[Antibacterial mechanism of Brevinin-2GHk, an antimicrobial peptide from Fejervarya limnocharis skin].
[Antibacterial mechanism of Brevinin-2GHk, an antimicrobial peptide from Fejervarya limnocharis skin].
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DOI：
10.12122/j.issn.1673-4254.2021.11.09
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发表时间：
2021-11
影响因子：
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通讯作者：
W. Xiong;J. Chai;J. Wu;M. Tian;Weili Lu;X. Xu
中科院分区：
文献类型：
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作者：
W. Xiong;J. Chai;J. Wu;M. Tian;Weili Lu;X. Xu
目的研究蛙皮抗菌肽Brevinin-2GHk（BR2GK）的二级结构、理化性质和抗菌活性，并探讨其抗菌机制。方法合成BR2GK，用高效液相色谱（HPLC）纯化，质谱鉴定。用圆二色谱法测定了BR2GK的二级结构和理化性质。采用二倍稀释法测定BR2GK的抑菌活性，并采用激光扫描共聚焦显微镜（LSCM）和扫描电镜（SEM）对其抑菌机理进行探讨。在红细胞中分析BR2GK的溶血活性。采用等温滴定量热法（ITC）和表面等离子体共振成像（SPRi）检测BR2GK与脂多糖（LPS）的结合，并采用生化试剂盒评价BR2GK的抗氧化活性。结果合成的BR2GK经纯化后纯度大于95%，具有阳离子抗菌肽的基本特征。BR2GK在SDS溶液中主要由α-螺旋结构组成，具有广谱抗菌活性。抗菌活性测定结果表明，BR2GK对多种细菌均有抑制和杀灭作用，对金黄色葡萄球菌的最低抑菌浓度（MIC）为2.76 μ mol/L。激光共聚焦显微镜和扫描电镜观察表明，活性浓度的BR2GK引起细菌细胞膜损伤，细胞肿胀、收缩、变形，细胞内物质大量渗出，但不引起溶血。ITC结果显示，BR2GK与LPS的结合亲和力KD为18.2 ± 0.8 μ mol/L。抗氧化实验表明，BR2GK具有较强的清除NO、ABTS和DPPH的能力。结论BR2GK是一种广谱抗菌肽，具有较强的抗氧化能力，不引起溶血，并能与LPS结合。BR2GK对今后设计合成抗菌活性更强、细胞毒性更低的抗菌肽具有重要价值。
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.