Liu Mei-Yi, Zhang Yi-Rong, Zhang Jian-Hua, Miao Liang, Dang Yun-Fei, Fei Chen-Jie, Li Chang-Hong, Chen Jiong
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, China; Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Meishan Campus, Ningbo, 315832, China; Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Meishan Campus, Ningbo, 315832, China.
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Meishan Campus, Ningbo, 315832, China; Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Meishan Campus, Ningbo, 315832, China.
Fish Shellfish Immunol. 2023 May;136:108703. doi: 10.1016/j.fsi.2023.108703. Epub 2023 Mar 21.
NK-lysin (NKL) is a positively charged antimicrobial peptide with broad-spectrum bactericidal activities. In this study, the cDNA sequence of NKL (TmNKL) from black scraper (Thamnaconus modestus) was cloned, which encodes a predicted polypeptide of 150 amino acids that contains a surfactant protein B domain with three disulfide bonds. Phylogenetically, TmNKL was most closely related to its teleost counterpart from tiger puffer (Takifugu rubripes). Expression analysis demonstrated that TmNKL transcripts were constitutively expressed in all tested tissues, with the highest expression levels in the gills. Its expression was significantly upregulated in the gills, head kidney, and spleen after infection with Vibrio parahaemolyticus. A linear peptide (TmNKLP40L) and a disulfide-type peptide (TmNKLP40O) were further synthesized and results showed that disulfide bonds are not essential for bactericidal activities of TmNKL, and that both forms of TmNKL exhibited potent bactericidal activities against 4 gram- negative bacteria, including V. parahaemolyticus, V. alginolyticus, Edwardsiella tarda, and V. harveyi. Observed antimicrobial activities are likely due to the effects of TmNKLP40L and TmNKLP40O treatment on disrupting the integrity of both inner and outer membrane of V. parahaemolyticus, resulting in hydrolysis of bacterial genomic DNA. Damaged cell membranes and leakage of intracellular contents were further confirmed using scanning and transmission microscopy. Moreover, administration of 1.0 μg/g TmNKLP40L or TmNKLP40O significantly decreased bacterial load in tissues and thus, pronouncedly enhanced the survival of V. parahaemolyticus-infected fish. Overall, our results demonstrated that TmNKL is a potent innate effector and provides protective effects against bacterial infection.
NK溶素（NKL）是一种带正电荷的抗菌肽，具有广谱杀菌活性。在本研究中，克隆了黑鳃梅童鱼（Thamnaconus modestus）的NKL（TmNKL）的cDNA序列，其编码一个预测的由150个氨基酸组成的多肽，该多肽包含一个具有三个二硫键的表面活性蛋白B结构域。在系统发育上，TmNKL与其来自虎河豚（Takifugu rubripes）的硬骨鱼对应物关系最为密切。表达分析表明，TmNKL转录本在所有测试组织中组成性表达，在鳃中表达水平最高。在用副溶血性弧菌感染后，其在鳃、头肾和脾脏中的表达显著上调。进一步合成了线性肽（TmNKLP40L）和二硫键型肽（TmNKLP40O），结果表明二硫键对于TmNKL的杀菌活性不是必需的，并且两种形式的TmNKL对4种革兰氏阴性菌均表现出强大的杀菌活性，包括副溶血性弧菌、溶藻弧菌、迟缓爱德华氏菌和哈维氏弧菌。观察到的抗菌活性可能是由于TmNKLP40L和TmNKLP40O处理对破坏副溶血性弧菌内膜和外膜的完整性产生了影响，导致细菌基因组DNA水解。使用扫描和透射显微镜进一步证实了细胞膜受损和细胞内容物泄漏。此外，以1.0μg/g的剂量施用TmNKLP40L或TmNKLP40O可显著降低组织中的细菌载量，从而显著提高副溶血性弧菌感染鱼的存活率。总体而言，我们的结果表明TmNKL是一种强大的先天效应物，并对细菌感染提供保护作用。
