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Chlamydia psittaci Plasmid-Encoded CPSIT_P7 Elicits Inflammatory Response in Human Monocytes via TLR4/Mal/MyD88/NF-?B Signaling Pathway.

ABSTRACT: The chlamydial plasmid, an essential virulence factor, encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. However, the virulence factors and the molecular mechanisms of Chlamydia psittaci are not well understood. In the present study, we investigated the roles and mechanisms of the plasmid-encoded protein CPSIT_P7 of C. psittaci in regulating the inflammatory response in THP-1 cells (human monocytic leukemia cell line). Based on cytokine arrays, CPSIT_P7 induces the expression of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in THP-1 cells. Moreover, the expression levels of IL-6, IL-8, and MCP-1 stimulated by CPSIT_P7 declined after silencing of the Toll-like receptor 4 (TLR4) gene using small interfering RNA and transfection of a dominant negative plasmid encoding TLR4 (pZERO-hTLR4). We further demonstrated that transfection with the dominant negative plasmid encoding MyD88 (pDeNy-hMyD88) and the dominant negative plasmid encoding Mal (pDeNy-hMal) could also abrogate the expression of the corresponding proteins. Western blot and immunofluorescence assay results showed that CPSIT_P7 could activate nuclear factor ?B (NF-?B) signaling pathways in THP-1 cells. Altogether, our results indicate that the CPSIT_P7 induces the TLR4/Mal/MyD88/NF-?B signaling axis and therefore contributes to the inflammatory cytokine response.

SUBMITTER: Chen Q

PROVIDER: S-EPMC7744487 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Chlamydia psittaci Plasmid-Encoded CPSIT_P7 Elicits Inflammatory Response in Human Monocytes via TLR4/Mal/MyD88/NF-κB Signaling Pathway.

Chen Qian Q Li Yumeng Y Yan Xiaoliang X Sun Zhenjie Z Wang Chuan C Liu Shuangquan S Xiao Jian J Lu Chunxue C Wu Yimou Y

Frontiers in microbiology 20201203

The chlamydial plasmid, an essential virulence factor, encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. However, the virulence factors and the molecular mechanisms of Chlamydia psittaci are not well understood. In the present study, we investigated the roles and mechanisms of the plasmid-encoded protein CPSIT_P7 of C. psittaci in regulating the inflammatory response in THP-1 cells (human monocytic leukemia cell line). ...[more]

PMID: 33343522

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Project description:The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-κB was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-κB. Furthermore, inhibition of TLR2, MyD88, and NF-κB in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-κB signaling pathway.

Project description:The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine secretion, strongly suggesting their involvement in the rPmp18.1-induced IL-1β cytokine secretion. Taken together, these results indicate that C. abortus Pmp18.1 induces IL-1β secretion by TLR4 activation through the MyD88, NF-κB as well as the Caspase-1 signaling pathways and may be a potential C. abortus vaccine candidate. The vaccine potential of Pmp18.1 will subsequently be evaluated in an appropriate animal model, using VCG as an immunomodulator, following immunization and challenge.

Project description:The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions.IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

Dataset Information

Chlamydia psittaci Plasmid-Encoded CPSIT_P7 Elicits Inflammatory Response in Human Monocytes via TLR4/Mal/MyD88/NF-?B Signaling Pathway.

Publications

<i>Chlamydia psittaci</i> Plasmid-Encoded CPSIT_P7 Elicits Inflammatory Response in Human Monocytes via TLR4/Mal/MyD88/NF-κB Signaling Pathway.

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