源自蜡样芽孢杆菌抗菌肽DB16的筛选及其对金黄色葡萄球菌的抑菌机制

Selection of Antimicrobial Peptide DB16 from Bacillus cereus and Its Antibacterial Mechanism against Staphylococcus aureus

本实验发现当与蜡样芽孢杆菌混合培养时,金黄色葡萄球菌的生长受到抑制。结合生物信息学方法从蜡样芽孢杆菌DeadBoxATP依赖性RNA解旋酶中预测、筛选得到抗菌肽DB16(RKLLQFAKKLGIVFTK)。抗菌肽DB16对金黄色葡萄球菌的最低抑菌质量浓度(minimum inhibitory concentration,MIC)为31.25μg/mL,可在0.5 h内完全抑制金黄色葡萄球菌的生长;抗菌肽DB16在磷酸盐缓冲溶液中呈现无规卷曲结构,在十二烷基硫酸钠环境中转变为α-螺旋结构。采用荧光探针、流式细胞术、透射电子显微镜、DNA凝胶电泳以及圆二色谱等方法探究抗菌肽DB16对金黄色葡萄球菌的抑菌机制,结果表明,抗菌肽DB16能够改变细菌细胞膜通透性,造成细胞内容物外泄,同时进入胞内与金黄色葡萄球菌基因组DNA结合,影响正常DNA的复制,最终抑制菌体的生长繁殖。此外,对哺乳动物红细胞的处理表明,DB16在8×MIC条件下仍无溶血性。综上,源自蜡样芽孢杆菌的抗菌肽DB16在金黄色葡萄球菌防控方面具有很大应用潜力。

In this study,it was found that when co-cultured with Bacillus cereus,the growth of Staphylococcus aureus was inhibited.The antimicrobial peptide DB16(RKLLQFAKKLGIVFTK)was predicted and selected from the ATP-dependent RNA helicase of B.cereus by bioinformatics.The minimum inhibitory concentration(MIC)of the antimicrobial peptide DB16 against S.aureus was 31.25μg/mL,which could completely inhibit the growth of S.aureus within 0.5 h.DB16 presented a random coiled structure in phosphate buffered saline(PBS),and turned into anα-helix structure in sodium dodecyl sulfate(SDS).Fluorescence probe,flow cytometry,transmission electron microscopy(TEM),DNA gel electrophoresis and circular dichroism(CD)spectroscopy were used to explore the antibacterial mechanism of DB16 against S.aureus.The results showed that the antibacterial peptide DB16 could change the permeability of the bacterial cell membrane,thereby causing the leakage of cellular contents,while entering the cells to combine with the genomic DNA of S.aureus,affecting normal DNA replication,and finally inhibiting the growth and reproduction of bacteria.In addition,DB16 at 8×MIC showed no hemolysis on mammalian red blood cells.In conclusion,the antimicrobial peptide DB16 from B.cereus has great potential for the prevention and control of S.aureus.