Screening And Preliminary Function Study Of Proteins Caused By PORF5 Implicated In The Pathogenesis Of C. Trachomatis

pORF5 plamsid protein of Chlamydia trachomatis(C.trachomatis) is the only plasmid protein secreted into the cytoplasm of the infected cells, Much evidence showed that p ORF5 was closely related to virulence. However, the molecular mechanism of p ORF5 in the pathogenesis of C.trachomatis remained unknown. The aim of the present study was to understand the roles of p ORF5-interacted proteins in the pathogenesis of C.trachomatis.This study could provide important information for further understanding the pathogenesis of C. trachomatis. The p ORF5-expressed lentivirus vector and control lentivirus vector were constructed, and transfected to HEK 293 T cell with packing plasmid to produce recombinant lentivirus, the lentivirus supernatant was collected to infect He La cells. p ORF5 stable transfected He La cell line and control He La cell line were sorted by flow cytometry. The m RNA and protein levels of p ORF5 expressed in infected-cells were indentifed by Quantitative real-time PCR(q RT-PCR) and Western blotting. The whole cell proteome profile of the p ORF5-He La cells and the control He La cells were compared by the i TRAQ quantitative proteomics approach. The differentially expressed protein datebase was established.The function of differentially expressed proteins was analyzed by bioinformatic method, such as DAVID online analysis software, STRING online analysis software and KEGG pathway database software. sh RNA- HMGB1 lentivirus vector was constructed to produce the sh RNA-HMGB1 lentivirus with three plasmid lentiviral packaging system, the lentivirus supernatant was collected to infected p ORF5-He La cells to establish the HMGB1 stable interfered celll line and control cell line. The expression of the autophagy-related protein Beclin-1 and LC-3 were detected through q RT-PCR and Western blotting. Apoptosis rate and cell cycle were detected by flow cytometry. The experimental results were as follows: 1. The stable He La cell line expressed p ORF5 protein and the control cell line were established successfully. 2. 314 differentially expressed proteins were identified via i TRAQ labeling coupled with 2D LC–MS/MS, 159 of which were upregulated, 155 of which were downregulated. Functional classification of the identified proteins according to GO using the DAVID software demonstrated that the differentially expressed proteins were classified into three categories: molecular function, cellular component and biological process. The molecular function proteins mainly included proteins with binding transcription factor activity such as nucleophosmin, RNA polymerase ⅡActivator p15, and nucleic acid binding transcription factor activity such as high mobility group protein B1, high mobility group protein B2. The cellular component proteins mainly included cell part function proteins such as chloride ion channel protein 1, annexin A1, and cell junction such as α-actinin-4, integrin α-6. Among the proteins with biological process function, a part of proteins took part in the cell reproduction, such as transferrin receptor protein-1, 60 S acidic ribosomal protein P2, a part of proteins involved incell immune response such as complement C9, complement decay-accelerating factor, and a part of proteins took part in cell death such as keratin, inhibiting protein-1. Through the STRING online software, there were direct and indirect interactions among the differentially expressed proteins. The KEGG pathway annotations of the dysregulated proteins demonstrated that the differential proteins were associated with multiple signal pathways, which were mainly involved in ribosome pathway, spliceosome, ECM receptor interaction, MAPK signaling pathway and p53 signaling pathway. 3. Among the 9 differential proteins, PARK7, HMGB1, HMGB2, HBA1 and HIST1H1 C were confirmed upregulated, CDKN2 A, SFN, KRT7 and CLIC1 were confirmed downregulated through q RT-PCR. HMGB1 and PARK7 were confirmed upregulated via Western blotting in p ORF5-He La cells, which were consistent with the proteomics results. 4. The m RNA expression of the autophagy-related protein Beclin-1 and LC-3 in the HMGB1 stable interfered p ORF5-He La cell line were reduced by 62% and 89.8% respectively, when compared with the control cell line. The Western blotting result showed that the expression of the protein Beclin-1 in the HMGB1 stable interfered p ORF5-He La cell line decreased obviously, and the ratio of LC3-Ⅱ/LC-3Ⅰin the HMGB1 stable interfered p ORF5-He La cells was less than that of the control group, LC3-Ⅱ was also decreased in the HMGB1 stable interfered p ORF5-He La cells. The results of flow cytometry showed that the apoptosis rate of the HMGB1 stable interfered p ORF5-He La cells increased by 18.84%, and silencing of HMGB1 in p ORF5-He La cells altered the dynamics of the cell cycle and changed the balance at the same time, the cells in G2 and S were increased by 4.44% and 3.4% respectively. The results suggest: 1. The differentially expressed protein database which caused by p ORF5 was established successfully, 314 proteins have been identified, in which 155 proteins were upregulated, 159 proteins were downregulated. The proteins identified were related with cellular immune response, biological regulation and biological adhesion. 2. p ORF5 induced autophagy and inhibited apoptosis through upregulating host cell protein HMGB1.