Chlamydia trachomatis induces lncRNA MIAT upregulation to regulate mitochondria-mediated host cell apoptosis and chlamydial development.

Chlamydia trachomatis persistent infection is the leading cause of male prostatitis and female genital tract diseases. Inhibition of host cell apoptosis is the key to maintaining Chlamydia survival in vivo, and long noncoding RNAs (lncRNAs) play important roles in its developmental cycle and pathogenesis. However, it is not clear how lncRNAs regulate persistent Chlamydia infection. Here, using a microarray method, we identified 1718 lncRNAs and 1741 mRNAs differentially expressed in IFN-γ-induced persistent C. trachomatis infection. Subsequently, 10 upregulated and 5 downregulated differentially expressed lncRNAs were verified by qRT-PCR to confirm the reliability of the chip data. The GO and KEGG analyses revealed that differentially regulated transcripts were predominantly involved in various signalling pathways related to host immunity and apoptosis response. Targeted silencing of three lncRNAs (MIAT, ZEB1-AS1 and IRF1) resulted in increased apoptosis rates. Furthermore, interference with lncRNA MIAT caused not only an obvious downregulation of the Bcl-2/Bax ratio but also a marked release of cytochrome c, resulting in a significantly elevated level of caspase-3 activation. Meanwhile, MIAT was involved in the regulation of chlamydial development during the persistent infection. Collectively, these observations shed light on the enormous complex lncRNA regulatory networks involved in mitochondria-mediated host cell apoptosis and the growth and development of C. trachomatis.

Chlamydia trachomatis plasmid-encoded protein pORF5 activates unfolded protein response to induce autophagy via MAPK/ERK signaling pathway.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular organism that depends on nutrients from the host cell for their replication and proliferation. Therefore, the interaction between this pathogen and host induces sustained endoplasmic reticulum (ER) stress in the infected cells. Unfolded protein response (UPR) has been demonstrated to be activated by chlamydial secreted effectors, allowing host cells to recover from the stressful state. In this study, we attempted to explore the role of the only secreted plasmid-encoded protein pORF5 of C. trachomatis between UPR and autophagy induction. The results showed that three branches of UPR (PERK, IRE1, and ATF6) were activated by pORF5. pORF5-induced autophagy was repressed by UPR inhibitors GSK2606414 and 4µ8C, while the autophagy inhibition was failed to influence pORF5-induced UPR significantly. MAPK/ERK inhibitor PD98059 partially suppressed the pORF5-induced autophagy, but had little effect on UPR, indicating that pORF5 actives UPR to induce autophagy via the MAPK/ERK signaling pathway. These observations provide clues on how the host maintains the cellular homeostasis during C. trachomatis infection.

Chlamydia trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis via the PI3K-AKT-mediated MDM2-p53 axis.

Chlamydia trachomatis, the most common human pathogen that causes trachoma and sexually transmitted disease, has developed various strategies for inhibiting host cell apoptosis. Activation of the PI3K (phosphoinositide 3-kinase)/AKT-mediated MDM2 (murine double minute 2)-p53 pathway plays a prominent role in the apoptosis resistance arising from C. trachomatis infection. However, the precise upstream mechanisms by which C. trachomatis activates this pathway have not been adequately investigated. Here, we reveal that the secreted C. trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis in HeLa cells. This process requires the activation of the PI3K/AKT signaling pathway, thereby leading to phosphorylation and nuclear entry of MDM2, and p53 degradation. PI3 K inhibitor LY294002 and MDM2 inhibitor Nutlin-3a block Pgp3-induced inhibition of HeLa cell apoptosis, suggesting a critical role for the PI3K/AKT pathway and its effect on the MDM2-p53 axis in Pgp3 anti-apoptotic activity.

LncRNA ZEB1-AS1/miR-1224-5p / MAP4K4 axis regulates mitochondria-mediated HeLa cell apoptosis in persistent Chlamydia trachomatis infection.

Persistent infection of Chlamydia trachomatis is thought to be responsible for the debilitating sequelae of blinding trachoma and infertility. Inhibition of host cell apoptosis is a persistent C. trachomatis infection mechanism. ZEB1-AS1 is a long non-coding RNA (lncRNA), which was up-regulated in persistent C. trachomatis infection in our previous work. In this study, we investigated the role of ZEB1-AS1 in persistent infection and the potential mechanisms. The results showed that ZEB1-AS1 was involved in the regulation of apoptosis, and targeted silencing of ZEB1-AS1 could increase the apoptosis rate of persistently infected cells. Mechanically, interference ZEB1-AS1 caused an apparent down-regulation of the Bcl-2/Bax ratio and the repression of the mitochondrial membrane potential with the remarkable release of cytochrome c, resulting in the significant elevation level of caspase-3 activation. Meanwhile, the luciferase reporter assay confirmed that ZEB1-AS1 acted as a sponge for miR-1224-5p to target MAP4K4. The regulatory effect of miR-1224-5p/MAP4K4 on persistent infection-induced antiapoptosis was regulated by ZEB1-AS1. In addition, ZEB1-AS1 inhibited the apoptosis of Chlamydia-infected cells by activating the MAPK/ERK pathway. In conclusion, we found a new molecular mechanism that the ZEB1-AS1/miR-1224-5p/MAP4K4 axis contributes to apoptosis resistance in persistent C. trachomatis infection. This work may help understand the pathogenic mechanisms of persistent C. trachomatis infection and reveal a potential therapeutic strategy for its treatment.

