Chlamydia Trachomatis CT622 Effector Protein Inhibits HeLa Cell Apoptosis Through Erk/p38 MAPK Signaling Pathway

Objective:Chlamydia trachomatis(Ct)is a prokaryotic microorganism with a unique biphasic developmental cycle.It is one of the most common pathogens causing sexually transmitted diseases worldwide,and its infection is also the main cause of clinical infertility.Chlamydia trachomatis needs to be parasitized in eukaryotic cells throughout its life cycle.It interacts with host cells by secreting effector proteins to manipulate key signaling pathways in host cells,and has evolved effective strategies to inhibit host cell apoptosis during infection.to complete its own growth and development.Type Ⅲ secretion system(T3SS)is the key to the virulence of Ct,and mediates the interaction with the host through the secretion of effector proteins.The important role,however,of CT622 protein on host cell proliferation,apoptosis and related signaling pathways remains unclear.In this study,HeLa cells were used as a cell model to explore the regulatory effect of Chlamydia trachomatis effector protein CT622 on host cell apoptosis,and to study the key signaling pathways that CT622 participates in regulating host cell apoptosis pathways,and further elucidate the role of CT622 in the pathogenesis of Ct.effect.In this study,HeLa cells were used as target cells to explore the regulation of C.trachomatis effector protein CT622 on host cell apoptosis,and to screen the key signaling pathways that CT622 plays in regulating apoptosis,so as to further clarify the function of CT622 in the pathogenesis of Ct.Methods:1.The prokaryotic expression recombinant vector pGEX-6p-1/CT622 was constructed using the Ct D-type genome as a template;the recombinant vector was induced and expressed by IPTG,the GST-CT622 fusion protein was purified with Glutathione Sepharose 4B beads,and the GST tag was removed by PreScission Protease for purification the CT622 protein.2.After the purified CT622 protein was treated with endotoxin,HeLa cells were stimulated with PBS and CT622 at a concentration of 0,5,10,15,20 and 25 μg/mL for 24 h,and the cell viability(%)was detected by CCK-8 detection.3.HeLa cells were treated with the above different concentration gradients of CT622 for 20h,and TNF-α apoptosis inducer was added to continue treatment for 4h.Western blot was used to detect the expression levels of cleaved caspase-3,Bax and Bcl-2.The apoptosis of cells in each group was detected by flow cytometry and Hoechst 33258 fluorescence staining.4.HeLa cells were treated with the optimal concentration of CT622 for 0,15,30,60 and 120 min,and Western blot was used to detect the phosphorylation of p38 MAPK and Erk1/2 signaling pathways.5.HeLa cells were treated with 30μM PD98059(Erk1/2 pathway)inhibitor and SB203580(p38 pathway)inhibitor for 0.5h,CT622 was added to stimulate for 20h,and then TNF-α was added to continue treatment for 4h,and the cells were detected Caspase-3,Bax and the expression level of Bcl-2.Then flow cytometry and Hoechst 33258 fluorescence staining were used to detect the apoptosis of cells in each group before and after adding PD98059 or SB203580 inhibitor.Results:1.After induction,expression and purification,the CT622 protein was successfully obtained.2.The results of CCK-8 showed that the CT622 protein prepared in this study had no obvious toxic effect on HeLa within a certain concentration range and could promote cell proliferation.3.Western blot results showed that when HeLa cells were stimulated by CT622,the expression level of Bcl-2 was up-regulated,and the expression levels of Cleaved caspase-3 and Bax were down-regulated.Compared with the apoptosis-inducing group,the morphology of the group was normal,and the number of apoptotic bodies was less.The results of flow cytometry also found that the apoptosis rate of cells in the TNF-α-treated protein group was significantly lower than that in the TNF-α-induced apoptosis group(P<0.05).4.Western blot detected that the phosphorylation levels of Erkl/2 and p38 were significantly up-regulated after CT622 stimulation,but the phosphorylation levels of SAPK/JNK did not change.After inhibiting the Erkl/2 and p38 pathways,the cells were stimulated with CT622,and the results of flow cytometry and Hoechst 33258 staining showed that,regardless of whether they were treated with TNF-α,the number of apoptotic bodies increased and the apoptotic rate was increased after CT622 stimulation.were increased.After inhibiting the Erkl/2 or p38 pathway,the expression level of Cleaved caspase-3 and the ratio of Bax/Bcl-2 were significantly up-regulated in CT622-treated cells regardless of whether they were treated with TNF-α.Conclusions:1.Chlamydia trachomatis effector protein CT622 can inhibit the apoptosis of HeLa cells.2.CT622 up-regulates Bcl-2 by activating Erk1/2 and p38MAPK signaling pathways,and down-regulates the expression of pro-apoptotic proteins such as Bax and Cleaved caspase-3 to regulate HeLa cell apoptosis.