Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, 10900, Thailand.
Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, 10900, Thailand.
Fish Shellfish Immunol. 2018 Sep;80:357-375. doi: 10.1016/j.fsi.2018.06.026. Epub 2018 Jun 15.
Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded β-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 10 and 1 × 10 CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system.
抗脂多糖因子 (ALF) 是一种与免疫相关的蛋白质，在甲壳动物抵御入侵病原体的免疫防御机制中起着至关重要的作用。本研究中发现了三种不同的巨型河虾 ALF（Mr-ALF3、Mr-ALF8 和 Mr-ALF9）。基于序列分析，Mr-ALF3 和 Mr-ALF9 分别被鉴定为甲壳动物 ALF 的新成员（分别属于 F 组和 G 组）。结构上，每个新鉴定的 Mr-ALF 都包含三个α-螺旋，它们与一个四链β-折叠结构相对，该结构具有 LPS 结合基序，通常与细菌的细胞壁成分结合。使用定量实时 RT-PCR（qRT-PCR）的组织表达分析表明，Mr-ALF3 在大多数组织中表达，在心脏和血细胞中表达最高。Mr-ALF8 基因在心、血细胞、中肠、肝胰腺和后肠中高度表达，而 Mr-ALF9 基因在心和血细胞中适度表达。通过 qRT-PCR 在鳃、肝胰腺和血细胞中研究了 Mr-ALFs 对嗜水气单胞菌和冷热温度的转录反应。我们发现，当实验虾暴露在极端温度（25 和 35°C）下时，所有 Mr-ALF 在所有测试组织中均明显受到抑制。此外，用 2 种不同浓度的嗜水气单胞菌（1×10 和 1×10 CFU/ml）处理后，所有检测组织中这些基因的表达水平均显著诱导，尤其是注射后 12 和 96 小时。最后，对每个 Mr-ALF 的 LPS 基序肽的结合活性分析表明，Mr-ALF3 的 LPS 肽与两种致病性革兰氏阴性菌嗜水气单胞菌和哈维弧菌以及非致病性革兰氏阳性菌巨大芽孢杆菌具有最强的粘附性。结果还表明，Mr-ALF8 和 Mr-ALF9 肽对类似测试细菌具有温和的抗菌作用。根据本研究获得的信息，明确鉴定了新型 ALF 基因。对其在致病和温度应激下的反应分析表明，这些 ALF 具有结合和抗菌活性，以及由此产生的生理效应，表明它们在虾免疫系统中具有至关重要的功能作用。
