Screening Of Pathogenic Molecules And Related Pathways Of Virulence Factor Tc0668 In Chlamydia Trachomatis Mouse Pneumonia Strain

TC0668 in Chlamydia muridarum is an important chromosomally encoded virulence factor that is associated with eliciting significant oviduct inflammation and inducing hydrosalpinx in the upper genital tract of mice.The standard C.muridarum laboratory strain,Nigg II,were serially sub-cultured under special conditions in vitro,and these attenuated strains that had markedly reduced pathogenicity were found to have a mutation in the tc0668 gene.TC0668 protein is a special structural protein which is distributed at the Chlamydia elementary body?EB?/reticulate body?RB?membrane,also exists in the cytoplasm of inclusion body.The translation and transcription of TC0668 were detected at 4 hours and peaked at 16 hours during the life cycle in vitro,C.muridarum bearing a single tc0668 gene mutation may have decreased urogenital pathogenicity that is explained by the effects of the mutation on the regulation of inflammation-related cytokine secretion.However,the molecular mechanism and function of TC0668 in inducing pathogenicity are still unknown.iTRAQ is an advanced quantitative proteomics research technique which has been developed in recent years.iTRAQ technology is a labeled proteomics technology with high sensitivity,good detection parallelism and stability,wide application range and high detection flux.As is known to all,iTRAQ has obvious advantages compared to the traditional proteomics technology and has been applied to detect the pathogenesis of various pathogens.ObjectivesUsing isotope labeling relative and absolute quantitative?iTRAQ?technique to establish TC0668^wt-HeLa cell line and TC0668^mut-HeLa cell line of whole cell protein expression spectrum,to build differentially expressed protein database and to screen relevant signaling pathways that may be involved in the pathogenesis of TC0668.MethodsThis research use iTRAQ technique combined with dimensional liquid chromatography tandem mass spectrometry?LC-MS/MS?technology to establish TC0668^wt?TC0668 null mutant?-HeLa cell line and TC0668^mut?TC0668 single mutant?-HeLa cell line of whole cell protein expression spectrum.The software Mascot2.2 and Proteome Discoverer1.4 were used for identification and quantitative analysis of differentially expressed protein to build related protein database.Blast2GO software,CytoScape software and KAAS software were applied to carry out molecular function annotation of GO,protein interaction and KEGG signal pathway analysis on differentially expressed proteins,so as to predict the biological functions of differentially expressed proteins.qRT-PCR was used to verify the mRNA expression levels of some differentially expressed proteins and Western blotting was used to detect the protein levels of differentially expressed proteins and key molecule s of signaling pathways.ResultsStable expression of HeLa cells infected with TC0668 single mutant C.muridarun strains and control cells infected with TC0668 null mutant strians were successfully established.A total of 550 proteins were identified by iTRAQ labeling combined with LC-MS/MS mass spectrometry at 18 hours?The mid-late stages of Chlamydia developmental cycle?after infection,with 222 proteins up-regulated and328 proteins down-regulated.Through the GO function and KEGG pathway analysis,it was found that the functions of these differentially expressed proteins were mainly related to binding,catalytic activity,transport molecule activity,molecular functional activity and transcriptional regulation activity.GO functional annotation showed that the functions of differentially expressed proteins mainly involved molecular functions,cell components and biological processes.Biological processes include regulation of plasminogen activation,negative regulation of defense response,keratinization and lipid reaction negative regulation.Molecular functions include combination of growth factors,receptor activity,cholesterol and so on.Cell components include membrane components high-density lipoprotein particles,keratin fibers and so on.The analysis results of STRING software showed that there were direct and indirect interactions among the differentially expressed proteins.The results of KEGG pathway analysis showed that differentially expressed proteins were related to multiple signaling pathways,mainly involving complement and coagulation cascade,PPAR signaling pathway,Fc gamma-mediated phagocytosis,MAPK signaling pathway,PI3K-AKt signaling pathway,NF-?B signaling pathway and NOD like receptor signaling pathway.qRT-PCR confirmed that the expressions of IFI16,TRAFD1 and MAPKAPK2 in TC0668-HeLa cells were up-regulated,and the expressions of SRPRB,JAK1,BCAP31,THBS1,ITPR1,PMM1 and HLA-DQB1 were down-regulated,which were consistent with the proteomic results.Western blotting results showed that,compared with mutant group,the expression level of NF-?B in null mutant infected cell lines increased gradually following the time of infection.There was no significant difference in the level of p-ERK between the two groups.Western blotting verified the expression level of JAK1 protein,which increased with the extension of infection time in the TC0668 null mutant group,but gradually decreased in the TC0668mutant group.The phosphorylation level of STAT3 protein which is the substrate of JAK1,in the TC0668 mutant group was higher than that in the TC0668 null mutant group The expression level of PI3K was significantly higher than that of the mutant group.Moreover,as an important molecule in the PI3K signaling pathway,p53 was also significantly lower than that of the mutant group at mid-late stages of postinfection.The activation of NF-?B in HeLa cells infected by TC0668^mutut and TC0668^wtt strains was detected by IFA.In the null mutant group,NF-?B gradually entered the nucleus over the increase of infection time,while in the mutant group,NF-?B entering the nucleus at 24 h was weaker than that in the null mutant groupConclusion1.The database of host differentially expressed proteins induced by TC0668 was successfully constructed,and the host differential proteins caused by TC0668 differences are related to cell process,metabolic process,immune response and biological regulation.2.TC0668 may play a pathogenic role in the host through PI3K,NF-?B and other signaling pathways.