Signaling Pathway Of Autophagy In RAW264.7 Cells Induced By Chlamydia Psittaci Plasmid Protein CPSIT_p7

Objective:Chlamydia psittaci?C.psittaci?is an obligate intracellular parasite that causes respiratory infections in humans and birds.Plasmid protein is an important virulence factor of Chlamydia,and little research has been done on its exact pathogenic mechanism.The aim of this study was to investigate the autophagy and related signaling pathways induced by C.psittaci plasmid protein in RAW264.7 cells,and to provide an important experimental basis for further elucidation of the pathogenesis of C.psittaci infection.Methods:The recombinant plasmid pET28a-CPSIT_p7 which had been constructed in the previous study was inoculated into solid medium,and the positive colonies were selected for expansion culture.The protein was purified by Ni²⁺NTA affinity chromatography column and concentrated by ultrafiltration tube to obtain CPSIT_p7 protein.After removal of endotoxin by affinity chromatography,RAW264.7 cells were stimulated with CPSIT_p7 protein at concentrations of 0.1?g/mL,1?g/mL,5?g/mL,and 10?g/mL for 18 h,using DPBS as a negative control.LC3 fluorescent spots were detected by indirect immunofluorescence,and protein expression levels of LC3,Beclin1 and SQSTM1/p62 were detected by Western blot.RAW264.7 cells were stimulated with CPSIT_p7?10?g/mL?for 30 min,60 min,90 min,and120 min respectively,and the phosphorylation levels of ERK,JNK,p38,and Akt were detected by Western blot.CPSIT_p7 stimulated RAW 264.7cells for 18 h,and RT-qPCR detected the transcription levels of Toll-like receptor?TLR1?,TLR2,TLR4 and TLR6 in RAW264.7 cells.The expression of TLR2 in RAW264.7 cells was silenced by TLR2 siRNA,the silencing effect of TLR2 siRNA was identified by Western blot.The level of autophagy was detected by Western blot and indirect immunofluorescence after CPSIT_p7 stimulation.After ERK signaling pathway was inhibited and CPSIT_p7 stimulation,the autophagy-related indicators and phosphorylation levels of ERK,JNK and p38 were detected by Western blot.Result:1.Purified CPSIT_p7 plasmid protein was analyzed by SDS-polyacrylamide gel electrophoresis,and the obtained protein band was consistent with the molecular weight of its protein.2.RAW264.7 cells were stimulated with CPSIT_p7 at concentrations of 0.1?g/mL,1?g/mL,5?g/mL and 10?g/mL for 18h.Western blot results showed that the expression levels of LC3 and Beclin1 were increased compared with the DPBS control group,while expression level of p62 protein decreased.After 10?g/mL concentration of CPSIT_p7 stimulated RAW264.7 cells for 18h,the indirect immunofluorescence assay showed that the fluorescence spots of LC3increased significantly.3.After stimulating RAW264.7 cells with 10?g/mL CPSIT_p7 for30 min,60 min,90 min and 120 min respectively,Western blot analysis showed that the phosphorylation levels of ERK,JNK,p38 and Akt were significantly increased,the the expression levels of of ERK,JNK and Akt phosphorylation reached a peak level at 60 min,and the p38phosphorylation reached a peak level at 120 min.4.RT-qPCR results showed that the expression of TLR2 transcript in RAW264.7 cell was significantly increased,the expression of TLR4transcript was slightly increased,while the expressions of TLR1 and TLR6 transcripts remained unchanged after stimulation with plasmid protein CPSIT_p7.5.RAW264.7 cells transfected with TLR2 SiRNA were stimulated with CPSIT_p7 at concentration of 10?g/mL.Western blot results represented that the levels of LC3 and beclin1 decreased,while p62protein levels increased compared to the control group.In addition,the indirect immunofluorescence assay showed that the fluorescence spots of LC3 decreased significantly.6.After pretreatment of RAW264.7 cells with PD98059 for 1 hour,RAW264.7 cells were stimulated with 10?g/mL CPSIT_p7 for 12 hours.Western blot analysis showed that the decreased expression levels of LC3and Beclin1 in PD98059-pretreated CPSIT_p7 stimulation group was compared with the PD98059-unpretreated stimulation group.Identically,the ERK phosphorylation level was decreased,but JNK and p38phosphorylation levels were unaffected.Conclusion:TLR2 mediates autophagy through ERK signaling pathway in Chlamydia psittaci plasmid protein CPSIT_p7-stimulated RAW264.7 cells.