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Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis,so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis.Methods HeLa cells were divided into two groups:carbonyl cyanide m-chlorophenyl hydrazone (CCCP,an apoptosis inducer) group was stimulated by CCCP for 30 minutes,and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes.Then,Western blot analysis was performed to determine the expression of apoptosisrelated proteins Bcl-2,Bax and caspase-3,JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells,and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay.To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein,HMGB 1 shRNA and control RNA were separately transfected into the HeLa cells,which were then stimulated by pORF5 plasmid protein and CCCP.Then,the protein expression of Bcl-2,Bax,activated caspase-3 was determined,and cytochrome c release was analyzed.Data were compared between two groups by using paired t test.Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential,and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3;t =6.95,P ＜ 0.01).The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t =8.62,P ＜ 0.01),while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79％ ± 10％ (t =9.23,P ＜ 0.01) and 75％ ± 8％ (t =4.26,P ＜ 0.05) respectively compared with the CCCP group.Compared with the control RNA transfection group,the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t =11.23,P ＜ 0.01),increased cytochrome c release,decreased Bcl-2 expresson (t =7.19,P ＜ 0.05) and increased Bax expression (t =13.06,P ＜ 0.01) after stimulation with pORF5 and CCCP.Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.
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Objective:To study the relationship between apoptosis and the pORF5 protein of Chlamydia trachomatis,and further to explore its molecular mechanisms,which could lay a foundation for chlamydial pathogenic mechanisms.Methods:pGEX-6p/pORF5 recombinant expression vector was transformed to XL1-blue E.Coli to express GST-pORF5 fusion protein,and GST-pORF5 fusion protein was purified with Glutathione Sepharose 4B Beads,and cleaved to get pORF5 protein without GST tag by PreScission protease.The pORF5 protein was used to stimulate HeLa cells at different concentrations,then Western blot was used to evaluate the ex-pression of Bax,Bcl-2 and phosphorylation of PI3K/Akt at different time points,Hoechst staining and Flow cytometry were applied to measure the apoptosis of HeLa cells.Before treated with pORF5 protein for 24 h,HeLa cells were pretreated with PI3K inhibitor LY294002 for 1 h,the expression of Bax,Bcl-2 and the phosphorylation of Akt were evaluated by Western blot,apoptosis rates were also determined.Results:The pORF5 protein changed the expression of Bax and Bcl-2 in dose-and time-dependent manners,pORF5 increased the expression of Bcl-2 and decreased the expression of Bax at the concentration of 10 μg/ml,and there was obvious change at concentration of 15 μg/ml for 24 h.The apoptosis rates of pORF5 treated group were reduced by 27.3% and 8.4% respectively when compared with TNF-α treated group(P<0.01) and untreated group (P<0.05).Akt was phosphorylated after stimulated with pORF5 protein for 15 min,and reached its peak at 30 min.PI3K/Akt inhibitor led to the decrease of the expression of Bcl-2 and phosphorylation of Akt and increase of the expression of Bax,furthermore,PI3K/Akt inhibitor reversed pORF5-mediated anti-apoptosis, the apoptosis rate in LY294002 treated group was increased by 13.0%,when compared with the control group(P<0.01).Conclusion:pORF5 protein could inhibit apoptosis through activating PI3K/Akt signal pathway by induction of Bcl-2 and suppression of Bax.
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Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha(TNF-α). Methods The recombinant lentiviral expression vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa (pORF5-HeLa) cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20μg/L TNF-αand fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup (pORF5-HeLa cells:35.5%± 4.5%vs. 9.5%± 1.5%, t=13.53, P<0.01;control HeLa cells:63.6%± 5.8%vs. 7.9%± 0.9%, t=32.36, P<0.01). Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA expression of Bax(72.8%)and Caspase 3(84.5%)(t = 35.29, 42.25, respectively, both P < 0.01), as well as in Bax protein expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(t = 87.12, 18.93, respectively, both P <0.01). Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.
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Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.
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Objective:To investigate the immunogenicity of pORF5 plasmid protein,and further to screen and identify its im-munodominant domian.Methods: 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamHⅠand NotⅠrestriction endonucleases.After identification by PCR and sequencing,the recombinant plasmids were transformed into XL1 Blue E.coli to express the GST fusion proteins.ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein.10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method.Results: pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human.The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de-stroyed.F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was.However,no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies.Conclusion:pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope,and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation.The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.
