Toxoplasma gondii ROP18 suppresses IFN-γ/STAT1 pathway by targeting NMI in HEK293T cells
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- 英文论文题名：Toxoplasma gondii ROP18 suppresses IFN-γ/STAT1 pathway by targeting NMI in HEK293T cells
- 论文作者：Jing Xia ; Hongjuan Peng
- 英文论文作者：Jing Xia ; Hongjuan Peng ; Department of Pathogen Biology ; Guangdong Provincial Key Laboratory of Tropical Disease Research ; School of Public Health ; Southern Medical University
- 年：2016
- 作者机构：Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University;
- 英文论文关键词：Toxoplasma gondii ; ROP18 ; NMI ; IFN-γ/STAT1 pathway
- 会议召开时间：2016-08-08
- 会议录名称：中国动物学会寄生虫学专业委员会第十届全国寄生虫学青年工作者学术研讨会论文摘要集
- 英文会议录名称：The Abstracts of the Tenth Symposium for Yong Scientists of China Society of Parasitology
- 语种：英文
- 分类号：R382.5
- 学会代码：JISC
- 会议名称：中国动物学会寄生虫学专业委员会第十届全国寄生虫学青年工作者学术研讨会
- 会议地点：中国四川成都
- 主办单位：中国动物学会寄生虫学专业青年委员会
- 学会名称：中国动物学会寄生虫学专业委员会
- 页数：1
- 文件大小：341k
- 原文格式：D
- 会议级别：全国
摘要
Objectives: Several studies have shown that Toxoplasma gondii has developed means to effectively inhibit the host interferon gamma(IFN-γ)-induced the signaling intermediate signal transducer and activator of transcription 1(STAT1)-dependent transcription, however, the precise molecular mechanisms of inhibition remain unclear, and the parasite effectors responsible for this inhibition remain unknown. To assess the role of rhoptry protein 18(ROP18), which is a major virulence factor of T. gondii, involved in the suppression of IFN-γ/STAT1 pathway, we performed a high-throughput screening of ROP18 interacting human cell proteins using a non-induced bimolecular fluorescence complementation(Bi FC) technique. Methods: We screened the ROP18 interacting human cell proteins by using the non-induced Bi FC method. Retroviruses expressing ROP18 fused with the N-terminal fragment of yellow fluorescent protein(YFPn), and 18,000 human cell proteins fused with the C-terminal fragments of yellow fluorescent protein(YFPc) were packaged to infect HTC75 cells. The yellow fluorescence was analyzed and the positive yellow fluorescent cells were sorted out by flow cytometry. To further confirm the interaction between ROP18 and N-myc(and STAT) interactor(NMI), COS-7 cells were transiently transfected with p ECFPN1-ROP18-3×FLAG and/or p EYFPC1-NMI-HA for a fluorescence resonance energy transfer(FRET) assay and a co-immunoprecipitation(Co-IP) assay. Results: Among the 18,000 human cell proteins screened, 1784 of them showed an interaction with ROP18. Gene ontology analysis revealed that nearly one third of the interacting proteins were related to response to stimulus, and other related biology processes included cell communication, cell signaling, biosynthetic process and so on, indicating that ROP18 might be involved in several complex molecular mechanisms to enhance the intracellular survival and persistence of the parasite. Particularly, NMI, which has been reported to significantly augment the activity of IFN-γ/STAT1 pathway in mammalian cells, was found a strong binding partner with ROP18 with the highest reads among all the positive cells sorted out by Bi FC method. In addition, our results of FRET assay and Co-IP assay both confirmed the interaction between ROP18 and NMI. Furthermore, in dually transfected COS-7 cells, the overexpression of ROP18 decreased the level of NMI in a dose-dependent fashion.Conclusion: T. gondii virulence factor ROP18 was a potential effector to inhibit the human IFN-γ/STAT1 pathway by targeting the STAT1 potentiator, NMI.