Chlamydia trachomatis Pgp3 protein regulates oxidative stress via activation of the Nrf2/NQO1 signal pathway.

AIM: Chlamydia trachomatis has evolved various strategies to alleviate oxidative stress of host cells to maintain their intracellular survival. However, the exact mechanism of anti-oxidative stress of C. trachomatis is still unclear. The activation of nuclear factor erythroid 2-related factor 2/quinone oxidoreductase (Nrf2/NQO1) signal pathway has been identified as an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. Pgp3 is a pivotal virulence factor of C. trachomatis involved in intracellular survival. The aim of this study is to explore the role of Pgp3 on Nrf2/NQO1 signal pathway against oxidative stress. MAIN METHODS: After HeLa cells were stimulated with Pgp3 protein, Nrf2 location and the inclusion bodies of C. trachomatis were detected by indirect immunofluorescence, western blotting and Oxidative stress assay kits were used to separately determine the protein expression and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) before and after the interference of Nrf-2 and NQO1. KEY FINDINGS: Pgp3 promoted the nuclear translocation of Nrf2 to increase NQO1 expression and reduced oxidative stress induced by LPS to contribute to the survival of C. trachomatis. Inhibition of Nrf2/NQO1 signal pathway with Nrf2 inhibitor and down-regulation of NQO1 with siRNA-NQO1 suppressed oxidative stress resistance induced by Pgp3. SIGNIFICANCE: Here we found that Pgp3 alleviated oxidative stress to promote the infectivity of C. trachomatis through activation of Nrf2/NQO1 signal pathway, which provided a novel understanding of the effects of Pgp3 in the pathogenesis of C. trachomatis.

Long Non-Coding RNA FGD5-AS1 Induced by Chlamydia trachomatis Infection Inhibits Apoptosis via Wnt/ß-Catenin Signaling Pathway.

Background: Chlamydia trachomatis (Ct) is one of the most common bacterial sexually transmitted infection (STI) pathogens in the world, but the exact pathogenic mechanism still needs to be further elucidated. Long non-coding RNAs (lncRNAs) have become vital regulators in many biological processes. Their role in the interaction between Ct and host cells has not been reported. Methods: Microarrays were used to study the expression profiles of lncRNAs and mRNAs in HeLa cells at 12, 24, and 40 h post-infection (hpi). Differentially expressed lncRNAs and mRNAs were verified by RT-qPCR. Coding-non-coding (CNC) network analysis showed co-expression molecules of selected lncRNA. Western blot, flow cytometry, and indirect immunofluorescence were used to detect the effect of lncRNA FGD5-AS1 on apoptosis during Ct infection. Results: Compared with the uninfected group, the number of differential lncRNAs were 2,130, 1,081, and 1,101 at 12, 24, and 40 hpi, and the number of differential mRNAs was 1,998, 1,129, and 1,330, respectively. Ct induced differential expression of large amounts of lncRNAs and mRNAs in HeLa cells, indicating that lncRNAs may play roles in the pathogenesis of Ct. RT-qPCR verified six differential lncRNAs and six differential mRNAs, confirming the reliability of the microarray. Among these molecules, lncRNA FGD5-AS1 was found to be upregulated at 12 and 24 hpi. Coding-non-coding (CNC) network analysis showed that co-expressed differential molecules of FGD5-AS1 at 12 and 24 hpi were enriched in the DNA replication and Wnt signaling pathway. The downregulation of FGD5-AS1 decreased the expression of ß-catenin and inhibited the translocation of ß-catenin and the DNA replication, while it promoted apoptosis of the host cells. Conclusions: DNA replication and apoptosis of host cells were affected by upregulating FGD5-AS1 via Wnt/ß-catenin pathway during Ct infection. This study provides evidence that lncRNAs are involved in the coaction between Ct and hosts, and provides new insights into the study of lncRNAs that regulate chlamydial infection.

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.

Antiapoptotic activity of Chlamydia trachomatis Pgp3 protein involves activation of the ERK1/2 pathway mediated by upregulation of DJ-1 protein.

