vaccine targets.Methods:Prokaryotic expression of NGO2105 protein passenger domain truncated protein and preparation of polyclonal antiserum,for localization analysis.Construction of gonococcal ngo2105 gene deletion mutants by deletion mutation method,and ngo2105gene complement strains were constructed by pCTS32 plasmid.Flow cytometry and western blot were used to analyze the surface localization and secretion characteristics of NGO2105 protein.In order to verify whether its serine protease activity is involved in the cleavage and secretion of passenger domain,point mutation technology was used to mutate serine 267 to alanine,the serine is the main amino acid in the triad structure catalyzed in NGO2105 protease activity.The passenger domain of Hbp autotransporter protein in E.coli was fused with the translocator domain of NGO2105 protein,and the surface location of the passenger domain of Hbp protein was analyzed to verify whether the translocator domain of NGO2105 protein can transport heterologous proteins.Using cervical cancer epithelial cells to investigate the role of NGO2105 protein in the adhesion and invasion of N.gonorrhoeae,and to study the adhesion inhibition ability of anti-NGO2105 antibody and anti-passenger antibody.An estrogen-treated mouse N.gonorrhoeae vaginal infection model was used to study the role of NGO2105 protein in N.gonorrhoeae colonization and the role of anti-NGO2105 antibodies in inhibiting N.gonorrhoeae colonization.Results:After successfully expressing and purifying the NGO2105 protein passenger domain truncated protein,polyclonal antiserum with a titer of 5.12×10~5 can be obtained after immunizing mice,which can meet the requirements of subsequent experiments.The ngo2105 gene deletion mutant strains and the ngo2105 gene complement strains were successfully constructed.Flow cytometry and western blot analysis showed that NGO2105protein was localized on the surface of the bacteria,then cleavaged and secreted into the external environment by autocatalytic hydrolysis.Successfully construced the NGO2105S267 point mutation strains,western blot confirmed that the passenger domain of the NGO2105 protein cannot be hydrolyzed and secreted into the culture medium after serine point mutation,flow cytometry analysis showed that NGO2105 protein could still be located on the surface of bacteria after point mutation,it was suggested that the serine protease activity of NGO2105 protein mediated the autologous cleavage and secretion of the passenger domain.The fusion protein of Hbp protein passenger domain and NGO2105protein translocator domain was successfully constructed,flow cytometry and immunofluorescence microscopy showed that the Hbp protein passenger domain was localized on the bacterial surface,it was suggested that the NGO2105 protein translocator domain has the function of transporting heterologous proteins.Cell adhesion and invasion experiment results showed that the adhesion and invasion ability of N.gonorrhoeae ngo2105 gene deletion mutant strains were significantly lower than that of WT strains,and the anti-NGO2105 antibodies and anti-passenger antibodies could inhibit the adhesion of gonococci.The results of mouse vaginal colonization experiments showed that the ability of N.gonorrhoeae ngo2105 gene deletion mutant strains to colonize in vivo was significantly lower than that of WT strains,and anti-NGO2105 antibody could inhibit the vaginal colonization ability of N.gonorrhoeae.Conclusion:This study confirms that NGO2105 is a new serine protease autotransporter in N.gonorrhoeae,which is consistent with the characteristics of autotransporters.NGO2105 plays an important role in the adhesion and invasion of N.gonorrhoeae and intravaginal colonization in mice,and it is an important virulence factor in the pathogenesis of N.gonorrhoeae.Anti-NGO2105 antibody can effectively inhibit the adhesion ability of N.gonorrhoeae and colonization ability in mice,suggesting that the NGO2105 protein may be a potential protein vaccine target.