pORF5 Plasmid Protein Research Articles

Chlamydia trachomatis Plasmid Protein pORF5 Up-Regulates ZFAS1 to Promote Host Cell Survival via MAPK/p38 Pathway.

Long non-coding RNAs (lncRNAs) have been demonstrated to play essential roles in many diseases. However, few studies have shown that lncRNAs take part in the pathogenesis of Chlamydia trachomatis (C. trachomatis). Here, we used a lncRNA microarray to detect the global lncRNA expression profiles in HeLa cells transfected with pORF5 plasmid protein, an important virulence factor for C. trachomatis. The differentially expressed lncRNAs and mRNAs screened by microarray were selected for validation by quantitative real-time PCR. The up-regulated lncRNA zinc finger antisense 1 (ZFAS1) was presumed to involved in MAPK pathways by bioinformatics analysis. Inhibition of ZFAS1 decreased the apoptotic rate of pORF5 and reduced the infectivity of C. trachomatis, and MAPK/p38 pathway was involved in anti-apoptotic effect induced by ZFAS1. Therefore, the present study confirmed that pORF5 up-regulates ZFAS1 to promote host cell survival via MAPK/p38 pathway and influences the infectivity of C. trachomatis.

Mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein in Chlamydia trachomatis via high-mobility group box 1 protein: a preliminary study

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 apoptosis-related 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, HMGB1 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. Key words: Chlamydia trachomatis; Apoptosis; Proto-oncogene proteins c-bcl-2; Bcl-2-associated X protein; Caspase-3; High mobility group box protein 1; pORF5 plasmid protein

Chlamydia trachomatis plasmid-encoded protein pORF5 protects mitochondrial function by inducing mitophagy and increasing HMGB1 expression.

Chlamydia trachomatis, an obligate intracellular pathogen, has various effective strategies to regulate host cell death signalling pathways that ensure completion of their growth cycle. Mitochondrial autophagy (mitophagy) is responsible for elimination of dysfunctional and impaired mitochondria, and this process plays a critical role in cell survival via restriction of the mitochondrial apoptotic pathway. However, the specific molecular mechanisms are not entirely understood. In the present study, we observed that pORF5 plasmid protein of C. trachomatis plays a crucial role in attenuating mitochondrial dysfunction and apoptosis. Knockdown high mobility group box 1 (HMGB1) by lentivirus suppressed pORF5-induced mitophagy and increased apoptosis, implying that pORF5 may participate in cell death signalling pathways via up-regulation of HMGB1. Thus, we concluded that up-regulation of HMGB1 is a pivotal event for C. trachomatis that manipulates mitophagy and apoptosis in order to establish a favourable environment supportive of Chlamydial growth, which should further promote our understanding of Chlamydial pathogenic mechanisms.

PORF5 plasmid protein promotes Chlamydia trachomatis infection through inhibition of the antibacterial peptide LL37: a preliminary study

Objective To investigate whether pORF5 plasmid protein promotes Chlamydia trachomatis (Ct)infection through inhibition of the antibacterial peptide LL37,and to explore its potential molecular mechanism.Methods After expression and purification,the glutathione S-transferase (GST)-pORF5 fusion protein was digested with proteases to obtain pORF5 protein without GST tag.Some Hela cells were classified into four groups:Ct group infected with unpretreated Ct,pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours,LL37 group infected with Ct that had been pretreated with LL37 of 20 μg/ml for 2 hours,combination group infected with Ct that had been pretreated with pORF5 of 30 μg/ml and LL37 of 20 μg/ml for 2 hours.After additional culture,indirect immunofluorescence assay was performed to count the number of Ct inclusion-forming units (IFUs),and real-time,fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay (ELISA) to detect the protein and mRNA expressions of tumor necrosis factor-α (TNF-α) respectively.Some Hela cells were divided into three groups:blank control group remaining untreated,Ct group infected with unpretreated Ct,pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours.After 6 hours of additional culture,real-time,fluorescence-based quantitative PCR and ELISA were conducted to measure the mRNA and protein expressions of LL37,respectively.Results The concentration of IFUs was significantly higher in the Ct group,pORF5 group and combination group than in the LL37 group (3.8 × 105/ml,3.0 × 105/ml and 3.1 × 105/ml vs.2.0 × 105/ml,all P ＜ 0.05),with no significant differences between the Ct group,pORF5 group and combination group (P ＞ 0.05).The expressions of TNF-α mRNA and protein were significantly increased in the combination group compared with the LL37 group.The mRNA expression of LL37 was reduced by 19％ in the pORF5 group compared with the Ct group.Conclusions pORF5 plasmid protein could promote Ct infection by counteracting the antibacterial peptide LL37,which may be associated with the up-regulation of TNF-α expression and downregulation of LL37 expression in Ct-infected cells. Key words: Chlamydia trachomatis; pORF5 plasmid protein; LL37; Antimicrobial peptides

Development of ELISAs for the Detection of Urogenital Chlamydia trachomatis Infection Targeting the pORF5 Protein

ObjectiveTo 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. MethodsThe 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. ResultsTwo 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). ConclusionTwo 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.

PORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells

Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy. Pathogenesis of infection has been mostly attributed to excessive cytokine production. However, precise mechanisms on how C. trachomatis triggers this production, and which protein(s) stimulate inflammatory cytokines remains unknown. In the present study, the C. trachomatis pORF5 protein induced tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) in dose- and time-dependent manners in the THP-1 human monocyte cell line. We found that intracellular p38/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)/MAPK signaling pathways were required for the induction of TNF-α, IL-1β and IL-8. Blockade of toll-like receptor 2 (TLR2) signaling reduced induction levels of TNF-α, IL-8 and IL-1β. We concluded that the C. trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.

Preliminary study on genital tract immune injury in mice induced by pORF5 plasmid protein of Chlamydia trachomatis

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. Key words: Chlamydia trachomatis; pORF5 plasmid protein; Immune injury; Cytokine

Purification and immunological characteristics of monoclonal antibody 2H4 against Chlamydia trachomatis pORF5 plasmid protein

Objective To 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.Conclusion 2H4 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. Key words: Chlamydia trachomatis; pORF5 plasmid protein; Monoclonal antibody; Immunological characteristics

Preliminary study on biological characterization of Chlamydia trachomatis plasmid protein p ORF5

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. Key words: Chlamydia trachomatis; Plasmid; Secreted protein; pORF5