Chlamydial Trachomatis Plasmid-encoded Protein PORF5 Induces Mitophagy And Inhibits Apoptosis By Up-regulation Of HMGB1

Objective: Chlamydia trachomatis,an obligate intracellular pathogen,has various effective strategies to regulate host cell death signaling pathways,accordingly ensures completion of their growth cycle.Mitophagy is responsible for elimination of dysfunctional and impaired mitochondria,and this process plays a critical role in cell survival via restriction of the mitochondrial apoptotic pathway.However,the accurate molecular mechanism remains not entirely understood.This research was designed to explore the relationship between Chlamydia trachomatis plasmid-encode protein p ORF5 and HMGB1,and its effects on mitochondrial autophagy and apoptosis,and further elucidate the role of p ORF5 in the pathogenesis of Chlamydia trachomatis.Methods: The recombinant plasmid pGEX-6p/pORF5 was inoculated into LB solid medium,and the positive colony was cultured and expanded.The GST-pORF5 fusion protein was purified by glutathione-agarose 4B,then,the GST tag was cleaved by PreScission Protease to get the pORF5 protein.After removal of endotoxin,HeLa cells were stimulated with 2μg/ml,4μg/ml,6μg/ml,8μg/ml and 10μg/ml p ORF5 plasmid protein for 0h,6h,12 h,18h and24 h respectively,and PBS was used as negative control.After processing,the level of HMGB1 was analyzed by Western blot.Subsequently,the JC-1fluorescent probe was applied to detect the mitochondrial membrane potential of HeLa cells,and the expression levels of autophagy-related proteins LC3,Beclin-1,SQSTM1/p62 and apoptosis-related proteins Bcl-2,Bax and Caspase-3 were detected by Western blot.Meanwhile,the proportion of cellswith LC3 and SQSTM1/p62 punctae,parkin protein translocation and cytochrome c release from mitochondria were analyzed by indirect immunofluorescence.HMGB1 shRNA was used to stably interfere with the expression of HMGB1 in HeLa cells.Western blot was used to identify the effect of interfering with HMGB1.After pORF5 protein treatment,the mitochondrial membrane potential of HeLa cells was detected by JC-1fluorescent probe,the autophagy and apoptosis related molecules were detected by Western blot and indirect immunofluorescence.Results:1.The identification of pORF5 protein was detected by 12% SDS-PAGE and Western blot,there was a significant protein band at the corresponding position,which was consistent with the expected molecular weight of the fusion protein.2.HeLa cells were stimulated with 2μg/ml,4μg/ml,6μg/ml,8μg/ml and10μg/ml pORF5 plasmid protein for 0h,6h,12 h,18h and 24 h respectively.Western blot results showed that the effects of pORF5 induced HMGB1 expression were both dose-dependent and time-dependent.Interestingly,the peak level of HMGB1 protein was appeared at the concentration of pORF5 reached 4μg/ml.3.The results of JC-1 fluorescence staining showed that pORF5 plasmid could attenuate CCCP-induced decrease of mitochondrial membrane potential to some extent.4.HeLa cells were treated with pORF5 at the concentrations of 2,4,6,8and 10μg/ml for 18 hours,Western blot analysis showed that the expression of LC3 and Beclin-1 were increased and the expression of SQSTM1/p62 was decreased.There was an increase in the amount of LC3 punctae formation following pORF5 plasmid protein treatment by immunofluorescence analysis.Moreover,our results demonstrated that LC3 apparently accumulated on mitochondria with pORF5 treatment compared to control,the fluorescentintensity levels of SQSTM1/p62 punctae were also blunted.Furthermore,the mitochondria were surrounded by parkin following pORF5 plasmid protein administration.5.HeLa cells were treated with different concentrations of pORF5 protein for 18 hours,the level of Bcl-2 was increased,while the expression of Bax and cleaved Caspase-3 were decreased.Immunofluorescence analysis results showed that less cytochrome c released from the mitochondria following pORF5 plasmid protein treatment compared with PBS.6.Western blot results showed that the levels of LC3 and Beclin-1 were decreased and the level of SQSTM1/p62 was increased after knocked down the expression of HMGB1;JC-1 fluorescence probe showed that mitochondrial membrane potential also decreased.7.Western blot results showed that the expression level of Bcl-2 was decreased and the expression of Bax was increased in sh-HMGB1 HeLa cells.Meanwhile,the amount of cytochrome c release was increased.Conclusions: Chlamydia trachomatis plasmid protein pORF5 induce d mitophagy and inhibited apoptosis by up-regulation HMGB1.