抗菌肽buforin Ⅱ衍生物抑制细菌核酸合成的机制研究

Antibacterial peptides buforin Ⅱ analogues on bacteria by inhibition of DNA synthesis

目的探究抗菌肽buforin Ⅱ的衍生物buforin Ⅱ-A(BF2-A)和buforin Ⅱ-B(BF2-B)对细菌的胞内抑菌作用机制。方法体外用原子力显微镜观察抗菌肽与基因组DNA的结合情况,荧光光谱分析肽与基因组DNA的结合方式。体内用透射电镜观察抗菌肽作用后金黄色葡萄球菌细胞膜超微结构的变化,流式细胞仪分析肽对金黄色葡萄球菌细胞周期的影响。最后通过凝胶阻滞实验推测肽与金黄色葡萄球菌DNA合成相关基因的结合引起了细胞周期的改变。结果肽与基因组DNA发生了结合,与溴化乙锭(EB)竞争性嵌入基因组DNA;BF2-A/BF2-B与金黄色葡萄球菌作用后几乎在不破坏细胞膜的情况下渗透进入胞内,与DNA合成相关基因发生了结合,特异性阻滞细胞周期中的DNA合成阶段;肽与DNA合成相关基因发生了结合。另外,所有的实验结果显示了BF2-B穿透细胞、与DNA结合的能力及对细胞周期的影响强于BF2-A。结论 BF2-A/BF2-B与DNA穿过金黄色葡萄球菌细胞膜后在细胞内通过与DNA结合,特异性的将细胞阻滞在了细胞周期的DNA合成期而发挥了抑菌作用,而且BF2-B的上述作用强于BF2-A。

Objective To study the intracellular action of analogues buforin II-A （BF2-A） and buforin II-B （BF2-B） of the antimicrobial peptide buforin II on bacteria. Methods In vitro, the bond of genomic DNA with BF2-A/BF2-B and the change of DNA structure after the binding was investigated with atomic force microscope （AFM） and fluorescence spectra respectively, and the competitive intercalation of BF2-A/BF2-B and ethidium bromide （EB） into genomic DNA were analyzed by fluorescence spectra. In vivo, transmission electron microscope （TEM） observed the cell membrane ultrastructure of Staphylococcus aureus treated by BF2-A/BF2-B. Then flow cytometry analyzed the change of bacterial cell cycle after treated by BF2-A/BF2-B. Finally, binding action between peptide and genes related to DNA synthesis that was harvested by PCR were researched by gel retardation assay. Results BF2-A/ BF2-B bond to DNA. Both the peptides could weaken the fluorescence intensity of EB-DNA complex. BF2-A/BF2-B penetrated into cell without destroying the cell membrane. Bacterial cell cycle after interactions of BF2-A/BF2-B with bacteria specifically changed and BF2-A/BF2-B binded with key genes. Besides, all the experiments showed that BF2-B was stronger than BF2-A in the DNA-binding, membrane penetration and blocking the cell cycle. Conclusion BF2-A/BF2-B penetrated into bacteria, and block DNA synthesis phase of cell cycle of bacteria by binding to genes related to DNA synthesis specifically. Above effects of BF2-B are better than BF2-A.