Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
Fish Shellfish Immunol. 2011 Apr-May;30(4-5):1095-108. doi: 10.1016/j.fsi.2011.02.004. Epub 2011 Feb 17.
The serum of rabbitfish (Siganus oramin) has been confirmed previously to have killing effect to Cryptocaryon irritans, an important marine ciliate protozoan that causes a disease referred to as "marine white spot disease". Herein, we find the serum of the rabbitfish also shows antibacterial activity against both gram-positive and gram-negative bacteria and has killing effect on two other parasites: Trypanosoma brucei brucei, Ichthyophthirius multifiliis. Results of scanning electron microscopy indicated that after treating with rabbitfish serum, the surface of the Staphylococcus aureus was wrinkled and pores were formed on the surface of Escherichia coli. Serum of the rabbitfish possesses a strong killing effect to Ichthyophthirius multifiliis in vitro, causing a similar effect as to C. irritans. The serum of rabbitfish also showed strong killing effect to T. b. brucei in vitro, with the minimus trypanocidal titre (MTT) only to be 1.5% in 1 h. Results of laser confocal fluorescence microscopy indicated that rabbitfish serum could also induce cell rupture of T. b. brucei. A novel antimicrobial protein (SR-LAAO) was isolated from the serum of rabbitfish by using ultrafiltration, reversed phase high performance liquid chromatography (RP-HPLC) and Native polyacrylamide gel electrophoresis (Native-PAGE). Results of gel overlay assay showed that the protein could act alone to inhibit the growth of S. aureus and E. coli. Results of western blot and automated Edman degradation showed that it was the same as the antiparasitic protein (APP) reported before to have killing effect on C. irritans. Full length cDNA sequence of the SR-LAAO was cloned. BLAST research suggested that the cDNA of SR-LAAO has a close similarity with a number of L-amino acid oxidases (LAAOs) and possesses two conserved motifs that exist in LAAOs. Combined, these results demonstrate that this protein which has antimicrobial activity to some pathogenic organisms was a novel LAAO found in the serum of rabbitfish. Immunohistochemical analysis demonstrated tissue specific expression and localization of SR-LAAO in the spleen, kidney, gill and blood of the rabbitfish, but was not found in other tissues. These results suggest that this protein may contribute considerably to the host non-specific immune defense mechanism to combat microbes of the rabbitfish and has the potency for using in future drug development.
先前已经证实,兔鱼(Siganus oramin)血清对引起“海水白点病”的重要海洋纤毛虫原生动物—— Cryptocaryon irritans 具有杀伤作用。在此,我们发现兔鱼血清还对革兰氏阳性菌和革兰氏阴性菌具有抗菌活性,并对另外两种寄生虫:布氏锥虫(Trypanosoma brucei brucei)、多子小瓜虫(Ichthyophthirius multifiliis)具有杀伤作用。扫描电子显微镜的结果表明,用兔鱼血清处理后,金黄色葡萄球菌的表面起皱,大肠杆菌表面形成孔。兔鱼血清对多子小瓜虫具有很强的体外杀伤作用,效果类似于 Cryptocaryon irritans。兔鱼血清对布氏锥虫(Trypanosoma brucei brucei)也具有很强的体外杀伤作用,在 1 小时内最小杀锥虫效价(MTT)仅为 1.5%。激光共聚焦荧光显微镜的结果表明,兔鱼血清也能诱导布氏锥虫的细胞破裂。通过超滤、反相高效液相色谱(RP-HPLC)和天然聚丙烯酰胺凝胶电泳(Native-PAGE)从兔鱼血清中分离到一种新型抗菌蛋白(SR-LAAO)。凝胶覆盖测定结果表明,该蛋白可单独抑制金黄色葡萄球菌和大肠杆菌的生长。Western blot 和自动 Edman 降解的结果表明,它与先前报道的对 Cryptocaryon irritans 具有杀伤作用的抗寄生虫蛋白(APP)相同。克隆了 SR-LAAO 的全长 cDNA 序列。BLAST 研究表明,SR-LAAO 的 cDNA 与许多 L-氨基酸氧化酶(LAAOs)具有密切的相似性,并具有 LAAOs 中存在的两个保守基序。综合这些结果表明,这种对一些致病生物具有抗菌活性的蛋白质是在兔鱼血清中发现的一种新型 LAAO。免疫组织化学分析表明,兔鱼的脾脏、肾脏、鳃和血液中组织特异性表达和定位 SR-LAAO,但在其他组织中未发现。这些结果表明,该蛋白可能对兔鱼的非特异性免疫防御机制对抗微生物具有重要作用,并有可能在未来的药物开发中使用。