南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (11): 1657-1663.doi: 10.12122/j.issn.1673-4254.2021.11.09

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蛙皮肤来源抗菌肽Brevinin-2GHk的抗菌作用机制

熊唯琛，柴金为，吴洁娜，田茂林，卢万成，徐学清   

  1. 南方医科大学药学院，广东 广州 510515
  • 出版日期:2021-11-20 发布日期:2021-12-10

Antibacterial mechanism of Brevinin-2GHk, an antimicrobial peptide from Fejervarya limnocharis skin

XIONG Weichen, CHAI Jinwei, WU Jiena, TIAN Maolin, LU Wancheng, XU Xueqing   

  1. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
  • Online:2021-11-20 Published:2021-12-10

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摘要： 目的 探讨Brevinin-2GHk（BR2GK）的二级结构、理化性质及抗菌活性，并揭示其抗菌作用机制。方法 合成并鉴定BR2GK；圆二色谱测定BR2GK的二级结构和理化性质；二倍稀释法测定BR2GK的抗菌活性；共聚焦激光扫描显微镜（LSCM）和扫描电子显微镜（SEM）研究BR2GK的杀菌方式；小鼠红细胞检测BR2GK的溶血活性；等温滴定量热实验和表面等离子共振成像检测BR2GK与脂多糖的结合；生化试剂盒检测BR2GK的抗氧化能力。结果 合成的BR2GK经高效液相色谱（HPLC） 纯化，纯度≥95%，并通过质谱进行确定。圆二色谱分析显示BR2GK具有阳离子抗菌肽的基本特征，在SDS溶液中以α-螺旋结构为主。抗菌活性测定显示BR2GK对多种菌株均具有抑制和杀灭活性，对金黄色葡萄球菌的最低抑菌浓度（MIC）达到2.76 μmol/L。LSCM和SEM观察到BR2GK使细菌细胞膜受损，细菌肿涨、收缩和变形，并渗出大量内容物，但在具有抗菌活性的浓度下对红细胞无溶血作用。等温滴定量热实验显示BR2GK与脂多糖的结合亲和力KD为18.2±0.8 μmol/L。抗氧化测试结果显示，BR2GK以浓度依赖的方式清除NO（P<0.01）。BR2GK对ABTS和DPPH自由基清除的反应快速，当浓度为40 μmol/L时，在 1 min内分别清除了73.51%的ABTS和90.32%的DPPH。结论 BR2GK具有良好的广谱抗菌活性，且不会导致溶血，此外还能结合脂多糖，并具有较强的抗氧化能力；BR2GK对设计合成具有更强抗菌活性和更低细胞毒性的抗菌肽具有重要的指导意义。

关键词: 两栖动物；抗菌肽；Brevinin-2GHk；抗菌机制；抗菌

Abstract: Objective To investigate the secondary structure, physicochemical properties and antibacterial activity of Brevinin-2GHk (BR2GK), an antimicrobial peptide from frog skin, and explore its antibacterial mechanism. Methods BR2GK was synthesized, purified with high performance liquid chromatography (HPLC) and identified using mass spectrometry. Circular dichroism was used to determine the secondary structure and physicochemical properties of BR2GK. Two-fold dilution method was used to determine the antibacterial activity of BR2GK, and its antibacterial mechanism was explored using laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). The hemolytic activity of BR2GK was analyzed in red blood cells. Isothermal titration calorimetry (ITC) and surface plasmon resonance imaging (SPRi) were employed to detect the binding of BR2GK to lipopolysaccharide (LPS), and the antioxidant activity of BR2GK was evaluated using biochemical kits. Results The synthesized BR2GK, with a purity exceeding 95% after purification, had the basic characteristics of cationic antimicrobial peptides. BR2GK consisted mainly of α-helical structure in SDS solution and exhibited a broad-spectrum antibacterial activity. Antibacterial activity assay showed that BR2GK had inhibitory and killing activity against a variety of strains with a minimum inhibitory concentration (MIC) of 2.76 μmol/L against Staphylococcus aureus. Observation with LSCM and SEM showed that BR2GK at an active concentration caused bacterial cell membrane damage, cell swelling, contraction, deformation, and massive exudation of intracellular contents without causing hemolysis. ITC showed that the binding affinity KD of BR2GK to LPS was 18.2±0.8 μmol/L. The antioxidant test showed that BR2GK was capable of effectively scavenging NO, ABTS and DPPH. Conclusion BR2GK, as a broad-spectrum antibacterial peptide with also a strong antioxidant capacity, does not cause hemolysis and is capable of binding to LPS. BR2GK has an important value for future design and synthesis of antimicrobial peptides with stronger antibacterial activity and lower cytotoxicity.

Key words: amphibian; antimicrobial peptide; Brevinin-2GHk; antibacterial mechanism; antibacterial