The Effect Of Chlamydiaphage PhiCPG1Capsid Protein Vp1on The Chlamydia Trachomatis

Chlamydia trachomatis(C.t) infections are among the most prevalent sexually transmitted diseases, frequently leads to male urethritis and female mucopurulent cervicitis,sequelae such as pelvic inflammatory disease, ectopic pregnancy, infertility but the effect of antibiotic treatment is not satisfactory. phiCPGl which is the second chlamydiaphage,gained access to growing chlamydiae by attaching to the EB. The infection of phiCPGl could attenuate chlamydial infection by arresting chlamydial developmental cycle at the reticulate body stage, making abnormally enlarged RB appear and reducing the infectious EB yield. This phenomenon also occurs in condition of gamma interferon treatment,which demonstrates that chlamydiaphage has potentially therapeutic value in clinic. Vp1is the main structure protein of chlamydiaphage, it maybe play an important role on chlamydiaphage’s infections.[Objective] To express and purify recombinant Vpl from phiCPGl in GPIC, and to investigate the effect of Vpl on C.t after Vpl was co-cultured with C.t (reference strains and clinical strains).[Methods] Vpl protein (the final concentration53μg/ml) and equal amount of Chlamydia trachomatis standard strains (E/UW-5/Cx and D/UW-3/Cx) or clinical strains, which had been incubated with Vpl protein at the concentration of53μg/ml for3hrs at room temperature, were inoculated into McCoy. After cell culture, idione stain and transmission electron microscope were used to observed the effect of Vpl on the chlamydia trachomatis. The effect of Vpl protein on the cell line McCoy was determined by MTT assay, the responses of Escherichia coli BL21and DH5a toward Vpl protein were determined using broth microdilution assays; Recombinant MOMP or EB of chlamydia trachomatis were injected into New Zealand rabbits to product antibodies, confocal fluorescence microscope technique with anti-MOMP antibody and anti-Vpl monoclonal antibody was used to detecte whether Vpl and inclusion of chlamydia trachomatis were located in the same place of McCoy; Far-Western blot was utilized to investigated the binding protein of Vpl on chlamydial outer membrane.[Results] We showed that after co-culture Vpl had obviously inhibitive effect on C.t, the inhibition ratios were about30-70%in clinical strains,72%in reference strain D and78%in E, respectively. There was no effect of Vp1on McCoy cells and bacteria BL21and DH5a. More importantly, abnormally enlarged RBs were observed after Vpl-treatment and Vpl could arrest chlamydial developmental cycle using electron microscope.Polyclonal antibodies were get after recombinant MOMP or EB of chlamydia trachomatis were injected into New Zealand rabbits,respectively, the titer of antibodies was to1:12800. The anti-MOMP antibody was more specific than the anti-EB antibody, because the anti-EB antibody could react with McCoy without inclusion.Vpl and chlamydia trachomatis were located in the same place of McCoy by confocal fluorescence microscope.Pmp I was the Vpl-binding protein on the chlamydial outer membrane by Far-Western blot.[Conclusion] The recombinant Vpl from phiCPGl in GPIC has obviouslly inhibitive effect on the growth of chlamydia trachomatis,and Vpl can bind to PmpI on the outer membrane of chlamydia trachomatis. These findings provide the first experimental evidence that Vpl protein is the important virulence factor of chlamydiaphages and the impact was carried out by binding to its specific receptor. The data demonstrate marked improvement in understanding the process of chlamydiaphage’s infection.