Wei Xiaoyuan, Wang Limin, Sun Wanwei, Zhang Ming, Ma Hongyu, Zhang Yueling, Zhang Xinxu, Li Shengkang
Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou, 515063, China; Marine Biology Institute, Shantou University, Shantou, 515063, China.
Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou, 515063, China; Marine Biology Institute, Shantou University, Shantou, 515063, China.
Dev Comp Immunol. 2018 Jul;84:213-229. doi: 10.1016/j.dci.2018.02.016. Epub 2018 Feb 21.
As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.
作为模式识别受体，C型凝集素（CTLs）通过识别并结合病原体表面保守的病原体相关分子模式（PAMPs），在甲壳类动物的免疫系统中发挥重要作用。在本研究中，从拟穴青蟹血细胞中鉴定出一种新的CTL，即SpCTL-B。SpCTL-B cDNA全长1278 bp，开放阅读框（ORF）为348 bp。预测的SpCTL-B蛋白含有一个单一的碳水化合物识别结构域（CRD）。SpCTL-B转录本分布于所有检测组织中，在肝胰腺中表达水平最高。用副溶血性弧菌、脂多糖（LPS）、聚肌胞苷酸（polyI:C）和白斑综合征病毒（WSSV）刺激后，血细胞和肝胰腺中SpCTL-B的mRNA水平上调。通过镍亲和层析纯化的重组SpCTL-B（rSpCTL-B）与金黄色葡萄球菌、β-溶血性链球菌、大肠杆菌、嗜水气单胞菌、溶藻弧菌的结合活性比与副溶血性弧菌和酿酒酵母的结合活性更强。rSpCTL-B对革兰氏阳性菌（金黄色葡萄球菌、β-溶血性链球菌）和革兰氏阴性菌（大肠杆菌、副溶血性弧菌、嗜水气单胞菌、溶藻弧菌）表现出广谱的微生物凝集活性，且呈钙依赖性。rSpCTL-B的凝集活性可被D-甘露糖和LPS抑制，但不能被D-果糖和半乳糖抑制。抗菌试验表明，rSpCTL-B对所有检测的革兰氏阳性菌和革兰氏阴性菌均有生长抑制作用。当通过RNA干扰使SpCTL-B沉默时，拟穴青蟹的细菌清除能力下降，血细胞中5种抗菌肽（AMPs）（SpCrustin、SpHistin、SpALF4（抗脂多糖因子）、SpALF5和SpALF6）的转录水平显著降低。在我们的研究中，敲低SpCTL-B可在mRNA转录水平和蛋白质翻译水平下调拟穴青蟹中SpSTAT的表达。同时，拟穴青蟹血细胞中SpCTL-B RNA干扰后，吞噬率和3种吞噬相关基因的表达均下降。总体而言，我们的结果表明，SpCTL-B可能作为一种模式识别受体（PRR），通过影响拟穴青蟹中AMPs的表达和血细胞的吞噬作用，在对病原体感染的免疫反应中发挥作用。
