FC8衍生肽对临床多重耐药菌的抗菌效应及作用机制

The antibacterial effect of FC8-derived peptides against clinically multidrug-resistant bacteria and their action mechanisms

摘要

目的 通过赖氨酸替换策略设计抗菌肽FC8的衍生肽,并探讨其对临床耐药菌的作用机制。方法 采用位点突变法将抗菌肽FC8中第7、10、21位的谷氨酰胺残基分别替换为赖氨酸,构建系列FC8衍生肽。通过圆二色光谱(circular dichroism, CD)检测肽的二级结构;采用微量肉汤稀释法测定最低抑菌浓度(minimum inhibitory concentration, MIC);利用CCK-8法评估其对BESA-2B细胞的细胞毒性。通过N-苯基-1-萘胺(N-phenyl-1-naphthylamine, NPN)荧光探针实验与脂质体模型实验分析肽与细胞膜的作用机制,并以凝胶阻滞分析评估肽与细菌基因组DNA的结合能力。结果 CD光谱结果显示,FC8及其衍生肽在磷酸盐缓冲液(phosphate buffered saline, PBS)中为无规卷曲结构,而在50%三氟乙醇环境中呈现α-螺旋结构。抗耐药鲍曼不动杆菌(Acinetobacter baumannii)方面,FC8、Q10K、Q7K/Q10K与Q7K/Q10K/Q21K肽的几何平均MIC值(geometric mean MIC, GM)分别为6.5、4.9、4.5、3.7 μmol/L;抗耐药金黄色葡萄球菌(Staphylococcus aureus)的GM值分别为4.0、2.6、2.5、2.0 μmol/L。各衍生肽对BESA-2B细胞的半数抑制浓度(half maximal inhibitory concentration, IC₅₀)分别为120.9、118.2、103.9、80.5 μmol/L。治疗指数(therapeutic index, TI)计算结果显示,Q10K衍生肽的TI最高。NPN荧光探针实验与脂质体模型实验结果提示,FC8及其衍生肽可有效破坏耐药菌细胞膜;凝胶阻滞分析显示,FC8及其衍生肽与细菌基因组DNA结合能力显著。结论 适度增加FC8肽的正电荷有助于增强其膜靶向性以及与DNA结合能力,从而显著提高其抗菌活性和TI。FC8衍生肽通过破坏细胞膜和结合细菌DNA的双重靶点机制,实现对临床耐药菌的有效杀菌作用。

Abstract

Objective To design a series of antimicrobial peptide FC8 derivatives through lysine substitution and to investigate their mechanisms against clinically drug-resistant bacteria. Methods Glutamine residues at positions 7, 10, and 21 of the parent peptide FC8 were individually or combinatorially replaced with lysine to generate FC8 derivatives. Circular dichroism (CD) spectroscopy was used to evaluate the secondary structure of the peptides. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. The cytotoxicity of peptides against BESA-2B cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. The interaction between the peptides and bacterial membranes was analyzed using the N-phenyl-1-naphthylamine (NPN) fluorescence probe and liposome leakage models. DNA binding ability was assessed using gel retardation assays. Results CD spectroscopy revealed that FC8 and its derivatives exhibited a random coil structure in phosphate-buffered saline (PBS), whereas they presented an α-helical structure in 50% trifluoroethanol. Against multidrug-resistant Acinetobacter baumannii, the geometric mean MICs (GM) of FC8, Q10K, Q7K/Q10K, and Q7K/Q10K/Q21K were 6.5, 4.9, 4.5, and 3.7 μmol/L, respectively. For methicillin-resistant Staphylococcus aureus, the corresponding GM were 4.0, 2.6, 2.5, and 2.0 μmol/L. The half-maximal inhibitory concentrations (IC₅₀) for BESA-2B cells were 120.9, 118.2, 103.9, and 80.5 μmol/L, respectively. Among all the derivatives, Q10K displayed the highest therapeutic index (TI). NPN and liposome assays demonstrated that FC8 and its derivatives disrupted bacterial membranes. Gel retardation analysis confirmed strong binding of the peptides to bacterial genomic DNA. Conclusion Moderate enhancement of peptide cationicity improves membrane-targeting and DNA-binding abilities, thereby significantly increasing the antibacterial selectivity and TI of FC8 derivatives. FC8-derived peptides exert potent bactericidal activity against clinically resistant bacteria through a dual-target mechanism involving disruption of the cell membrane and binding to bacterial DNA.

关键词

抗菌肽 / FC8 / 鲍曼不动杆菌 / 金黄色葡萄球菌 / α-螺旋 / 荧光光谱 / 凝胶阻滞分析 / 多重耐药菌

Key words

antimicrobial peptide / FC8 / A.baumannii / S.aureus / α-helix / fluorescence spectrum / gel retardation assay / multidrug-resistant bacteria

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基金资助

黑龙江省卫生健康委课题项目(20210202020363)