实验海洋生物学重点实验室知识库

长牡蛎（Crassostrea gigas）是我国重要的海水养殖经济贝类，但夏季大规模死亡事件时有发生，造成的经济损失直接影响了我国牡蛎养殖产业的可持续发展。已有研究证实，牡蛎疱疹病毒（Ostreid Herpevirus -1，OsHV-1）是造成长牡蛎大规模死亡的重要病原微生物。因此，深入研究长牡蛎抗病毒免疫防御机制对探索病毒防治方法有重要的意义。Toll-样受体（Toll-like receptor，TLR）是一类重要模式识别受体（Pattern recognition receptor，PRR），在识别以及清除病原微生物中有重要作用。TLR信号通路在脊椎动物中研究较为透彻，然而在无脊椎动物的研究中还处于探索阶段。已有研究发现，在若干无脊椎动物中TLR基因有明显的扩张现象，在基因组中预测到了上百个TLR同源序列（长牡蛎，小头虫，紫海胆），暗示TLR通路在无脊椎动物中可能存在功能分化。目前关于软体动物TLR信号通路的研究有限，所得结果较为简单零散。深入了解这些基因在通路中的作用机制有助于我们更好地认识TLR信号通路在抗病毒天然免疫防御过程中的作用。

本研究利用分子生物学和细胞生物学等方法对长牡蛎TLR信号通路关键基因的作用机制进行了探究。大量研究表明在脊椎动物中TLR通过TIR-TIR结合作用招募接头分子。我们首先以OsHV-1爆发期表达上调的4个TLR基因为研究对象，通过酵母双杂交文库筛选了可能与之存在相互作用的蛋白。结果发现，这4个TLR蛋白只有CgTLR-27513筛选到的接头分子蛋白中有TIR结构域，因此我们后续实验则以该TLR基因为主要研究对象。而另外三个TLR筛选到的潜在互作蛋白通过功能注释发现，这些蛋白主要参与了细胞迁移、呼吸作用和细胞因子运输，因此，我们推测长牡蛎TLR基因功能表现出多样性。

筛选到的与CgTLR-27513互作蛋白中有3个具有TIR结构域的蛋白，其中两个是已经被克隆鉴定的接头分子MyD88，另一个是只具有TIR结构域的MyD88-like蛋白，将其命名为CgMyD88s。我们发现，CgMyD88-1和CgMyD88-2能够自身或相互之间结合形成同源或异源二聚体参与CgTLR-27513介导的抗病毒免疫过程。除此之外，CgMyD88-1在LPS及poly(I:C)刺激下在细胞质中凝聚成团，而CgMyD88-2在LPS及poly(I:C)刺激下在细胞膜和细胞质中均有凝结，说明CgMyD88-1和CgMyD88-2在功能上存在分化。结合实验室以往数据，CgMyD88s在OsHV-1 μvar刺激后呈现先下降后上升的表达模式，与CgMyD88-1和CgMyD88-2的表达模式正好相反。免疫共沉淀结果显示，CgMyD88s只能够与CgTLR-27513结合而不能与CgMyD88-1以及CgMyD88-2结合。干扰CgMyD88s表达后，CgMyD88-1和CgMyD88-2均有明显的表达上调，并且CgMyD88s不仅不能激活NF-κB的转录还会抑制CgMyD88-1和CgMyD88-2蛋白对NF-κB的转录激活作用。因此，我们推测CgMyD88s与CgMyD88-1以及CgMyD88-2蛋白存在竞争作用。

其次，我们克隆了MyD88下游IRAK家族基因，并根据结构特征将其命名为CgIRAK4。该基因在OsHV-1 μvar，溶藻弧菌和poly(I:C)刺激下均有明显的表达上调，并且能够通过与CgMyD88-1和CgMyD88-2的死亡结构域结合参与到TLR介导的免疫过程。然而CgIRAK4并不能激活NF-κB的转录活性，还会抑制CgMyD88-1和CgMyD88-2蛋白对NF-κB的转录激活作用。说明CgIRAK4参与了MyD88依赖型TLR信号通路的负调控过程。

最后，我们首次在长牡蛎中克隆了MyD88非依赖型信号通路（TLR-TRIF）下游的TBK1和IKKε基因，这两个基因在OsHV-1 μvar，溶藻弧菌和poly(I:C)刺激下均有明显的表达上调。CgTBK1在胞质中表达且需要与CgSTING结合激活IFN-β的转录活性；CgIKKε自身能够诱导NF-κB及IFN-β启动子的激活，而CgIKKε激酶结构域截短体则会丧失该激活作用。虽然在无脊椎动物中还没有MyD88非依赖型信号通路的相关报道，但是存在TLR-TRIF下游的TBK1和IKKε基因参与天然免疫过程。

综上所述，本研究首次提出长牡蛎TLR基因可能通过细胞迁移和呼吸作用辅助抗病毒天然免疫过程。梳理了由CgTLR-27513，CgMyD88-1，CgMyD88-2，CgMyD88s和CgIRAK4参与的长牡蛎TLR抗病毒天然免疫通路的信号机制。克隆了MyD88非依赖型信号通路下游的TBK1和IKKε基因并对其在天然免疫过程中的功能进行了初步探究。这些结果丰富了对无脊椎动物TLR信号通路功能的理解，为深入探索无脊椎动物免疫系统提供了理论基础。

