| Background Chlamydia trachomatis(C.t)urogenital tract infection is the most common sexually transmitted disease in recent years.About 2/3 of C.t infected people have no symptoms,but the infection is sustainable and causes a series of serious complications,such as pelvic inflammation,ectopic pregnancy and tubal infertility in women,urethritis and epididymitis in men.The concealment of symptoms increases the probability of transmission of infection in the population.Because of the special biological characteristics of C.t,the therapeutic effect of antibiotics is not satisfactory.So far,there is no vaccine with effective immune protection.C.t is internationally recognized as a threat to human public health.According to the latest research on the importance of biological microenvironment to human body,accurate biological treatment for specific pathogenic bacteria without affecting other bacteria groups is the ideal target of international pursuit,but so far it is not yet.The severe microbial resistance situation has prompted us to explore and establish new effective mechanisms and specific methods that may,to some extent,provide valuable treatment options for unmet clinical needs.The effect of Vp1 protein on C.t was found to be inhibited / destroyed by 65%-92%.The effect has been verified in the model of genital tract infection of C.t in mice,and the protein has no inhibitory effect on other common genital tract bacteria.This may be a new mechanism of bacteriophage inhibiting and destroying its corresponding host.Objectives To explore the inhibition of Chlamydia virus CPG1 capsid protein Vp1 of C.t even damage effect and mechanism,providing a new and highly specific treatment methods for clinical chronic refractory infection of C.t,which different from the traditional concept of phage display new mechanisms of host bacteria.Contents After the incubation of Chlamydia virus CPG1 capsid protein Vp1 in the process of C.t infection to host cells,changes of host cell protein levels of p53 and Mcl-1,cIAP-2 and Puma transcription level of;inhibitory effect of blocking PmpI by PmpI polyclonal antibody after inhibition of Vp1;the change of C.t incA and cadd at the transcriptional level after incubation with Vp1.Methods The recombinant plasmid Vp1-Pet30a(+)was induced to express Vp1 protein and purified and refolded.A small amount of purified Vp1 was used for SDS-PAGE electrophoresis.After refolding,the Vp1 protein was quantified and filtered for subsequent use.The purified Vp1 protein acted on the host cells infected with different serotypes of C.t(D,E and G),and the number of inclusion bodies was detected by iodine staining and indirect immunofluorescence assay.The total protein of the cells was extracted,the level of p53 protein was determined by Western Blot method,and the total RNA of the cells after the extraction was extracted and reverse transcribed into cDNA.The changes of Mcl-1,cIAP-2,Puma and incA in the transcription level of HeLa cells were determined.The cytotoxic effects of C.t on HeLa cells before and after Vp1 intervention were measured by flow cytometry,and the apoptosis levels of HeLa cells before and after intervention were compared.To demonstrate the effective inhibition of C.t by Vp1.Detection of protein interaction between PmpI and Vp1 using Far-Western Blot technique.The effect of blocking PmpI protein on Vp1 inhibition was observed by iodine staining after PmpI protein was blocked by PmpI polyclonal antibody.The time of inhibition of C.t growth by Vp1 was analyzed,and the changes in the transcription level of Chlamydia toxic protein CADD were analyzed by qPCR.Results After HeLa cells were infected by C.t,the level of p53 protein decreased,Mcl-1 and cIAP-2 genes increased at transcription level,and puma gene decreased at transcription level.When Vp1 was infected with C.t D strain,E strain and G strain infected with HeLa cells,the inclusion body after iodine staining was significantly reduced compared with that in the untreated group.After Vp1 intervention,the p53 protein levels and the mRNA levels of Mcl-1,cIAP-2 and Puma of HeLa cells were recovered.These results indicate that Vp1 protein can effectively inhibit the growth cycle of C.t.incA is also elevated at transcriptional level.IncA is secreted by type III secretion system,indicating that type III secretion system is disrupted.Type III secretion system may be the downstream signaling pathway of PmpI protein.After the PmpI polyclonal antibody blocked the PmpI protein on the surface of C.t,the inhibitory effect of Vp1 protein was decreased.CADD protein increased at the transcriptional level.Conclusions PmpI protein is the binding site of phage Chlamydia virus CPG1 capsid protein Vp1 in the outer membrane of C.t.It is speculated that PmpI will transduct the stimulation into C.t after Vp1 combine with PmpI.This signal may induce the reverse disruption of CADD to C.t transducted by the type III secretion system or two component transduction system.Thus,C.t is suppressed or even destroyed. |
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