Bacillus pumilus HN-10抗菌肽P-1对粉红单端孢的抑菌机理

Bacillus pumilus HN-10抗菌肽P-1是一种对粉红单端孢（Trichothecium roseum）具有很强抑菌作用的肽，但其抑菌机理尚不明确。本研究从细胞膜通透性、蛋白合成和核酸合成3 方面研究其抑菌机理。通过大分子释放量和电导率研究抗菌肽P-1对粉红单端孢细胞膜通透性的影响；经胞内蛋白含量测定及胞内蛋白十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析研究抗菌肽P-1对蛋白合成的影响；采用琼脂糖凝胶电泳和荧光光谱分析抗菌肽P-1对粉红单端孢DNA的凝胶阻滞作用及与溴化乙锭（ethidium bromide，EB）竞争性结合作用，并通过DNA和RNA相对含量来研究粉红单端孢核酸合成是否受到抑制。结果表明：抗菌肽P-1可引起粉红单端孢细胞膜通透性增加，胞内电解质和大分子物质外泄；同时，菌体蛋白合成受到抑制，尤其对分子质量在43.0～97.4 kDa之间的蛋白最为明显；抗菌肽P-1对DNA没有明显阻滞作用，但其能与EB竞争性结合DNA，使核酸合成受到抑制，导致菌体代谢紊乱，影响蛋白质的正常表达，进而起到抑菌作用。

Antimicrobial Mechanism of Antimicrobial Peptide P-1 from Bacillus pumilus HN-10 against Trichothecium roseum

Antimicrobial peptide P-1 from Bacillus pumilus HN-10 has strong antifungal activity against Trichothecium roseum, but its mechanism of action remains unclear. In this study, we investigated this topic from the perspectives of cell membrane permeability, protein synthesis and nucleic acid synthesis. Firstly, the effect of antimicrobial peptide P-1 on the membrane permeability of T. roseum was studied by measuring the release of macromolecules and electrical conductivity. Secondly, the effect on protein synthesis was studied by measuring the expression of intracellular and intracellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Finally, in order to prove whether nucleic acid synthesis was inhibited by antimicrobial peptide P-1, agarose gel retardation assay was performed and the competitive binding of antimicrobial peptide P-1 with ethidium bromide (EB) to T. roseum DNA and RNA contents were analyzed by fluorescence spectroscopy. The results showed that the antimicrobial peptide P-1 could increase the membrane permeability of T. roseum, leading to leakage of intracellular electrolytes and macromolecules. Meanwhile, T. roseum protein synthesis was inhibited, especially for proteins with molecular masses between 43.0 and 97.4 kDa. The antifungal peptide showed no DNA gel retardation, but it could compete with EB for binding to DNA and inhibit nucleic acid synthesis, resulting in fungal metabolism disorder and abnormal protein expression and eventually exerting antimicrobial activity.