来自穿膜肽的新肽的抗菌活性及抑菌机制

Antibacterial activity and mechanisms of a new peptide derived from cell-penetrating peptide

【目的】研究基于穿膜肽和抗菌肽构效关系改造获得的新肽P7的抗菌活性及其对大肠杆菌(E.coli)的抑菌机制。【方法】微量稀释法和溶血实验分析P7的抑菌活性及其对正常细胞的细胞毒性;采用膜荧光探针、流式细胞术和扫描电镜分析P7对E.coli膜通透性、膜完整性的影响和细胞超微结构变化;通过激光共聚焦分析P7在E.coli细胞中的定位;凝胶阻滞实验测定P7与E.coli基因组DNA结合能力。【结果】P7比母肽显示更强的抑菌活性,最低抑菌浓度范围为4-32μmol/L,且在作用浓度范围内具有较弱的溶血活性。P7可以增加E.coli外膜和内膜的通透性,使E.coli细胞膜的完整性和细胞表面结构受损。同时P7可以穿过E.coli细胞膜在细胞质聚集并与基因组DNA结合。【结论】P7通过增加E.coli内外膜通透性,穿过细胞膜与胞内DNA结合发挥抑菌活性。

[Objectivel We studied the antibacterial activities and mechanism of a new peptide P7, according to the structure-activity relationships of eell-penetraling peptide and antimicrobial peptide. [ Methods ] The antimicrobial activities and cytotoxieity of P7 were examined using the microdilution and hemolysis analysis. The effects of P7 on lhe outer anti plasma membrane permeability, membrane integrity and morphology of E. coli cells were analyzed by the membrane fluorescent probe, flow eytometry and scanning electron microscopy. Localization of the P7 onto the E. coli cells was determined by using a confocal laser-scanning microscopy. The DNA binding activity of P7 was evaluated by electrophoretic mobility shift assay. [ Results] P7 possessed stronger antimicrohial activity than ppTG20. The inhibitory eoncentration was in the range from 4 to 32 l, zmol/L where P7 shown low hemolysis. P7 eouht increase the outer and plasma membrane permeability, induce the cell membrane integrity loss and cells structure damage. P7 penetrated the cell membrane, aecumulated inside the cytoplasm and interacted with DNA. [ Conclusion] P7 exerted its antibacterial activity by increasing cell membrane permeability, penetrated the cell membrane and binding to DNA.