Zhuang Ze-Rui, Yang Xiao-Dong, Huang Xia-Zi, Gu Hui-Xian, Wei Han-Yin, He Yi-Jun, Deng Li
Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
Fish Shellfish Immunol. 2017 Jul;66:240-253. doi: 10.1016/j.fsi.2017.04.011. Epub 2017 Apr 19.
The present study reports the identification, and characterization of three new putative piscidin paralogues, ecPis-2, ecPis-3 and ecPis-4, from orange-spotted grouper (Epinephelus coioides). The cDNA of the three piscidins with the 207, 216, and 231 nt open reading frame encoded respectively a 68-, 71-, and 76-amino acid preprotein consisting of the predicted signal peptide, and putative mature peptide and prodomain. The phylogenetic analysis indicated that multiple piscidin paralogues in one fish species are highly diversified, the analysis suggested that the piscidins should be a family belonging to the superfamily of ancient cationic, linear, and amphipathic host defence peptides widespread across invertebrate and vertebrate taxa comprising insect cecropins and ceratotoxins, and the amphibian dermaseptins. The synthetic putative mature peptides, ecPis-2S, ecPis-3S and ecPis-4S, had strong activities against bacterial and fungal species. EcPis-3S exhibited powerful activity against the infective stage of Cryptocaryon irritans, theronts. The full length ecPis-2 and ecPis-4 by removal of signal peptide, ecPis-2L and ecPis-4L respectively, had potency against bacterial, fungal and parasitic species. The peptide ecPis-2S was proved to exist in spleen of orange-spotted grouper by HPLC followed by ESI-LCMS analysis. Basal transcriptions of ecPis-2, ecPis-3 and ecPis-4 were detected not only in the potential sites of pathogen entry such as gills, skin and intestine, but also in tissues such as head kidney, trunk kidney, blood cells, and spleen with highly abundant immune cells, however different paralogues expressed constitutively with different levels in the tissues. In addition, the expression of ecPis-2, ecPis-3 and ecPis-4 was upregulated in orange-spotted grouper challenged by Vibrio Parahaemolyticus, in different tissues at different time point after bacteria injection. These results support ecPis-2, ecPis-3 and ecPis-4 being the important immune-related genes in orange-spotted grouper innate immune system and playing multifunctional and complementary roles following their structural and functional diversification, and expression pattern difference. Finally, this study facilitates the evaluation of ecPis-2S, 2L, ecPis-3S, and ecPis-4S, -4L as potential templates of therapeutic agents against pathogens.
本研究报告了从点带石斑鱼（Epinephelus coioides）中鉴定并表征了三种新的假定杀鱼菌素旁系同源物，即ecPis-2、ecPis-3和ecPis-4。这三种杀鱼菌素的cDNA分别具有207、216和231个核苷酸的开放阅读框，编码由预测的信号肽、假定的成熟肽和前结构域组成的68、71和76个氨基酸的前体蛋白。系统发育分析表明，一种鱼类中的多个杀鱼菌素旁系同源物高度多样化，该分析表明，杀鱼菌素应属于一个超家族，该超家族是广泛存在于无脊椎动物和脊椎动物类群中的古老阳离子、线性和两亲性宿主防御肽，包括昆虫杀菌肽和角毒素，以及两栖动物皮肤抗菌肽。合成的假定成熟肽ecPis-2S、ecPis-3S和ecPis-4S对细菌和真菌具有很强的活性。EcPis-3S对刺激隐核虫的感染阶段，即游动孢子，表现出强大的活性。去除信号肽后的全长ecPis-2和ecPis-4，分别为ecPis-2L和ecPis-4L，对细菌、真菌和寄生虫具有活性。通过高效液相色谱（HPLC）和电喷雾电离液相色谱-质谱联用（ESI-LCMS）分析，证明肽ecPis-2S存在于点带石斑鱼的脾脏中。不仅在病原体进入的潜在部位如鳃、皮肤和肠道，而且在头肾、躯干肾、血细胞和含有大量免疫细胞的脾脏等组织中检测到ecPis-2、ecPis-3和ecPis-4的基础转录，然而不同的旁系同源物在组织中的组成性表达水平不同。此外，在副溶血性弧菌攻击的点带石斑鱼中，ecPis-2、ecPis-3和ecPis-4的表达在细菌注射后的不同时间点在不同组织中上调。这些结果支持ecPis-2、ecPis-3和ecPis-4是点带石斑鱼先天免疫系统中重要的免疫相关基因，并且在其结构和功能多样化以及表达模式差异后发挥多功能和互补作用。最后，本研究有助于评估ecPis-2S、2L、ecPis-3S和ecPis-4S、-4L作为抗病原体治疗剂的潜在模板。
