Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.

中文翻译：

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PvML1 通过识别 LPS 和调节虾的 AMP 表达来抑制细菌感染

Toll 和 Toll 样受体 (TLR) 在先天免疫中发挥重要作用果蝇和哺乳动物。最近的研究表明虾中存在 Toll 介导的免疫信号通路。然而，由于缺乏关键的识别分子，如肽聚糖识别蛋白，甲壳类动物中Toll信号通​​路的识别和激活机制仍然知之甚少。在这里，一个新的 MD2 相关脂质识别 (ML) 成员命名为光伏ML1 的特点是南美白对虾. 我们发现光伏ML1 与人类 MD2 具有相似的 3D 结构，可以特异性识别参与 LPS 介导的 TLR4 信号传导的脂多糖 (LPS)。PVML1在血细胞中高表达，并在后显着上调副溶血性弧菌挑战。此外，结合和凝集测定表明光伏ML1 对 LPS 及其关键部分脂质 A 以及弧菌细胞和结合光伏ML1与细菌细胞导致细菌凝集，提示光伏ML1 可能在与 LPS 相互作用后充当潜在的病原体识别蛋白。此外，涂层副溶血性弧菌与重组光伏ML1促进细菌清除体内并提高了受细菌攻击的虾的存活率。RNAi实验进一步证实了这一结果。的击倒PVML1显着抑制了血淋巴中细菌的清除，降低了感染虾的存活率。同时，沉默的PVML1严重损害了一些抗菌肽 (AMP) 的表达。这些结果证明了细菌清除介导的显着相关性光伏带有 AMP 表达式的 ML1。有趣的是，我们发现光伏ML1 与细胞外区域相互作用光伏Toll2，之前已被证明通过调节 AMP 表达参与细菌清除。总之，拟议的抗菌模型由光伏ML1 可以描述如下。光伏ML1通过与LPS结合作为革兰氏阴性菌的潜在识别受体，然后被激活光伏Toll2 介导的信号通路通过与光伏Toll2 通过产生特定的 AMPs 来消除入侵的细菌。该研究为无脊椎动物Toll信号通​​路的识别和激活机制以及ML成员的防御功能提供了新的见解。