拟穴青蟹MD2同源物介导抗弧菌免疫信号途径的识别和激活机制研究

Vibriosis greatly restricts the development of the aquaculture of shrimp and crabs. The lack of full knowledge about the immune mechanism against Vibrio limited the prevention and control of vibriosis. LPS is the important component on Vibrio surface, and can induce the immune response of animals. The mammalian MD2 can recognize LPS and is critical for the activation of TLR4 signaling pathway. Previous study proved that Scylla paramamosain Toll could mediate the anti-Vibrio response, but the recognition and activation mechanism is unclear. We identified a MD2 homolog (SpMD2) and found it can recognize LPS and modulate the induction of antimicrobial peptide expression by Vibrio infection. We speculate SpMD2 is located upstream of SpToll and mediates the recognition and activation of TLR Pathway. Besides, we found the mud crab LGBP might bind and present LPS, and strengthen the TLR pathway. To confirm the speculation, this program is planned to study the possibility that the formation of LPS-SpMD2-SpToll complex, to investigate whether the complex can activate the TLR pathway and regulate the antimicrobial peptide expression, and to explore whether SpLGBP can present LPS to TLR pathway to strengthen the activation. This study will illustrate the specific recognition and activation mechanism of the anti-Vibrio signaling pathway mediated by SpMD2, and provide new insights to the prevention and control of vibriosis.

弧菌疾病严重危害虾蟹养殖业发展，目前对甲壳动物抗弧菌免疫机制缺乏足够了解,限制了对弧菌病的防治。LPS是弧菌表面重要组分，能诱导动物的免疫应答。哺乳动物MD2能识别LPS，是激活TLR4信号途径必需蛋白。我们的前期研究证明拟穴青蟹SpToll途径参与抗弧菌反应，但识别和激活机制并不清楚。我们在青蟹中鉴定到MD2同源物SpMD2，其可结合LPS并影响弧菌对抗菌肽的诱导，推测其可能位于SpToll上游，参与该途径的识别和激活。另外，我们还发现青蟹LGBP可能结合并提呈LPS，增强上述途径的激活效果。为证实该推测，本项目拟研究LPS、SpMD2和SpToll形成复合体的可能性及互作方式，明确上述复合体是否激活信号途径而调控抗菌肽表达，探明SpLGBP提呈LPS及其增强激活信号途径的可能性。本项目预期阐明SpMD2介导的抗弧菌信号途径的特异识别和激活机制，为虾蟹弧菌疾病防治提供新的理论支撑。

目前对甲壳动物抗弧菌免疫机制缺乏足够了解，限制了弧菌病的防治。哺乳动物MD2识别LPS，是激活TLR4信号途径并诱导抗感染免疫必需蛋白。我们从青蟹中鉴定到与人MD2类似的分子SpMD2。SpMD2与LPS及其Lipid A特异性结合。干扰SpMD2表达，降低包括SpALF2在内的多种抗菌肽表达水平，导致血淋巴清除弧菌能力降低、弧菌数量增加；过表达SpMD2，促进SpALF2高表达，提示SpMD2可调控抗菌肽表达。对青蟹9个Tolls进行分析，发现SpToll6与人TLR4相似。多种结合实验结果显示，SpMD2与SpToll6胞外区以浓度依赖方式结合，SpMD2+LPS复合体也与SpToll6特异性结合，并且SpMD2与SpToll6能在细胞表面共定位，表明LPS-SpMD2-SpToll6能形成三元复合体。干扰SpToll6表达，抑制了SpALF2表达，并导致血淋巴清除弧菌能力减弱、弧菌数量增加，表明SpToll6参与抗弧菌免疫。向过表达SpToll6的细胞培养液中添加LPS+SpMD2，提高了荧光素酶活性（SpALF2高表达），提示SpMD2位于SpToll6信号通路上游。由此，青蟹体内可能存在LPS-SpMD2-SpToll6参与的‘核心版’抗革兰氏阴性菌免疫信号途径。其中，SpMD2作为辅助识别受体，通过识别LPS并结合SpToll6参与该信号途径的识别和激活。SpLGBP可与LPS特异性结合，并且结合活性显著高于SpMD2，提示其不具备向SpMD2传递LPS的能力。SpLGBP作为模式识别受体，既参与酚氧化酶系统激活，也参与SpToll6抗菌信号途径激活，但其参与激活SpToll6通路能力弱于SpMD2，表明SpLGBP很可能通过其它方式激活SpToll6信号通路。本研究揭示了SpToll6抗弧菌信号途径的特异识别和激活机制，为虾蟹弧菌疾病防治提供了新的理论支撑。