鲎源抗菌肽对大肠杆菌的抑杀机理

Mechanisms of tachyplesin I against Escherichia coli

【目的】研究鲎源抗菌肽鲎素对大肠杆菌抑杀的作用机理,为防治大肠杆菌引起的肠道疾病提供新的潜在抗菌药物。【方法】利用牛津杯法和微量MH肉汤稀释法测定其抗菌活性,激光共聚焦扫描显微镜观察鲎素对大肠杆菌的结合、分布及杀伤过程,透射电镜观察鲎素对大肠杆菌超微结构的影响,并采用琼脂糖凝胶阻滞电泳研究其对大肠杆菌基因组DNA和RNA的影响。【结果】鲎素对大肠杆菌的最小抑菌浓度与最低杀菌浓度分别为5 mg/L和20 mg/L;激光共聚焦扫描显微镜和透射电镜观察发现鲎素能快速作用于细胞表面,并发生聚集现象,随着作用时间的延长能导致细胞膜结构的破坏和细胞内含物的释放;通过琼脂糖电泳结果显示鲎素也能够作用于大肠杆菌基因组DNA,并呈浓度依赖关系,10 mg/L鲎素对基因组DNA无明显影响,80 mg/L鲎素能导致DNA断裂;凝胶阻滞电泳显示鲎素也能与基因组DNA和RNA发生结合。【结论】研究结果为深入探讨鲎素抑杀大肠杆菌的分子机制提供重要的理论依据。

[Objective] The role mechanism of tachyplesin I on Escherichia coli was investi-gated to provide potential novel antibacterial drugs for treating disease caused by enteropa- thogenic bacteria. IMethods] Antimicrobial activity for tachyplesin I for Escherichia coli was determined using Oxford cup and micro-broth dilution methods. The combination, distribution and killing process of tachyplesin I against Escherichia coli were observed under laser confo- cal scanning microscopy. Ultrastructure of tachyplesin I against Escherichia coli was observed under transmission electron microscope. The genomic DNA and RNA binding ability of tachyplesin I was examined by agarose gel retardation electrophoresis in this experiment. IResults] The minimum inhibitory concentration and minimum bactericidal concentration was 5, 20 mg/L, respectively. The results of laser confocal scanning microscopy and transmission electron microscope indicated that tachyplesin I penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm immediately after being added to the cells. Later, mem- brane damage and cellular content outflow was seen. And tachyplesin I had effect on genomic DNA in a concentration-dependent manner. The low concentrations of tachypesin I （10 mg/L） had no obvious effect on DNA, while the high concentrations of tachypesin I （above or equal to 80 mg/L） could lead to break in genomic DNA. Tachypesin I could combine with genomic DNA and RNA. [Conclusion] The results provide a theoretical basis to further understand sterilization mechanism of tachyplesin I at molecular level.