The Mechanisms Of Chlamydiaphage PhiCPG1 Capsid Protein Vp1 On The Chlamydia Trachomatis

ObjectivesExplore the inhibition mechanisms of Vp1 protein on Chlamydia trachomatis. ContentsInvestigate the changes of Vp1 on Chlamydia trachomatis in microscopy and immunofluorescence microscopy. Investigate the changes of p-ERK1/2, ERK1 and ERK2, inflammatory factors IL-1 and IL-8 in MEK/ERK pathways in different time after Vp1 on Chlamydia trachomatis. Investigate the difference effect of Vp1 and Azithromycin on Chlamydia trachomatis. Investigate the gene changes of Type â…¢ secretion system after Vp1 on Chlamydia trachomatis. Investigate the influence in growth cycle after Vp1 on Chlamydia trachomatis. MethodsVp1 was expressed by recombinant plasmid Vp1-Pet30a(+), identified and purified by SDS-PAGE and Western Blotting, renaturated by dialysis, and prepared for usage after quantification and sterilization. Vp1 and Azithromycin were cultured with Chlamydia trachomatis in Mc Coy cells. We observed the inhibition effects of both Vp1 and Azithromycin in microscopy and immunofluorescence microscopy. Total proteins were extracted in different groups and time and tested the changes of p-ERK1/2 and total ERK1/2 by Western Blotting. Total RNA were extracted in different groups and tested the changes of ERK1 and ERK2 gene by Realtime-PCR method. Culture medium was extracted in different group and time and tested the level of IL-1 and IL-8 by ELISA.Total RNA in cells was extracted after 12 h, 24 h, 36 h, 48 h culture with Vp1 and Chlamydia trachomatis and tested by Realtime-PCR. The genes to be measured included Tarp(CT456), CT694, Inc A(CT119), Tep P(CT875), CT621, Cdsf(CT666), Hct A(CT743), Hct B(CT046), Omc A(CT444), Omc B(CT443) before and after Vp1 treated. Results1. Vp1 and Azithromycin inhibit Chlamydia trachomatis growth in Mc Coy cells. Inclusions decrease under the microscopy and immunofluorescence microscopy and degree is associated with concentration of Vp1 and Azithromycin. Vp1 may combine to the protein in Chlamydia trachomatis membrane and follow it moving into host cells. 2. Vp1 and Azithromycin make the expression of p-ERK1/2, ERK1 and ERK2 decrease, especially the ERK1. 3. Azithromycin inhibits Chlamydia trachomatis at early time of growth cycle, while Vp1 inhibits at late time. 4. IL-1 and IL-8 decrease after Vp1 and Azithromycin treated, accompany with inflammation relieve. IL-1 decreases at late time, while IL-8 decreases at early time in Vp1 treated group and at late time in Azithromycin treated group. 5. Type â…¢ secretion system of Chlamydia trachomatis is cut down after Vp1 treated. Cdsf(CT666)decreases at early time to limit the infection ability of EB. Tarp(CT456) and CT694 decrease at late time to limit the invasion ability. Inc A(CT119)increases at late time to make RB homotypic fusion. Cdsf(CT666)and CT621 decrease at late time to diminish the RB to EB. Tep P( CT875) decreases in whole cycle to interfere the signal pathway and immunological reactions between Chlamydia trachomatis and hosts. 6. Hct A(CT743), Hct B(CT046), Omc A(CT444)and Omc B(CT443)decrease in late time after Vp1 treated to inhibit RB to EB. ConclusionVp1 may inhibit Chlamydia trachomatis growth by restricting the process of RB to EB in late growth cycle.