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抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制

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[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肱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50 μg/mL;抑菌曲线表明,抗菌肱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.
Antimicrobial Mechanism of Antimicrobial Peptide LL-1 Against Klebsiella pneumoniae ATCC 700603
[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 50 μg/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 and β-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.

antimicrobial peptideKlebsiella pneumoniaeantimicrobial mechanismcell wallcell membrane

连凯琪、王宇航、曾雅静、周玲玲、张元臣、张明亮、张改平、王双山

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安阳工学院生物与食品工程学院,安阳 455000

河南农业大学动物医学院,郑州 450046

河南省兽用生物制品研发与应用国际联合实验室,安阳 455000

河南省太行山林业有害生物野外科学观测研究站,林州 456550

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抗菌肽 肺炎克雷伯菌 抗菌机制 细胞壁 细胞膜

国家自然科学基金青年项目河南省科技攻关计划项目安阳市重点研发与推广专项安阳工学院博士后创新实践基地启动经费资助

318021702121021100902023C01NY013BHJ2020005

2024

中国畜牧兽医
中国农业科学院北京畜牧兽医研究所

中国畜牧兽医

CSTPCD北大核心
影响因子:0.72
ISSN:1671-7236
年,卷(期):2024.51(5)
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