Chlamydial CT849 Protein Regulates Host Cell Apoptosis Through Activation Of ERK1/2 And P38 MAPK Signaling Pathway

Objective: To explore whether chlamydia CT849 protein has anti-apoptotic effect,and to further clarify whether CT849 protein can regulate host cell apoptosis and its molecular mechanism by activating the MAPK signaling pathway,and provide data analysis for further exploring of the pathogenic mechanism of Chlamydia trachomatis.Methods: : Inoculate the pGEX-6p-1/ct849 recombinant plasmid bacteria constructed in the early stage of this research group into LB solid medium for culture,and then expand the culture and express the GST-CT849 fusion protein after IPTG induction.After the protease removes the label,remove the internal The toxin obtains the CT849 protein,and the BCA method determines the CT849 protein concentration.After stimulating HeLa cells with 0,2,4,6,10,15 μg/m L CT849 protein for 0,4,6,8,12,and 24 h,western blot was used to detect the expression of apoptosis-related proteins Bax and Bcl-2 in infected cells,Hoechst33258 staining and flow cytometry were used to detect the apoptosis rate of HeLa cells.The phosphorylation levels of ERK1/2,JNK and p38 in HeLa cells were detected by western blot at different time points(0、5、 15、 30、60、 and 120 min)after CT849 protein was stimulated with the optimal concentration.HeLa cells were pretreated with 10 μM ERK1/2 specific inhibitor PD980595 and 5 μM p38 specific inhibitor SB203580 for 1h,and then stimulated with CT849 protein at the optimal time point.Western blot technology was used to detect the phosphorylation levels of ERK1/2,JNK,p38 and the expression levels of apoptosis-related proteins Bax and Bcl-2 in infected cells.Hoechst33258 staining and flow cytometry were used to detect cell apoptosis.Results:1.pGEX-6p-1/ct849 recombinant bacteria are induced by IPTG to express GST-CT849 recombinant protein,with a molecular weight of about 44 k Da;it is digested and purified to a relatively pure CT849 protein.2.The expression level of Bcl-2 and Bax protein changes in a certain time and concentration-dependent manner.HeLa cells were stimulated with different concentrations of CT849 recombinant protein,and it was found that when the concentration of CT849 protein was 10 μg/m L,the ratio of Bax/Bcl-2 was significantly reduced.Then HeLa cells were stimulated with 10 μg/m L CT849 protein at different time points.The results showed that when CT849 protein stimulated HeLa cells for 12 h,the expression of Bcl-2 increased and the expression of Bax decreased,and the ratio of Bax/Bcl-2 was found to decrease significantly.Hoechst staining showed that CT849 combined with apoptosis inducer group had a significantly lower apoptosis rate compared with the inducer positive group;the results of flow cytometry.It is consistent with the results of Hoechst immunofluorescence.3.Western blot results showed that HeLa cells stimulated with 10μg/m L CT849 protein,the phosphorylation levels of ERK1/2 and p38 were significantly increased.ERK began to be phosphorylated from 15 minutes after stimulation,and reached the peak at 30 minutes after stimulation,while p38 began to be phosphorylated from 30 minutes after stimulation,and reached the peak at 1 hour after stimulation.There was no significant change in the phosphorylation level of JNK.4.ERK1/2 inhibitor PD98059 and p38 inhibitor SB203580 can effectively inhibit its phosphorylation expression;compared with the CT849 protein stimulation group,after pretreatment with PD98059 or SB203580 inhibitor for 1 h.It was found that in infected HeLa cells,the expression of Bcl-2 protein decreased,the amount of Bax protein increased,and the Bax/Bcl-2 ratio decreased significantly.The apoptosis rate of the ERK1/2 inhibitor group or the p38 inhibitor group was increased,and the apoptosis phenomenon was more obvious.The results of flow cytometry found that it was compared with the DMSO/CT849 control condition.Compared with the CT849 control group,the apoptosis rate of the ERK1/2 inhibitor group or the p38 inhibitor group increased,which was consistent with the aforementioned results.Conclusion: Chlamydial CT849 protein can inhibit cell apoptosis by activating ERK1/2 and p38 MAPK signaling pathways.