PORF5Plasmid Protein Enhances Chlamydia Trachomatis Infection Through Inhibition Of LL37Antibacterial Peptide

Objective: Persistent infection of Chlamydia trachomatis(Ct）can lead to manyserious complication, but the mechanisms keep unclear. It has demonstrated that LL37can inhibit the Chlamydia trachomatis infection. The pORF5protein, encoded bychlamydial plasmid, is a secreted protein which is closely related to Chlamydialpathopoiesis. In the present study, we investigated whether the pORF5plasmidprotein promoted chlamydial infection through inhibition of LL37antibacterialpeptide, and its molecular mechanism. This study could provide experimental basisfor further study on the pathogenic mechanism of Ct infection.Methods：pGEX-6p-1/pORF5recombinant plasmid was transformed into E.coliXL1-Blue strain and induced to express GST-pORF5fusion protein by IPTG. Afterpurification, GST-pORF5fusion protein was digested with protease to cut the GSTtag to get pORF5protein. Then the pORF5protein was identified by usingSDS-PAGE and Western blotting. BCA was used to detect the concentration ofpORF5protein. After the endotoxin was removed, different concentrations of pORF5protein and/or LL37were used to stimulate HeLa cells, and detected the cell apoptosisby flow cytometry method28h later. Ct was pretreated with20μg/mL LL37and/or30μg/mL pORF5protein and then infected HeLa cells. After28h, indirectimmunofluorescence assay was used to examine the morphological changes ofinclusion body. Ct was pretreated with30μg/mL pORF5and/or20μg/mL LL37andthen infected HeLa cells, the expression of TNF-α was tested6h later. HeLa cellspretreated with30μg/mL pORF5and/or Ct were used to test the expression of LL376h later. Results:(1) The plasmid pGEX-6p-1/pORF5was transformed into XL1-Blue strainsuccessfully and the GST-pORF5fusion protein was expressed with highofficiency after the induction of IPTG, GST-pORF5was cleaved with PreScissionprotease to get pORF5protein without GST tag. The purity of pORF5fusionprotein was about95%.(2) pORF5protein could induce apoptosis with concentration dependence, when theconcentration of pORF5fusion protein was10μg/mL, the apoptosis rate was5.75%, when the concentration of pORF5protein was20μg/mL, the apoptosisrate was9.42%, when the concentration of pORF5protein was30μg/mL, theapoptosis rate was17.52%.(3) The pORF5protein reduced the inhibitory effect of LL37on Chlamydia infectionobviously. The chlamydial inclusion-forming units(IFUs) in the group pretreatedwith30μg/mL pORF5was3.00×10~5/mL, the IFUs in the group pretreated with20μg/mL LL37was2.00×10~5/mL, and the IFUs in the group pretreated with30μg/mL pORF5and20μg/mL LL37was3.07×10~5/mL, the IFUs in the controlgroup was3.80×10~5/mL. The IFUs in pORF5-pretreated group and LL37andpORF5-pretreated group were higher than that of group pretreated with LL37(P<0.05), but there were no statistically significant differences in IFUs amonggroup pretreated with pORF5and group pretreated with pORF5and LL37ascompared to control group(P>0.05).(4) pORF5decreased the expression of LL37to83%and increased the expression ofTNF-α by7.3times.Conclusions:(1) pORF5plasmid protein could reduce the inhibitory effect of LL37antibacterialpeptide on Chlamydial infection.(2) pORF5plasmid protein reduced the inhibitory effect of LL37by up-regulating the expression of TNF-α and dowen-regulating the expression of LL37in cellsinfected with Ct.