鸡毒支原体GroEL蛋白通过NF-κB信号通路诱导DF-1细胞释放IL-1β的研究
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摘要
鸡毒支原体(MG)脂质相关膜蛋白(LAMPs)在刺激宿主细胞天然免疫中起重要作用,GroEL蛋白是本实验室前期利用质谱鉴定筛选出的LAMPS中的关键组分。为研究GroEL蛋白诱导宿主细胞炎性反应的信号通路,本研究通过原核表达系统表达出MG的GroEL蛋白,利用激光共聚焦试验、荧光定量PCR和western blot方法分别检测GroEL蛋白的粘附特性、p65入核、磷酸化水平及IL-1β分泌情况。激光共聚焦试验结果显示,GroEL蛋白能够黏附于DF-1细胞表面并刺激DF-1细胞中p65从胞浆转入细胞核;western blot检测显示GroEL蛋白能够激活NF-κB信号通路促进p65磷酸化水平提高;荧光定量PCR检测显示GroEL蛋白能够促进IL-1β释放,当NF-κB信号通路被抑制后,IL-1β的释放显著下降(p<0.01)。本研究结果表明MG GroEL蛋白能够粘附于宿主细胞表面并通过激活NF-κB信号通路诱导宿主细胞IL-1β的释放,从而在炎症反应中发挥作用。本研究为深入研究MG致病机制提供了实验依据。
The lipid associated membrane proteins(LAMPs) of Mycoplasma gallisepticum(MG) play important roles in stimulating innate immunity of host cells, in the previous study, we have identified the GroEL protein is the key component of LAMPs by mass spectrometry analysis. In order to study the signaling pathway of host cell inflammatory reaction induced by GroEL protein, the GroEL protein was expressed in prokaryotic expression system, and the adhere property of GroEL protein, the p65 translocated into nuclear, the phosphorylation level of p65 and the release of IL-1β in DF-1 cells were detected by the confocal laser scanning microscopy, SYBR Green I real-time PCR, and western blot. The confocal laser scanning microscopy results showed that GroEL protein was able to adhere to the surface of DF-1 cells and stimulated the p65 translocated into nuclear.Western blot results showed the stimulation of GroEL protein activated the NF-κB signaling pathway and promote the phosphorylation level of p65. SYBR Green I real-time PCR detection showed GroEL protein promoted the release of IL-1β in DF-1 cells. While the NF-κB signaling pathway was inhibited, and the mRNA transcriptional level of IL-1β decreased significantly.These results suggested that GroEL protein of MG had the ability to adhere to the surface of DF-1 cells and play a role in inflammatory reaction by the release of IL-1β activated via the activation of NF-κB signaling pathway, and provided information for further study the pathogenic mechanism of MG.
The lipid associated membrane proteins(LAMPs) of Mycoplasma gallisepticum(MG) play important roles in stimulating innate immunity of host cells, in the previous study, we have identified the GroEL protein is the key component of LAMPs by mass spectrometry analysis. In order to study the signaling pathway of host cell inflammatory reaction induced by GroEL protein, the GroEL protein was expressed in prokaryotic expression system, and the adhere property of GroEL protein, the p65 translocated into nuclear, the phosphorylation level of p65 and the release of IL-1β in DF-1 cells were detected by the confocal laser scanning microscopy, SYBR Green I real-time PCR, and western blot. The confocal laser scanning microscopy results showed that GroEL protein was able to adhere to the surface of DF-1 cells and stimulated the p65 translocated into nuclear.Western blot results showed the stimulation of GroEL protein activated the NF-κB signaling pathway and promote the phosphorylation level of p65. SYBR Green I real-time PCR detection showed GroEL protein promoted the release of IL-1β in DF-1 cells. While the NF-κB signaling pathway was inhibited, and the mRNA transcriptional level of IL-1β decreased significantly.These results suggested that GroEL protein of MG had the ability to adhere to the surface of DF-1 cells and play a role in inflammatory reaction by the release of IL-1β activated via the activation of NF-κB signaling pathway, and provided information for further study the pathogenic mechanism of MG.
引文
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[2]Stipkovits L,Kempf I.Mycoplasmoses in poultry[J].Rev Sci Tech,1996,15(4):1495-1525.
[3]Yu Ying,Chen Ying,Wang Yang,et al.TLR2/MyD88/NF-κBsignaling pathway regulates IL-1βproduction in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs[J].Microb Pathog,2018,117:225-231.
[4]Ricci C,Ortore M G,Vilasi S,et al.Stability and disassembly properties of human nave Hsp60 and bacterial GroEL chaperonins.[J].Biophys Chem,2016,208:68-75.
[5]Calderwood S K,Mambula S S,Gray J R P J.Extracellular heat shock proteins in cell signaling and immunity[J].Ann N YAcad Sci,2007,1113:28-39.
[6]Breloer M,Dorner B,MoréS H,et al.Heat shock proteins as"danger signals":Eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells[J].Eur J Immunol,2001,31(7):2051-2059.
[7]Schmitt E,Gehrmann M,Brunet M,et al.Intracellular and extracellular functions of heat shock proteins:Repercussions in cancer therapy[J].J Leukoc Biol,2007,81(1):15-27.
[8]Davis K L,Wise K S.Site-specific proteolysis of the MALP-404lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans[J].Infect Immun,2002,70(3):1129-1135.
[9]Jenkins C,Geary S J,Gladd M,et al.The Mycoplasma gallisepticum OsmC-like protein MG1142 resides on the cell surface and binds heparin[J].Microbiology,2007,153(5):1455-1463.
[10]Noss E H,Pai R K,Sellati T J,et al.Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-k Da lipoprotein of Mycobacterium tuberculosis[J].Immunology,2001,167(2):910-918.
[11]Giotis E S,Ross C S,Robey R,et al.Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells[J].Sci Rep,2017,7(1):17485-17499.
[12]Indikova I,Much P,Spitkovits L,et al.Role of the GapA and CrmA cytadhesins of Mycoplasma gallisepticum in promoting virulence and host colonization[J].Infect Immun,2013,81(5):1618-1624.
[13]周长平,陈莹,李媛,等.鸡毒支原体热休克蛋白GrpE粘附特性的鉴定[J].中国预防兽医学报,2017,1(1):28-33.
[14]Eskra L,Mathison A,Splitter G.Microarray analysis of mRNAlevels from RAW264.7 macrophages infected with Brucella abortus[J].Infect Immun,2003,71(25):1125-1133.
[15]Arthur S,Ley S.Mitogen-activated protein kinase in innate immunity[J].Nature,2013,13(26):679-692.
[16]Gilmore T D.Introduction to NF-kappaB:Players,pathways,perspectives[J].Oncogene,2006,25(51):6680-6684.
[17]Ghosh S,May M J,Kopp E B.NF-κB and Rel proteins:Evolutionarily conserved mediators of immune responses[J].Annu Rev Immunol,1998,16:225-260.
[18]He Jun,You Xiao-xing,Zeng Yan-hua,et al.Mycoplasma genitalium-derived lipid-associated membrane proteins activate NF-kappaB through toll-like receptors 1,2,and 6 and CD14 in a MyD 88-dependent pathway[J].Clin Vaccine Immunol,2009,16(12):1750-1757.
[19]Wang Yang,Liu Su-li,Li Yuan,et al.Mycoplasma bovis-derived lipid-associated membrane proteins activate IL-1βproduction through the NF-κB pathway via toll-like receptor 2 and MyD 88[J].Dev Comp Immunol,2016,55:111-118.