| Objective:Chlamydia trachomatis(C.trachomatis,Ct)is a common obligate intracellular parasite,which can inhibit host cells in various strategies to achieve replication.Recent studies show that activation of PI3 K /AKT-mediated MDM2-p53 pathway plays a prominent part in development and apoptosis resistance of Chlamydia infection.However,the precise upstream mechanisms of how Ct activates this pathway remain unclear.Ct-secreted plasmid protein pORF5 is a major virulence factor and is closely associated with the pathogenicity of Ct.Here,our research was to investigate the relationship between Ct-secreted plasmid pORF5 and host cell apoptosis,and further to explore whether its precise molecular mechanisms was regulated by PI3K/AKT signaling pathway mediated MDM2-p53 axis.This research provides a basis for clarifying the role of pORF5 in the pathogenic mechanism of Ct.Methods: The recombinant plasmid pGEX-6p-1/pORF5 was transformed into E.coli XL1-blue strain,and 0.2mM IPTG was used to induce the expression of GST-pORF5 fusion protein.The fusion protein was purified by Glutathione Sepharose 4B and the GST tag was removedby PreScission Protease.The pORF5 plasmid protein was examined and identified by SDS-PAGE and Western blotting,its concentration was determined by BCA,finally,polymyxin B was applied to remove endotoxin.Western blotting was used to evaluate the expression of Bax and Bcl-2 in HeLa cells after stimulated with pORF5 at different concentrations(0,5,10,15,25μg/ml)and times(0,8,12,24h),as well as the expression of p53.Moreover,Hoechst staining and Flow cytometry were applied to measure the apoptosis of HeLa cells which were stimulated with pORF5 at the optimal concentration for the optimal time periods.After stimulation of HeLa cells with pORF5 at an optimal concentration for different time periods(0,5,15,30,60min),Western blotting was used to monitor the phosphorylation of AKT and MDM2.And indirect immunofluorescence was applied to detect the localization of MDM2.After pre-treatment with 20μM PI3 K inhibitor LY294002 for1 h,He La cells were stimulated with pORF5 at an optimal concentration for 30 min,Western blotting was used to examine the phosphorylation of AKT and MDM2,and indirect immunofluorescence was applied to detect the localization of MDM2.After pre-treatment with 20μM PI3 K inhibitor LY294002 and 10μM,20μM MDM2-p53 inhibitor for 1h,HeLa cells were stimulated with pORF5 at an optimal concentration for an optimal time periods,the expression of p53,Bax and Bcl-2 were examined by Western blotting,and Hoechst and Flow cytometry were applied tomeasure the apoptosis of HeLa cells.Results:1、Western blotting showed that the concentrations of p ORF5 reached to 10μg/ml,the expression of Bax and p53 was down-regulated,and the expression of Bcl-2 was up-regulated.And when the concentrations of pORF5 reached to 15μg/ml,the change of Bax,p53 and Bcl-2 expression was most obviously.Therefore,using a concentration of15ug/ml to stimulate HeLa cells for various times,in contrast the control group,when the stimulation time reached to 8h,the expression of Bax,p53 was down-regulated,the expression of Bcl-2 was up-regulated,when the stimulation time reached to 24 h,the change of Bax,p53 and Bcl-2expression was obviously.In contrast to the TNF-α positive group and the control group,Hoechst staining revealed that the apoptosis rate of pORF5-treated group decreased by 25.6%(P<0.001)and 6.13%(P<0.001)respectively.Flow cytometry suggested that the the apoptosis rate of p ORF5-treated group decreased by 27.55%(P<0.001)and 7.248%(P<0.001)respectively.2、Western blotting also showed that MDM2 and AKT were phosphorylated at 15 min,most prominently at 30 min,and the total MDM2 and AKT protein levels were almost not changed.And treatment with PI3 K inhibitor LY294002 on HeLa cells,the phosphorylation of MDM2 and AKT was obviously declined.3、Indirect immunofluorescence revealed that MDM2 translocated to the nucleus of HeLa cells in the presence of pORF5,and this translocation of MDM2 was blocked by LY294002.4、Pre-treatment with MDM2-p53 inhibitor Nutlin3 a and PI3 K inhibitor LY294002 on HeLa cells,Western blotting showed that the expression of Bax and p53 was obviously up-regulated,and the expression of Bcl-2 was obviously down-regulated.In contrast to the control group,the apoptosis rate of Nutlin3 a treatment group increased by10.47%(P<0.001),and the apoptosis rate of LY294002 treatment group increased by 13.02%(P<0.001)by Hoechst staining.Flow cytometry revealed that the apoptosis rate of Nutlin3 a treatment group increased by6.14%(P<0.01),and the apoptosis rate of LY294002 treatment group increased by 7.5%(P<0.01).Conclusions:Chlamydia trachomatis-secreted plasmid protein pORF5 inhibited host cell apoptosis via PI3K-AKT mediated MDM2-p53 axis... |
PDF Full Text Request