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Objective:To investigate whether pORF5 plasmid protein of Chlamydia trachomatis(Ct) induces 1L-1βand 1L-18 production in THP-1 cells,and its potential molecular mechanism.Methods:pORF5 plasmid protein was used to stimulate THP-1 cells at different concentrations(0,3,6,12,24,36 μg/ml),then the inflammatory cytokines IL-18 and IL-1βwere detected by ELISA at the time of 0,8,16,24,36 h;The mRNA expression of NALP3 inflammasome were detected by Realtime-PCR,and Caspase-1 activity was determined by Western blot analysis.THP-1 cells were transfected with siRNA targeting NALP3 and ASC gene for 24 h or pretreated with Caspase-1 inhibitor(Z-YVAD-FMK) for 30 min,and subsequently stimulated with pORF5(24 μg/ml) for 24 h,then secretion of IL-1βand IL-18 were analyzed by ELISA.Results: The pORF5 plasmid protein induced THP-1 cells to secrete IL-1βand IL-18 by dose-and time-dependent manners,production of IL-1βand IL-18 reached their peaks(491 pg/ml and 186 pg/ml) at concentration of 24 μg/ml,and the peak amount of IL-1βand IL-18 occurred at 24 h and 16 h post-stimulation respectively.pORF5 plasmid protein in-creased mRNA expression of NALP3 inflammasome and activated Caspase-1 in THP-1 cells.NALP3 siRNA,ASC siRNA and Z-YVAD-FMK reduced pORF5-induced IL-1βand IL-18 production when compared with control groups(P<0.05).Conclusion:pORF5 plasmid protein could induce THP-1 cells to produce IL-1βand IL-18 through NALP3 inflammasome activation,which may play an important role in the pathogenesis in Ct infection.
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<p><b>OBJECTIVE</b>To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections.</p><p><b>METHODS</b>The pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA.</p><p><b>RESULTS</b>Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6).</p><p><b>CONCLUSION</b>Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Chlamydia Infections , Diagnosis , Chlamydia trachomatis , Virulence , Enzyme-Linked Immunosorbent Assay , Methods , Urogenital System , MicrobiologyABSTRACT
Objective To investigate the immune injury in genital tract of BALB/c mice induced by plasmid protein pORF5 of Chlamydia trachomatis and its possible mechanism.Methods GST(glutathione-S-transferases)-pORF5 fusion protein was expressed and digested with PreScission Protease to obtain the target protein without GST tag.After further purification and endotoxin removal,pORF5 protein was injected into the posterior fornix of BALB/c mice on day 1,3 and 6,while the control groups were injected with PBS or GST protein respectively,and then all the mice were sacrificed on day 7 to evaluate genital tract gross pathology and histopathological characterization.The levels of TNF-α,IL-1β and IL-6 in serum,splenocytes culture supernatant and vaginal douche were detected by ELISA.Results Mice in pORF5 group developed different degrees of swelling in isthmic portion and ampulla of uterine tube,connective tissue adhesion and hydrosalpinx in the genital tract tissues,while the PBS group and the GST group did not show any obvious change.The inflammatory score showed that the genital tract pathology in pORF5 group was much more severe than PBS and GST control groups (P＜0.O1).The levels of TNF-α,IL-1β and IL-6 in vaginal douche and splenocytes culture supernatants in pORF5 group were obviously higher than those of PBS and GST groups (P＜0.05).The levels of TNF-α and IL-1β in serum were also higher than those of GST and PBS groups (P＜0.01).Conclusion pORF5 plasmid protein could induce pathological immune response in the genital tract of BALB/c mice,which may be associated with the increase of the production of the inflammatory cytokines TNF-α,IL-1β and IL-6 in BALB/c mice.
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ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.
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Objective To localize and characterize the plasmid protein pORF5 in the Chlamydia trachomatis(Ct) infected cells. Methods The open reading frame encoding for pORF5 protein from the Ct plasmid was amplified and cloned into the pGEX-6p vector. The recombinant plasmid pGEX-pORF5 was transformed into XL1-blue E. coli to express fusion protein with the glutathione-s-transferase (GST). After purified with Glutathione Sepharose 4B beads, the pORF5 fusion protein was used to immunize mice to make monoclonal and polyclonal antibody. The antibodies were used to localize the endogenous pORF5 protein and detect the expression pattern in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). At the same time, ELISA was used to determine whether pORF5 plasmid protein was expressed and immunogenic during Ct infection in humans. Results pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF. pORF5 also appeared in the RBs and EBs in small quantity. Athough pattern was similarly, pORF5 did not overlap with CPAF. pORF5 protein was strongly recognized antiserum in an ELISA. Conclusion The pORF5 plasmid protein was identified as a secreted protein with good immunogenicity, pORF5 gene was to express the endogenous target protein during human infection.