Objectives: Several studies have shown that Toxoplasma gondii has developed means to effectively inhibit the host interferon gamma(IFN-γ)-induced the signaling intermediate signal transducer and activator of transcription 1(STAT1)-dependent transcription, however, the precise molecular mechanisms of inhibition remain unclear, and the parasite effectors responsible for this inhibition remain unknown. To assess the role of rhoptry protein 18(ROP18), which is a major virulence factor of T. gondii, involved in the suppression of IFN-γ/STAT1 pathway, we performed a high-throughput screening of ROP18 interacting human cell proteins using a non-induced bimolecular fluorescence complementation(Bi FC) technique. Methods: We screened the ROP18 interacting human cell proteins by using the non-induced Bi FC method. Retroviruses expressing ROP18 fused with the N-terminal fragment of yellow fluorescent protein(YFPn), and 18,000 human cell proteins fused with the C-terminal fragments of yellow fluorescent protein(YFPc) were packaged to infect HTC75 cells. The yellow fluorescence was analyzed and the positive yellow fluorescent cells were sorted out by flow cytometry. To further confirm the interaction between ROP18 and N-myc(and STAT) interactor(NMI), COS-7 cells were transiently transfected with p ECFPN1-ROP18-3×FLAG and/or p EYFPC1-NMI-HA for a fluorescence resonance energy transfer(FRET) assay and a co-immunoprecipitation(Co-IP) assay. Results: Among the 18,000 human cell proteins screened, 1784 of them showed an interaction with ROP18. Gene ontology analysis revealed that nearly one third of the interacting proteins were related to response to stimulus, and other related biology processes included cell communication, cell signaling, biosynthetic process and so on, indicating that ROP18 might be involved in several complex molecular mechanisms to enhance the intracellular survival and persistence of the parasite. Particularly, NMI, which has been reported to significantly augment the activity of IFN-γ/STAT1 pathway in mammalian cells, was found a strong binding partner with ROP18 with the highest reads among all the positive cells sorted out by Bi FC method. In addition, our results of FRET assay and Co-IP assay both confirmed the interaction between ROP18 and NMI. Furthermore, in dually transfected COS-7 cells, the overexpression of ROP18 decreased the level of NMI in a dose-dependent fashion.Conclusion: T. gondii virulence factor ROP18 was a potential effector to inhibit the human IFN-γ/STAT1 pathway by targeting the STAT1 potentiator, NMI.
Objectives: Several studies have shown that Toxoplasma gondii has developed means to effectively inhibit the host interferon gamma(IFN-γ)-induced the signaling intermediate signal transducer and activator of transcription 1(STAT1)-dependent transcription, however, the precise molecular mechanisms of inhibition remain unclear, and the parasite effectors responsible for this inhibition remain unknown. To assess the role of rhoptry protein 18(ROP18), which is a major virulence factor of T. gondii, involved in the suppression of IFN-γ/STAT1 pathway, we performed a high-throughput screening of ROP18 interacting human cell proteins using a non-induced bimolecular fluorescence complementation(Bi FC) technique. Methods: We screened the ROP18 interacting human cell proteins by using the non-induced Bi FC method. Retroviruses expressing ROP18 fused with the N-terminal fragment of yellow fluorescent protein(YFPn), and 18,000 human cell proteins fused with the C-terminal fragments of yellow fluorescent protein(YFPc) were packaged to infect HTC75 cells. The yellow fluorescence was analyzed and the positive yellow fluorescent cells were sorted out by flow cytometry. To further confirm the interaction between ROP18 and N-myc(and STAT) interactor(NMI), COS-7 cells were transiently transfected with p ECFPN1-ROP18-3×FLAG and/or p EYFPC1-NMI-HA for a fluorescence resonance energy transfer(FRET) assay and a co-immunoprecipitation(Co-IP) assay. Results: Among the 18,000 human cell proteins screened, 1784 of them showed an interaction with ROP18. Gene ontology analysis revealed that nearly one third of the interacting proteins were related to response to stimulus, and other related biology processes included cell communication, cell signaling, biosynthetic process and so on, indicating that ROP18 might be involved in several complex molecular mechanisms to enhance the intracellular survival and persistence of the parasite. Particularly, NMI, which has been reported to significantly augment the activity of IFN-γ/STAT1 pathway in mammalian cells, was found a strong binding partner with ROP18 with the highest reads among all the positive cells sorted out by Bi FC method. In addition, our results of FRET assay and Co-IP assay both confirmed the interaction between ROP18 and NMI. Furthermore, in dually transfected COS-7 cells, the overexpression of ROP18 decreased the level of NMI in a dose-dependent fashion.Conclusion: T. gondii virulence factor ROP18 was a potential effector to inhibit the human IFN-γ/STAT1 pathway by targeting the STAT1 potentiator, NMI.
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