The Pacific oyster (Crassostrea gigas) is one of important marine aquaculture species in China, while summer mortality events have been reported occasionally. The economic losses directly affect the sustainable development of Chinese aquaculture industry. It has been studied that the Ostreid Herpevirus -1 (OsHV-1) is an important pathogen that causes the oysters’ mortality. Therefore, in-depth study of the anti-virus immune defense mechanism of Pacific oyster is of great significance to the exploration of virus prevention. Toll-like receptor (TLR) is an important pattern recognition receptor (PRR) in recognizing and eliminating pathogens. The mechanism of TLR signaling pathway has been studied more intensively in vertebrates and is still in an exploratory stage in the study of invertebrates. It has been found that the TLR genes have obvious expansion in several invertebrates, and hundreds of TLR homologous (Crassostrea gigas, Capitella capitata, Strongylocentrotus purpuratus) are predicted in the genome, suggesting that the TLRs in invertebrates have functional differentiation. At present, the research on the TLR signaling pathway in molluscs is poor, and the results obtained are relatively simple and fragmented. Therefore, an in-depth study of the mechanism of key genes in the TLR pathway helps us to better understand the role of oyster TLR signaling pathways in the anti-viral innate immune defense process.

In this study, molecular biology and cell biology methods and so on were used to investigate the mechanism of key genes in the oyster TLR signaling pathway. A large number of studies have shown that TLR recruits adaptor proteins through TIR-TIR binding in vertebrates. We first used the four TLR genes that were up-regulated in the outbreak period of OsHV-1 as the research objects, and screened proteins that might interact with them through the yeast two-hybrid library system. Our results show that only one of these four TLR proteins, CgTLR-27513, identified interacting proteins that containing TIR domain. Therefore, we use CgTLR-27513 gene as the main component in our subsequent experiments. By analyzing the function of the remaining three TLR potential interacting proteins, we found those predicted interacting proteins participate in cell migration, respiration and cytokines transport. Therefore, we speculate the oyster TLR genes are functional diversification.

There are 3 potential interacting proteins containing TIR domain screened by CgTLR-27513. Two of them are MyD88, adaptor proteins that have been cloned and identified, and the other is a MyD88-like protein with only a TIR domain named after CgMyD88s. We found that CgMyD88-1 and CgMyD88-2 can bind to each other to form homo- or heterodimerization to participate in CgTLR-27513-mediated antiviral immunity. In addition, CgMyD88-1 aggregated in the cytoplasm under stimulation with LPS and poly(I:C), whereas CgMyD88-2 condenses in the cell membrane and cytoplasm under both LPS and poly(I:C) stimulation. These results suggest that CgMyD88-1 and CgMyD88-2 are functional differences in TLR signaling pathway. Combined with the past data in laboratory, CgMyD88s exhibited a decreased and then increased expression pattern after stimulation with OsHV-1 μvar, which is the opposite of the two CgMyD88. The result of co-immunoprecipitation showed that CgMyD88s could only bind with CgTLR-27513 but not with two CgMyD88. After interference with CgMyD88s expression, both CgMyD88 expressions were significantly upregulated, and CgMyD88s not only failed to activate NF-κB transcription but also inhibited the activation of NF-κB by two CgMyD88 proteins. Therefore, we speculated that CgMyD88s competes with two CgMyD88 proteins.

Secondly, we cloned a member of IRAK family which acted as a downstream molecular of MyD88 genes and named it CgIRAK4 based on its structural features. CgIRAK4 mRNA was up-regulated after OsHV-1 μvar, Vibrio alginolyticus and poly(I:C) stimulation, and it was able to participate in the TLR-mediated immune process by binding to two CgMyD88 through death domain. However, CgIRAK4 is unable to activate the transcriptional activity of NF-κB, while it could inhibit the transcriptional activation of NF-κB by two CgMyD88 proteins. This indicates that CgIRAK4 is involved in the negative regulation of MyD88-dependent TLR signaling pathway.

Finally, we first cloned the TBK1 and IKKε genes which participate in MyD88-independent signaling pathway (TLR-TRIF) in oysters. Both of them are significant up-regulated after OsHV-1 μvar, Vibrio alginolyticus and poly(I:C) stimulation. CgTBK1 is expressed in the cytoplasm and needs to bind to CgSTING to activate the transcriptional activity of IFN-β; CgIKKε can induce the activation of NF-κB and IFN-β transcription by itself, whereas the truncation only containing kinase domain of CgIKKε loses this activation. Although there have been no reports of MyD88-independent signaling pathways in invertebrates, there are TLR-TRIF downstream TBK1 and IKKε genes involved in the innate immune process.

In summary, this study first proposed that the TLR genes of Pacific oyster may assist the antiviral natural immune process through cell migration and respiration. Set up the mechanism of the anti-viral innate immune TLR pathway of Pacific oysters involved in CgTLR-27513, CgMyD88-1, CgMyD88-2, CgMyD88s and CgIRAK4. Clone the downstream genes of MyD88-independent signaling pathway, CgTBK1 and CgIKKε, and investigate their function in the innate immunity initially. These results enrich the understanding of the function of invertebrate TLR signaling pathways, providing a theoretical basis for in-depth exploration of invertebrate immune system.