鱼类识别双链RNA的Toll样受体及其接头分子的系统解析——以草鱼为例

Toll-like receptor (TLR) gene family play a crucial role in the immune system. However, the ligands remain largely unclear, and the quantities and corresponding relations of their adaptors also keep indistinct for most teleost TLR members. In the present study, a case of grass carp with severe viral disease loss and genomic database is taken as an example. We focus on the comprehensive screening of TLRs recognizing viral associated molecular pattern (double-stranded RNA) and investigation of their adaptors. Our previous studies found that there are 21 TLR members in grass carp, and mRNA expressions of most TLRs are induced post viral infection. In this study, we firstly reconstruct and express LRR (leucine-rich repeat) domains of TLRs, surface plasmon resonance (SPR) and other techniques are employed to screen the TLRs of direct interaction with poly(I:C) (double-stranded RNA). Then, TLRs overexpression, poly(I:C) stimulation, transcriptome sequencing, knockdown expression, and other techniques are used to confirm the interactions through triggering the classical pathways or the newly identified important molecules. After that, the adaptors of TLRs are investigated by Co-immunoprecipitation (Co-IP), quantitative proteome (iTRAQ) and the cytoplasmic TIR database of grass carp. In the end, the reverse Co-immunoprecipitation, laser scanning confocal microscopy and RNAi techniques are performed to identify the adaptors. The present study will systematically clarify the TLRs recognizing dsRNA and their adaptors in teleost, and lay a solid foundation for further elucidating the antiviral immune regulatory network in fish.

Toll样受体（TLR）基因家族在免疫系统中发挥着至关重要的作用。然而，鱼类多数TLR识别配体谱系尚不清楚，接头分子的数量和对应关系尚不明确。本研究以病毒性疾病损失严重且有基因组数据库的草鱼为例，全面筛查识别病毒相关分子模式（双链RNA）的TLR及其接头分子。我们前期研究发现草鱼有21个TLR成员，大多数对病毒感染表达量有变化。本研究首先重组表达LRR结构域，利用表面等离子共振等技术逐一筛查与poly(I:C)（双链RNA）的直接互作；接着通过过表达TLR、poly(I:C)刺激、转录组测序、敲降表达等对经典通路和新发现重要分子的调控来确认互作；然后免疫共沉淀结合定量蛋白组学及草鱼胞内TIR数据库筛选接头分子；最后反向免疫共沉淀结合激光扫描共聚焦显微镜共定位等技术鉴定接头分子。本研究将系统夯实鱼类识别双链RNA的TLR成员及其接头分子，为进一步阐释鱼类抗病毒免疫调控网络奠定坚实的基础。

Toll样受体（TLR）感知微生物相关分子模式，激活先天性免疫系统，调控随后的获得性免疫应答，形成连接先天性免疫和获得性免疫的桥梁，在免疫系统中发挥着至关重要的作用。哺乳动物具有13个TLR，不同鱼类TLR数量不等，鱼类缺TLR6，有些为鱼类特有。我们通过配体受体直接互作及对下游信号的激活/抑制，从草鱼21个TLR成员中系统筛选到11个有效识别dsRNA的TLR成员（TLR2、TLR3、TLR4b、TLR5a、TLR5b、TLR7、TLR8a、TLR19、TLR21、TLR22a、TLR22b），表现出明显的扩张现象（人体仅有TLR3和TLR10识别dsRNA，发挥拮抗抗病毒作用），且出现基因新功能化。重点对TLR5a、TLR5b、TLR7、TLR19、TLR22a、TLR22b开展了抗病毒免疫信号通路的系统研究，主要创新性发现：1）阐明了鲤科鱼类特有的两种膜型TLR5（TLR5a/b）免疫功能：TLR5a/b定位于早期内体和溶酶体；TLR5a/b形成同源二聚体结合鞭毛蛋白但不传导信号，识别dsRNA分别负调控和正调控抗病毒免疫，草鱼TLR5a-G240和TLR5b-N547位点是结合dsRNA并“开关”抗病毒免疫的关键识别位点，草鱼TLR5a-T101及对应TLR5b-I99位点N-糖基化修饰对抗病毒免疫调控起到“开关”作用；TLR5a/b形成异源二聚体识别细菌鞭毛蛋白，激活抗细菌免疫；TLR5a/b以MyD88、Mal、TRIF为接头分子。2）鱼形动物（圆口类和鱼类）TLR7不仅识别病毒ssRNA，而且识别dsRNA，以MyD88为接头分子，经由SLC15A4/TASLa/b，通过促进IRF5/7和AP-1表达，上调IFN1、IFN3、IL-1β，从而激活抗病毒免疫。3）阐明了部分硬骨鱼类特有的TLR19的免疫信号通路：TLR19在内体识别dsRNA，以TRIF为接头分子，通过促进IRF3蛋白和磷酸化水平、抑制IRF7磷酸化水平，激活抗病毒免疫。4）革新了变温脊椎动物都具有的TLR22亚细胞定位（溶酶体）、接头分子（MyD88），草鱼TLR22a促进IRF7磷酸化发挥抗病毒作用，TLR22b抑制TLR7磷酸化发挥促进病毒增殖作用。本研究为建立鱼类自身TLR介导的抗病毒免疫调控网络提供强有力的实验支撑，为免疫系统演化和比较免疫学研究提供实验数据。

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