Huang Ying, Zhang Guosong, Ren Qian
College of Oceanography, Hohai University, Nanjing, China.
School of Agriculture and Bioengineering, Heze University, Heze, China.
Front Physiol. 2019 Jul 25;10:952. doi: 10.3389/fphys.2019.00952. eCollection 2019.
Tolls/Toll-like receptors (TLRs) play a key role in innate immunity by detecting the invading microbes and subsequently activating downstream signaling cascades. In this study, two new molluscan Toll members (designed as and ) were identified from triangle-shell pearl mussel (). The obtained full-length cDNA was 3207 bp consisting of a 2223 bp open reading frame (ORF) that encoded a peptide of 740 amino acids. cDNA is a 3216 bp molecule that contains an ORF of 2139 bp encoding a protein of 712 amino acids. The deduced and proteins share two common structures: extracellular leucine-rich repeat (LRR) domains and intracellular Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR results showed that and were mainly expressed in the hepatopancreas and the gills, and they responded rapidly to bacterial stimulation. RNA interference by dsRNA results revealed that and RNAi strongly decreased the expression of lysozyme () and defensin () in the gills of RNAi-treated mussels with challenge. As a pattern recognition receptor, the prokaryotic expressed the recombinant LRR domains of and (rHcToll6-LRR and rHcToll7-LRR) could bind to Gram-positive and Gram-negative bacteria and had a strong tendency to recognize lipopolysaccharide (LPS) and peptidoglycan (PNG). rHcToll6-LRR and rHcToll7-LRR exhibited a significant bactericidal activity against and . These findings provide useful information to characterize Tolls in mussels.
Toll样受体（Tolls/Toll-like receptors，TLRs）通过检测入侵的微生物并随后激活下游信号级联反应，在先天免疫中发挥关键作用。在本研究中，从三角帆蚌（Hyriopsis cumingii）中鉴定出两个新的软体动物Toll成员（分别命名为HcToll6和HcToll7）。获得的HcToll6全长cDNA为3207 bp，包含一个2223 bp的开放阅读框（ORF），编码一个740个氨基酸的肽段。HcToll7 cDNA是一个3216 bp的分子，包含一个2139 bp的ORF，编码一个712个氨基酸的蛋白质。推导的HcToll6和HcToll7蛋白具有两个共同结构：细胞外富含亮氨酸重复序列（LRR）结构域和细胞内Toll/白细胞介素-1受体（TIR）结构域。实时定量PCR结果表明，HcToll6和HcToll7主要在肝胰腺和鳃中表达，并且它们对细菌刺激反应迅速。双链RNA介导的RNA干扰结果显示，在经嗜水气单胞菌（Aeromonas hydrophila）攻毒的RNA干扰处理贻贝的鳃中，HcToll6和HcToll7 RNA干扰强烈降低了溶菌酶（LZM）和防御素（DEF）的表达。作为一种模式识别受体，原核表达的HcToll6和HcToll7的重组LRR结构域（rHcToll6-LRR和rHcToll7-LRR）能够结合革兰氏阳性菌和革兰氏阴性菌，并且具有强烈识别脂多糖（LPS）和肽聚糖（PNG）的倾向。rHcToll6-LRR和rHcToll7-LRR对嗜水气单胞菌和鳗弧菌（Vibrio anguillarum）表现出显著的杀菌活性。这些发现为表征贻贝中的Tolls提供了有用信息。
