Chlamydia Trachomatis Plasmid-encoded Pgp3 Induces IL-8 Production Through NOD1/RIP2 With The Independent Pathways Of ERK And P38

Objective: To unravel the mechanism of Chlamydia trachomatis plasmid-encoded protein Pgp3 inducing the production of IL-8 in HeLa cells and provide effective experimental evidence for understanding the mechanism of C.trachomatis pathogenesis.Method : In this paper,the constructed recombinant bacteria pGEX-pgp3/XL1-blue was induced to express using IPTG,and then purified by glutathione sepharose 4B to get the fusion proteins GST-Pgp3.Next,fusion proteins with GST tag was cut off by PreScission Protease with endotoxin-removing treatment to obtain the purified Pgp3 proteins.Pgp3 was identified by SDS-PAGE gel electrophoresis and western blot.Various concentration with 0 μg/mL,4μg/mL,12 μg/mL,24 μg/mL of purified Pgp3 were selected to stimulate HeLa cells on 0 h,6 h,12 h,24 h to detect IL-8 and RIP2 expression level via Real-time PCR and NOD1 transcriptional level and expression via Real-time PCR and western blot;then pick plasmid protein Pgp3 with the optimal concentration and timing to pretreat He La cells,ERKphosphorylating was detected via western blot as well as p38 on various timing 0 min,15 min,30 min,60 min were verified by western blot.After pretreated with NOD1 inhibitor(ML130),RIP2 inhibitor(GSK583),ERK inhibitor(PD98059)and p38 inhibitor(LY2228820)repectively,HeLa cells were stimulated by Pgp3 with optimal concentration and timing.western blot,Real-time PCR and ELISA were employed to detect whether the impact of IL-8 expression was modulated by NOD1/RIP2 with independent of ERK and p38 signal pathway or not.Result:1.GST-Pgp3 fusion proteins with about 54 kD molecular weight were expressed under the following induction condition: 30℃,4h,180 rpm and 0.2 mmol/L IPTG concentration by recombinant bacteria pGEX-pgp3/XL1-blue,and then GST tag of the proteins were cut off by PreScission Protease.The 28 kD Pgp3 proteins with purity beyond 95% were gained,which demonstrated via Western blot.2.In HeLa cells,the expression level of IL-8 can be elevated by Pgp3 plasmid proteins,and the optimal stimulating condition present dependence on concentration and timing.In this research,24 μg/ml and 24 h were thought to be the optimal parameter.3.Pgp3 could regulate the expression of NOD1 receptor from HeLa cells,both the transcriptional and expression level of NOD1 were elevated and presented perfectly while the stimulating concentration and time of Pgp3 is 24 μg/ml and 12 h,respectively.4.Pgp3 could regulate the expression the downstream molecule RIP2.Between the two groups stimulated with Pgp3,mRNA expression level of the one which was pretreated with NOD1 inhibitor was 26.87% lower than the group with Pgp3 stimulating only.5.ERK and p38 was phosphorylated on the timing of 30 min and 15 min after HeLa cells were stimulated by Pgp3.6.The induction of IL-8 for Pgp3 was modulated by NOD1 with independence of ERK and p38 phosphorylating in HeLa cells.The HeLa cells pretreated with NOD1inhibitor(ML130),RIP2 inhibitor(GSK583),ERK inhibitor(PD98059)and p38 inhibitor(LY2228820),and then added with Pgp3,mRNA transcription and expression level of IL-8 were lowered 65.70%,40.87%,31.47%,27.84% and 66.02%,34.77%,42.02%,24.58% than negative group,respectively.Conclusion:1.Pgp3 could induce the expression of IL-8 in HeLa cells.2.The induction of IL-8 for Pgp3 was modulated by NOD1/RIP2 with the independence of ERK and p38 pathways in HeLa cells.