Department of Life Sciences, College of Natural Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
Fish Shellfish Immunol. 2011 Feb;30(2):532-42. doi: 10.1016/j.fsi.2010.11.025. Epub 2010 Dec 15.
Lysozyme (muramidase) represents an important defense molecule of the fish innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of β-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of a g-type lysozyme (RBgLyz) was identified in the Oplegnathus fasciatus rock bream fish genome by means of multi-tissue normalized cDNA pyrosequencing using Roche 454 GS-FLX™ technology. RBgLyz is composed of 669 bp, with a 567 bp open reading frame that encodes 188 amino acids. Protein motif searches indicated that RBgLyz contains the soluble lytic transglycosylase domain involved in maintaining cell wall integrity. Furthermore, RBgLyz shares significant identity (81.4%) with Chinese perch Siniperca chuatsi. Quantitative real-time RT-PCR analysis results showed that RBgLyz transcripts are constitutively expressed in various tissues from healthy rock breams. In order to determine RBgLyz function in immunity, its expression was analyzed in head kidney following exposure to known immune stimulants or pathogens. RBgLyz transcripts were significantly up-regulated in response to challenge with lipopolysaccharide (LPS) and Edwardsiella tarda, as compared to non-injected control fish. Polyinosinic:polycytidylic acid (poly I:C) dsRNA stimulated a moderate expression of RBgLyz, as did Streptococcus iniae but to a lesser extent. There were no specific time-dependent effects on RBgLyz mRNA expression observed in response to rock bream iridovirus (RBIV) infection. Taken together, the gene expression results indicated that g-type lysozyme plays a role in the innate immune response to LPS, poly I:C, E. tarda and S. iniae in rock bream. Thus, we generated recombinant RBgLyz in an Escherichia coli expression system and characterized its antimicrobial activity. Our results indicated that recombinant RBgLyz had lytic activity against Gram-negative Vibrio salmonicida, Gram-positive Listeria monocytogenes, S. iniae and Micrococcus lysodeikticus. In addition, observations by scanning electron microscope (SEM) confirmed that the cell morphology of M. lysodeikticus was altered in the presence of recombinant RBgLyz.
溶菌酶（胞壁质酶）是鱼类先天免疫系统的重要防御分子。溶菌酶因其杀菌特性而闻名，它可以催化肽聚糖层中 N-乙酰氨基葡萄糖和 N-乙酰胞壁酸之间的β-（1,4）-糖苷键水解。在这项研究中，通过使用罗氏 454 GS-FLX™技术对多组织进行归一化 cDNA 焦磷酸测序，从 Oplegnathus fasciatus 石斑鱼基因组中鉴定出一种 g 型溶菌酶（RBgLyz）的完整编码序列。RBgLyz 由 669bp 组成，其中 567bp 的开放阅读框编码 188 个氨基酸。蛋白质基序搜索表明，RBgLyz 含有参与维持细胞壁完整性的可溶性溶菌转糖苷酶结构域。此外，RBgLyz 与中国鲈鱼 Siniperca chuatsi 具有显著的同源性（81.4%）。实时定量 RT-PCR 分析结果表明，RBgLyz 转录物在健康石斑鱼的各种组织中持续表达。为了确定 RBgLyz 在免疫中的功能，在头肾中分析了其在暴露于已知免疫刺激物或病原体后的表达。与未注射对照鱼相比，RBgLyz 转录物在 LPS 和爱德华氏菌的刺激下显著上调。多聚肌苷酸：多聚胞苷酸（poly I:C）dsRNA 刺激 RBgLyz 适度表达，而链球菌也有一定程度的表达，但程度较低。在石斑鱼虹彩病毒（RBIV）感染后，没有观察到 RBgLyz mRNA 表达的特定时间依赖性变化。总的来说，基因表达结果表明，g 型溶菌酶在石斑鱼对 LPS、poly I:C、爱德华氏菌和链球菌的先天免疫反应中发挥作用。因此，我们在大肠杆菌表达系统中生成了重组 RBgLyz，并对其抗菌活性进行了表征。我们的结果表明，重组 RBgLyz 对革兰氏阴性鲑鱼气单胞菌、革兰氏阳性单核细胞增生李斯特菌、链球菌和微球菌具有溶菌活性。此外，扫描电子显微镜（SEM）的观察结果证实，在存在重组 RBgLyz 的情况下，微球菌的细胞形态发生了改变。
