Study On The Pathogenic Mechanism Of Adhesion Protein 65 （TvAP65） In Trichomonas Vaginalis Infection

BackgroundTrichomonas vaginalis（T.vaginalis）,as a facultative anaerobic parasitic protozoa,is commonly parasitic on the urethra,prostate,testes,epididymis and subcutaneous tissue of foreskin of males as well as the vagina of females.Trichomoniasis is a sexually transmitted disease caused by the infection of T.vaginalis,which is widely prevalent worldwide.When adhering to the parasitic site,T.vaginalis,as an extracellular parasitic protozoa,can directly damage the epithelial cells and cause inflammation of tissues due to a combination of effects such as mechanical injury of pleomorphic pseudopodia walking and wrapping and flagellum movement,digestion of releasing hydrolase,phagocytosis and comprehensive effect of abscission factor.Thus,adhesion to the host cell surface is a key step in the parasitization and pathogenesis of T.vaginalis.Meanwhile,the T.vaginalis adhesion protein 65（TvAP65）plays an important role in the adhesion process as one of the major adhesion proteins.However,the pathogenic mechanism of TvAP65 in T.vaginalis infection is still unclear.PurposeOur study is to clarify the pathogenic role of TvAP65 in the process of T.vaginalis infecting host cells,evaluate the possibility of TvAP65 as a vaccine candidate antigen against T.vaginalis infection,and provide a theoretical basis for intervention of Trichomoniasis.Methods1.TvAP65 gene was cloned.The prokaryotic expression vector was constructed and the recombinant TvAP65 protein（r TvAP65）was expressed and purified.TvAP65 polyclonal antibody was prepared by immunizing rats.2.The immunogenicity of r TvAP65 protein and the molecular size of natural TvAP65 were analyzed by immunoblotting.3.The localization of TvAP65 in T.vaginalis trophozoites and the binding of r TvAP65 protein to human vaginal epithelial cells（VK2/E6E7）were analyzed by immunofluorescence.4.Liposome transfection and immunofluorescence were used to optimized conditions for T.vaginalis transfected with si RNA.The expression of TvAP65 in T.vaginalis trophozoites was knocked down by RNA interference.The primers were screened for detecting TvAP65 m RNA levels.The levels of TvAP65 m RNA and protein were detected by q PCR and Western blot for confirming the expression of TvAP65 decreased in T.vaginalis.5.After the expression of TvAP65 was knocked down,VK2 / E6E7 cells were infected with T.vaginalis labeled with fluorescent probes.The trophozoites adhered to VK2/E6E7 cells were counted under fluorescence microscope,in order to analyze the role of TvAP65 protein in the adhesion of T.vaginalis to host cells.6.MTT assay,flow cytometry and trypan blue staining were used to analyze the effects of T.vaginalis on the proliferation,apoptosis and death of VK2/E6E7 cells after knockdown of TvAP65 expression.7.Both mice actively immunized with r TvAP65 protein and passively immunized with TvAP65 polyclonal antibody were infected intraperitoneally by T.vaginalis trophozoites,and normal mice were infected intraperitoneally with T.vaginalis trophozoites blocked by TvAP65 polyclonal antibody.The survival time of all infected mice was observed to analyze the pathogenicity of trophozoites in infected mice after TvAP65 protein were blocked,and to evaluate the possibility of TvAP65 as a vaccine candidate antigen.Results1.The results of nucleic acid gel electrophoresis showed that the TvAP65 gene was successfully amplified and the prokaryotic expression vector p ET-32a-TvAP65 was constructed.The results of SDS-PAGE gel electrophoresis showed that the r TvAP65 was obtained and a high titer anti-TvAP65 polyclonal antibody was prepared.2.The results of Immunoblotting showed that the r TvAP65 protein was recognized by the serum of mice infected with T.vaginalis,while the molecular size of the natural TvAP65 protein was 70 k Da,slightly larger than the predicted molecular size（63.13 k Da）.3.Immunofluorescence analysis showed that TvAP65 protein was mainly localized on the surface of T.vaginalis trophozoites and could bind to VK2/E6E7 cells.4.The optimal duration of si RNA transfection using liposomes for T.vaginalis trophozoites was 24 h and the optimal concentration was 200 nmol.The q PCR and Western blot results showed that the expression of TvAP65 was successfully knocked down in trophozoites.5.Observation under microscope showed that the number of T.vaginalis trophozoites adhering to VK2/E6E7 cells was decreased significantly（P<0.001）,after the expression of TvAP65 was knocked down.6.The trophozoites significantly decreased the inhibition of VK2/E6E7 cells proliferation,and reduced VK2/E6E7 cell apoptosis and death（P<0.001）,after the expression of TvAP65 was knocked down..7.Compared with control groups,the survival time of mice actively immunized with r TvAP65 protein or passively immunized with TvAP65 polyclonal antibody,and mice infected with T.vaginalis trophozoites blocked by TvAP65 polyclonal antibody was significantly prolonged（P<0.01）.Conclusions1.As one of the adhesion proteins of T.vaginalis,TvAP65 was mainly located on the surface of T.vaginalis trophozoites and could bind to host cells.2.After the expression of TvAP65 was knocked down,the number of adhesion host cells of T.vaginalis was decreased,and the ability of trophozoites to inhibit host cell proliferation and induce host cell apoptosis and death was reduced.3.Animal challenge showed that the pathogenicity of trophozoites decreased after blocking TvAP65,and TvAP65 could be used as a vaccine candidate antigen against T.vaginalis infection.