| Objective:To investigate whether the pORF5 plasmid protein of Chlamydia trachomatis can activate the Wnt/?-catenin signaling pathway to inhibit host cell apoptosis and promote cell proliferation,which provides a basis for elucidating the role of pORF5 plasmid protein in the pathogenesis of Ct.Methods:The pGEX-6p-1/pORF5 recombined plasmid strain constructed earlier in our research group was inoculated and cultured in LB solid medium,and then expanded to express the GST-pORF5 fusion protein after induction with 0.2 mM IPTG.The pORF5 plasmid protein was collected and detoxified after purification and then digestion of GST with protease.The pORF5 plasmid protein with the concentration of 0?g/mL,5?g/mL,10?g/mL,15?g/mL and 20?g/mL was used to stimulate HeLa cells for 0 h?6 h?12 h?18 h?and 24 h,total cell protein and RNA were collected.Western blot and qRT-PCR were used to detect the expression of?-catenin protein in HeLa cells.The nuclear translocation of?-catenin protein was observed by IFA.HeLa cells were pretreated with 10?M Wnt inhibitor IWP2 for 1 h,and then treated with apoptosis-inducing agent TNF-?(25 ng/mL)for 4 h.Western Blot was used to detect the changes in expression of?-catenin protein and apoptosis-related proteins Bcl-2 and Bax;Apoptosis was detected using Hoechst 33258 fluorescence staining and flow cytometry;CCK-8 kit was used to detect the cell proliferation and the mRNA levels of cell adhesion molecule EpCAM and OLFM4 were detected by qRT-PCR after stimulated with pORF5 plasmid protein concentration for 8 h?12 h?24h?36 h.HeLa cells were infected with Ct serotype E for 36 h,after pretreated with 10?M Wnt inhibitor IWP2 for 1 h,then Chlamydial trachomatis infection rate was calculated.Results:1.pGEX-6p-1/pORF5 recombinant bacteria were induced to express GST-pORF5 fusion protein with a molecular weight of about 54 kDa,and pORF5 plasmid protein without GST tag was obtained after cleavage with protease.2.The expression of?-catenin protein reached peak when HeLa cells were stimulated with 15?g/mL pORF5 plasmid protein for 18 h;IFA results showed that?-catenin protein expressed obvious nuclear translocation in the pORF5 protein-stimulated group;The inhibitor IWP2of Wnt signaling pathway can reduce?-catenin expression.3.Bax was up-regulated and Bcl-2 was down-regulated in the IWP2/pORF5 group compared with the pORF5 protein-stimulated group;Hoechst staining results showed that apoptosis rate increased 20.3%(P<0.001)in IWP2/pORF5 group when compared with DMSO/pORF5group,and IWP2 obviously inhibited nuclear fragmentation and nuclear shrinkage.The flow cytometry results showed that the apoptosis rate increased 22.08%(P<0.001)when compared with DMSO/pORF5 group(P<0.001).4.Bcl-2 expression was up-regulated and Bax expression was down-regulated when HeLa cells were stimulated with 20?g/mL pORF5plasmid protein for 18 h.Hoechst staining results showed that apoptosis rate decreased 27.9%(P<0.001)and 5.4%(P<0.01)in pORF5/TNF-?group,respectively,when compared with TNF-?group and normal group.The flow cytometry results showed that the apoptosis rate in pORF5/TNF-?group decreased 23.26%(P<0.001)and 2.97%(P<0.05),respectively,compared with TNF-?group and normal group.5.The cell proliferation activity was the highest and increased significantly in pORF5 plasmid protein-stimulated group when compared with the IWP2 inhibitor group(P<0.01);qRT-PCR showed that the mRNA levels of EpCAM?OLFM4 and?-catenin in the pORF5 plasmid protein-stimulated group increased significantly when compared with the IWP2/pORF5 group.6.Inclusion bodies were observed by IFA,the results showed that the IFU of normal group and IWP2 group were 2.79×10~7/ml and1.16×10~7/ml,respectively,the IFU of the IWP2 group was significantly lower than that of the normal control group.Conclusion:Chlamydia trachomatis pORF5 plasmid protein increases Chlamydial infection rate by inhibiting host cell apoptosis and promoting cell proliferation through activating the Wnt/?-catenin signaling pathway. |
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