Chlamydia trachomatis has evolved strategies to prevent host cell apoptosis to evade the host immune defense. However, the precise mechanisms of antiapoptotic activity of C. trachomatis still need to be clarified. Pgp3, one of eight plasmid proteins of C. trachomatis, has been identified to be closely associated with chlamydial virulence. In this study, we attempted to explore the effects and the mechanisms of Pgp3 protein on apoptosis in HeLa cells; the results showed that Pgp3 increased Bcl-2/Bax ratio and prevented caspase-3 activation to suppress apoptosis induced by TNF-α and cycloheximide (CHX) through ERK1/2 pathway activation. Downregulation of DJ-1 with siRNA-DJ-1(si-DJ-1) reduced ERK1/2 phosphorylation and elevated apoptotic rate significantly in Pgp3-HeLa cells. However, inhibition of ERK1/2 signal pathway with ERK inhibitor PD98059 had little effect on DJ-1 expression. These findings confirm that plasmid protein Pgp3 contributes to apoptosis resistance through ERK1/2 signal pathway mediated by upregulation of DJ-1 expression. Therefore, the present study provided novel insights into the role of Pgp3 in apoptosis and suggested that manipulation of the host apoptosis response could be a new approach for the prevention and treatment of C. trachomatis infection.

Identification of proteins interacting with pORF5 in the pathogenesis of C. trachomatis.

OBJECTIVE: This study is to identify and investigate the proteins interacting with pORF5 implicated in the pathogenesis of C. trachomatis. METHODS: The isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis was applied to identify and quantify the differentially expressed proteins in the pORF5-transfected HeLa (pORF5-HeLa) cells and the control vector-transfected HeLa (vector-HeLa) cells. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the mRNA and protein expression levels. RESULTS: Totally 3355 proteins were quantified by employing biological replicates, 314 of which were differentially expressed between the pORF5-HeLa and vector-HeLa cells. Nine differentially expressed proteins (HIST1H1C, HBA1, PARK7, HMGB1, HMGB2, CLIC1, KRT7, SFN, and CDKN2A) were subjected to qRT-PCR, and two over-expressed proteins (HMGB1 and PRAK7) were subjected to the Western blot analysis, to validate the proteomic results. The results from the qRT-PCR and Western blot analysis were consistent with the findings from the proteomic analysis. Moreover, pORF5 could inhibit the TNF-α-induced apoptosis in HeLa cells. Through siRNA-mediated functional screening, the high-mobility group box 1 (HMGB1) was shown to be relevant to the inhibition of the apoptotic response in the host cells. CONCLUSION: Identification of key proteins interacting with pORF5 could contribute to the understanding and further exploration of the function of pORF5 in the pathogenic mechanisms of C. trachomatis.

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.

Chlamydial plasmid-encoded protein pORF5 induces production of IL-1ß and IL-18 via NALP3 inflammasome activation and p38 MAPK pathway.

The pathogenesis of Chlamydia-induced inflammation is poorly understood. pORF5 is the only secreted protein encoded by Chlamydial plasmid. This study aims to investigate the effects of pORF5 on the production of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) and the underlying mechanisms of these effects. THP-1 (a human acute monocytic leukemia cell line) cells were stimulated by pORF5 with or without pretreatment with Natch domain, Leucine-rich repeat and PYD-containing protein 3 (NALP3) siRNA, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) siRNA, cysteine aspartate-specific protease-1 (caspase-1) specific inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. IL-1ß, IL-18 and caspase-1 expression was detected through both ELISA and qRT-PCR. NALP3 and ASC expression was detected by qRT-PCR. The expression of caspase-1 and phosphorylated-p38 MAPK was detected by western blot analysis. pORF5 induced IL-1ß, IL-18, caspase-1 and NALP3 inflammasome expression in THP-1 cells. Caspase-1 inhibitor significantly reduced pORF5-induced IL-1ß and IL-18 expression. The siRNAs for NALP3 inflammasome significantly reduced pORF5-induced IL-1ß, IL-18 and caspase-1 expression. Furthermore, p38 MAPK inhibitor significantly reduced pORF5-induced IL-1ß, IL-18, caspase-1 and NALP3 inflammasome expression. pORF5 could induce production of IL-1ß and IL-18 via NALP3 inflammasome activation and p38MAPK pathway. pORF5 protein might play an important role in Chlamydia pathogenesis. This study provides a new insight into the molecular pathogenesis of Chlamydial diseases.

Localization and characterization of the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.

The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis. Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells. The open reading frame (ORF) encoding the CT440 protein from the C. trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase (GST) fusion protein in E. coli XL1-Blue. The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody. After verification by Western blot and immunofluorescence assay (IFA), the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells. Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection. The results showed that the hypothetical protein CT440 was localized in the C. trachomatis inclusion membrane, and was detectable 12 h after chlamydial infection. Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection. In summary, we have identified a new inclusion membrane protein that may be an important candidate for understanding C. trachomatis pathogenesis.

[Cloning and cellular localization of pORF8 plasmid protein of Chlamydia trachomatis].

OBJECTIVE: To clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells. METHODS: pORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA). RESULTS: The pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein). CONCLUSION: The plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.