抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制

Antimicrobial Mechanism of Antimicrobial Peptide LL-1 Against Klebsiella pneumoniae ATCC 700603

【目的】探明新抗菌肽LL-1对肺炎克雷伯菌(Klebsiella pneumoniae)的抗菌效果和机制。【方法】测定抗菌肽LL-1对肺炎克雷伯菌的最小抑菌浓度(minimum inhibitory concentration,MIC),通过抑菌曲线评价抗菌肽LL-1对肺炎克雷伯菌的体外抗菌效果;通过检测碱性磷酸酶(ALP)、β-半乳糖苷酶(β-galactosidase)、核酸、蛋白质的泄露,评价抗菌肽LL-1对肺炎克雷伯菌细胞膜和细胞壁的影响;通过核酸凝胶电泳检测抗菌肽LL-1与肺炎克雷伯菌DNA的结合作用;通过扫描电子显微镜观察抗菌肽LL-1对肺炎克雷伯菌菌体形态的影响。【结果】抗菌肽LL-1对肺炎克雷伯菌具有良好的抗菌活性,MIC为50μg/mL;抑菌曲线表明,抗菌肽LL-1对肺炎克雷伯菌具有生长抑制作用,且2 MIC的抗菌肽LL-1在6 h内能完全抑制肺炎克雷伯菌;抗菌肽LL-1能导致肺炎克雷伯菌的ALP和β-半乳糖苷酶发生泄漏,且1/4 MIC的抗菌肽LL-1即可导致其泄漏量极显著高于对照组(P<0.01);核酸和蛋白质泄露的检测结果进一步证实抗菌肽LL-1能导致肺炎克雷伯菌胞内容物外流;此外抗菌肽LL-1还能与肺炎克雷伯菌DNA结合;扫描电子显微镜观察显示,抗菌肽LL-1会导致肺炎克雷伯菌表面变得粗糙、菌体变形等。【结论】抗菌肽LL-1能增加肺炎克雷伯菌细胞壁和细胞膜的通透性,并结合其DNA,从而发挥抗菌作用,本研究结果将为深入研究抗菌肽LL-1对肺炎克雷伯菌的抗菌机制奠定基础。

【Objective】This experiment was aimed to study the antibacterial effect and mechanism of a novel antibacterial peptide LL-1.【Method】The minimum inhibitory concentration(MIC)of antibacterial peptide LL-1 against Klebsiella pneumoniae was detected,and the in vitro antibacterial effect of antibacterial peptide LL-1 against Klebsiella pneumoniae was evaluated through the antibacterial curve.Leakage of alkaline phosphatase(ALP),β-galactosidase,nucleic acid and protein were determined to evaluate the effect of antibacterial peptide LL-1 on the cell membrane and cell wall of Klebsiella pneumoniae.The binding of antibacterial peptide LL-1 to Klebsiella pneumoniae DNA was detected by nucleic acid gel electrophoresis.The effect of antibacterial peptide LL-1 on the morphology of Klebsiella pneumoniae was observed through scanning electron microscopy.【Result】Antibacterial peptide LL-1 had good antibacterial activity against Klebsiella pneumoniae,with a MIC of 50μg/mL.The antibacterial curve showed that 2 MIC of antibacterial peptide LL-1 could completely inhibit the growth of Klebsiella pneumoniae within 6 h.Antibacterial peptide LL-1 led to the leakage of ALP andβ-galactosidase of Klebsiella pneumoniae,and 1/4 MIC of antibacterial peptide LL-1 resulted in a extremely significantly higher leakage rate than the control group(P<0.01).The detection of nucleic acid and protein leakage further confirmed that antibacterial peptide LL-1 might cause the efflux of intracellular contents in Klebsiella pneumoniae.In addition,antibacterial peptide LL-1 could bind to the DNA of Klebsiella pneumoniae.The results of scanning electron microscope observation suggested that antibacterial peptide LL-1 caused the surface of Klebsiella pneumoniae to become rough,bacterial deformation.【Conclusion】The results showed that antibacterial peptide LL-1 increased the permeability of the cell wall and membrane in Klebsiella pneumoniae,and bound with the DNA of Klebsiella pneumoniae,and thus exerted antibacterial effects.This would lay the foundation for in-depth research on the antibacterial mechanism of antibacterial peptide LL-1 against Klebsiella pneumoniae.