摘要
为研究新型酪蛋白源抗菌肽BCp12对金黄色葡萄球菌的抑菌机理,本实验通过酶标仪、流式细胞仪、透射电子显微镜分析BCp12对金黄色葡萄球菌的壁膜损伤机制;采用荧光光谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究BCp12对菌体DNA结合及蛋白质合成的影响;利用赖氨酸乙酰化、琥珀酰化、2-羟基异丁酰化、丙二酰化4种泛抗体结合免疫印迹分析BCp12对菌体蛋白翻译后修饰的影响。结果表明:BCp12的最小抑菌质量浓度(minimum inhibitoryconcentration,MIC)为2 mg/mL,经质量浓度超过MIC的BCp12处理后的菌体细胞膜疏水性显著下降(P≤0.001),通透性增加,菌体形变严重,部分细胞内容物外泄形成空腔;BCp12与溴化乙锭竞争性结合菌体DNA,使核酸合成受到抑制,胞内蛋白质量浓度显著下降(P≤0.001),特别是分子质量为15~35 kDa的蛋白变化最为明显,说明BCp12可抑制菌体蛋白质的合成;金黄色葡萄球菌蛋白存在大量的赖氨酸乙酰化、琥珀酰化、丙二酰化修饰,经BCp12处理的菌体赖氨酸丙二酰化修饰水平明显下调,而对赖氨酸琥珀酰化和2-羟基异丁酰化修饰的影响不明显。本实验揭示了BCp12对金黄色葡萄球菌的多靶点抑菌机制,对新型乳源抗菌肽的应用和保障食品安全具有一定的意义。
In this research, to investigate the antibacterial mechanism of a novel casein-derived antibacterial peptide,BCp12, against Staphylococcus aureus, the mechanism of cell membrane and wall damage caused by BCp12 was analyzed using a microplate reader(MR), a flow cytometer(FC), and a transmission electron microscope(TEM). The effects of BCp12 on DNA binding and protein synthesis were studied using fluorescence spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The influence of BCp12 on protein post-translational modification(PTM) was analyzed by Western blot using pan anti-acetyl, anti-succinyl, anti-2-hydroxyisobutyryl and anti-malonyl lysine antibodies. The results showed that the minimum inhibitory concentration(MIC) of BCp12 was 2 mg/mL. Treatment with BCp12 at concentrations above the MIC decreased the cell surface hydrophobicity of S. aureus significantly(P ≤ 0.001), increased the cell membrane permeability, and caused the cells to deform severely and cellular contents leak out to form a cavity. BCp12 competed with ethidium bromide to combine with bacterial DNA,resulting in inhibition of nucleic acid synthesis and significantly decreased protein contents(P ≤ 0.001), especially for proteins with a molecular mass of 15–35 kDa, indicating that BCp12 can inhibit the synthesis of bacterial proteins.S. aureus proteins showed a large number of lysine acetylation, succinylation and malonylation. The level of malonylation was significantly down-regulated after treatment with BCp12, which, however, had no noticeable effect on the level of 2-hydroxyisobutyrylation or succinylation. The results of this study are meaningful for applying new milk-derived antimicrobial peptides and ensuring food safety.
作者
李钰芳
杨昆
顾韦维
赵琼
黄艾祥
施娅楠
LI Yufang;YANG Kun;GU Weiwei;ZHAO Qiong;HUANG Aixiang;SHI Yanan(College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2021年第23期34-41,共8页
Food Science
基金
国家自然科学基金地区科学基金项目(32060572)
云南省农林专项重点项目(2018FG001-011)
云岭产业技术领军人才项目(云发改人事(2014)1782号)。
