The Effects Of Penicillin G Induced Persistent Chlamydophila Psittaci Infection On Gene Expression And Host Cell Apoptosis

Objective: To establish the model of persistent C.psittaci infection in human cervicalcarcinoma epithelial cells （HeLa cells） in vitro and explore the effects of persistentC.psittaci infection on the mRNA expression levels of10C.psittaci genes and hostcell apoptosis，our studywill be contributed to elucidate the molecular mechanism ofpersistent C.psittaci infection.Methods: After human epithelial cells cultured in vitro and then infected withC.psittaci at a multiplicity of infection （MOI） of2for2h, the fresh Chlamydia culturemedium supplemented with various concentrations of penicillin G were exchanged forthe maintains of host cells infected C.psittaci for48h, the optimal concentration ofPenicillin G was determined by analyzing the infectivity of C.psittaci upon theinfluence of various concenrations of Penicillin G through indirectimmunofluorescence staining. After24h or48h of persistent C.psittaci infectioninduced by Penicillin G, the morphological characteristics of C.psittaci inclusion wereanalyzed through transmission electron microscopy and indirect immunofluorescencestaining. Besides, the recoverable ability of C.psittaci was analyzed with the removalof Penicillin G. A set of10genes （DnaA、DnaK、FtsW、FtsY、GrpE、RpsD、IncC、OmcB、CPSIT₀846、CPSIT₀042） with functions in cell division and replication、metabolism、cell membrane were selected from genome sequences of C.psittaci.qRT-PCR was used to quantitate the mRNA expression levels of genes at varioustime points （6h、12h、24h、48h、72h） during persistent C.psittaci infection. The16SrRNA gene served as internal standard for comparisons of gene transcription betweenacute and persistent infection. Besides, after24h of persistent C.psittaci infectioninduced by penicillin G for24h, the1μM apoptosis inducer Staurosporine anddissolvant were used for the treatment of cells for4h, the levels of apoptosis cells were determined by DNA Ladder analysis、AO/EB staining、Hoechst33258stainingand flow cytometry.Results:（1） C.psittaci would obtain the persistent features after the induction of Penicillin G,and the optimal concentration of Penicillin G for inducing persistent C.psittaciinfection was100U/mL.（2） Upon the persistent state, the morphological features of C.psittaci were obviouslydifferent from that in acute state. In acute infection group, C.psittaci inclusion bodywas large and various, and there were lots of dense EB particles and a little of RBparticles. However, the chlamydial inclusion size was reduced in penicillin G-inducedgroup, and the number of EB particles was significantly decreased, and there wereenlarged aberrant RB particles. Besides, with the removal of Penicillin G, C.psittacicould be reversed to productive state partically.（3） Compared with the acute state of C.psittaci infection, the mRNA expression levelsof following C.psittaci genes have been alerted in persistent state: the mRNAexpression levels of FtsW、FtsY and CPSIT₀042were significantly down-regulated（P value<0.01）, the mRNA expression levels of DnaA、DnaK、GrpE、RpsD、IncC andCPSIT₀846were down-regulated （P value <0.05）; however, the mRNA expressionlevel of OmcB in persistent state was as same as that in acute state （P value>0.05）.（4） With the treatment of Staurosporine, the percent of apoptosis cells in persistentC.psittaci infected cells were significantly lower than that in uninfected cells （P value<0.01）, and higher than that in acute infected cells （P value <0.05）.Conclusions:（1） The cell model of Penicillin G induced persistent C.psittaci infection in vitro wasestablished successfully.（2） During persistent C.psittaci infection induced by Penicillin G, the mRNAexpression levels of DnaA、DnaK、GrpE、RpsD、IncC、 CPSIT₀846、FtsW、FtsY and CPSIT₀042have been altered significantly, and these genes may beinvolved in the altered morphological features of C.psittaci during persistentinfection. （3） The anti-apoptosis ability of the host cells was enhanced by C.psittaci underpersistent state; however, the anti-apoptosis ability of persistent C.psittaci infectedcells was much less than that in acute infection.