抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制

doi:10.16431/j.cnki.1671-7236.2024.05.044

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (5): 2237-2244.doi: 10.16431/j.cnki.1671-7236.2024.05.044

抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制

连凯琪^1,2,3, 王宇航¹, 曾雅静¹, 周玲玲^1,3, 张元臣⁴, 张明亮^1,3, 张改平², 王双山^1,3

1. 安阳工学院生物与食品工程学院, 安阳 455000;
2. 河南农业大学动物医学院, 郑州 450046;
3. 河南省兽用生物制品研发与应用国际联合实验室, 安阳 455000;
4. 河南省太行山林业有害生物野外科学观测研究站, 林州 456550

收稿日期:2023-10-13 出版日期:2024-05-05 发布日期:2024-04-28
通讯作者: 张改平, 王双山 E-mail:zhanggaip@126.com;wangshuangshan2005@163.com
作者简介:连凯琪,E-mail:liankaiqi616@163.com。
基金资助:
国家自然科学基金青年项目(31802170);河南省科技攻关计划项目(212102110090);安阳市重点研发与推广专项(2023C01NY013);安阳工学院博士后创新实践基地启动经费资助(BHJ2020005)

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

LIAN Kaiqi^1,2,3, WANG Yuhang¹, ZENG Yajing¹, ZHOU Lingling^1,3, ZHANG Yuanchen⁴, ZHANG Mingliang^1,3, ZHANG Gaiping², WANG Shuangshan^1,3

1. School of Biotechnology and Food Science, Anyang Institute of Technology, Anyang 455000, China;
2. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
3. Henan Joint International Research Laboratory of Veterinary Biologics Research and Application, Anyang 455000, China;
4. Taihang Mountain Forest Pests Observation and Research Station of Henan Province, Linzhou 456550, China

Received:2023-10-13 Online:2024-05-05 Published:2024-04-28

摘要/Abstract

摘要： 【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50 μg/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。

关键词: 抗菌肽; 肺炎克雷伯菌; 抗菌机制; 细胞壁; 细胞膜

Abstract: 【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.

Key words: antimicrobial peptide; Klebsiella pneumoniae; antimicrobial mechanism; cell wall; cell membrane

中图分类号:

S859.79

连凯琪, 王宇航, 曾雅静, 周玲玲, 张元臣, 张明亮, 张改平, 王双山. 抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制[J]. 中国畜牧兽医, 2024, 51(5): 2237-2244.

LIAN Kaiqi, WANG Yuhang, ZENG Yajing, ZHOU Lingling, ZHANG Yuanchen, ZHANG Mingliang, ZHANG Gaiping, WANG Shuangshan. Antimicrobial Mechanism of Antimicrobial Peptide LL-1 Against Klebsiella pneumoniae ATCC 700603[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2237-2244.

参考文献

[1] OLIVEIRA S,MOURA R A,SILAVA K C,et al.Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11(ST437 and ST340) in urban rivers[J].Journal of Antimicrobial Chemotherapy, 2014,69(3):849-852.
[2] HOLDEN V I,BREEN P,HOULE S,et al.Klebsiella pneumoniae siderophores induce inflammation,bacterial dissemination,and HIF-1α stabilization during pneumonia[J]. mBio,2016,7(5):e01397-16.
[3] SHI Y F,WANG Y K,WANG Y H,et al.Metastatic infection caused by hypervirulent Klebsiella pneumoniae and co-infection with Cryptococcus meningitis:A case report[J].World Journal of Clinical Cases,2019,7(22):3812-3820.
[4] 盛席静,张凡,刘方佳,等.动物源肺炎克雷伯菌致病性与耐药性研究进展[J].中国畜牧兽医,2023,50(7):2966-2973. SHENG X J,ZHANG F,LIU F J,et al.Research progress on pathogenicity and drug resistance of Klebsiella pneumonia from animals[J].China Animal Husbandry & Veterinary Medicine,2023,50(7):2966-2973.(in Chinese)
[5] 胡付品,郭燕,朱德妹,等.2021年CHINET中国细菌耐药监测[J].中国感染与化疗杂志,2022,22(5):521-530. HU F P,GUO Y,ZHU D M,et al.CHINET surveillance of antimicrobial resistance among the bacterial isolates in 2021[J].Chinese Journal of Infection and Chemotherapy,2022,22(5):521-530.(in Chinese)
[6] 王哲红,吴桐忠,赵玉宾,等.石河子地区某规模化奶牛场肺炎克雷伯菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2021,48(9):3464-3472. WANG Z H,WU T Z,ZHAO Y B,et al.Isolation,identification and drug resistance analysis of Klebsiella pneumonia in a large-scale dairy farm in Shihezi area[J].China Animal Husbandry & Veterinary Medicine,2021,48(9):3464-3472.(in Chinese)
[7] ZHENG Z,GORDEN P J,XIA X,et al.Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S[J].Environmental Microbiology,2022,24(3):1183-1199.
[8] 谢兴凤,刘小青,代春梅,等.耐碳青霉烯酶肺炎克雷伯菌的流行病学特点及药物敏感性分析[J].四川医学,2023,44(3):279-284. XIE X F,LIU X Q,DAI C M,et al.Epidemiological characteristics and drug sensitivity analysis of carbapenemase resistant Klebsiella pneumonia[J].Sichuan Medical Journal,2023,44(3):279-284.(in Chinese)
[9] 熊巍.肺炎克雷伯菌的临床分布及耐药性探析[J].中国医药指南,2022,20(35):33-36. XIONG W.Clinical distribution and drug resistance of Klebsiella pneumoniae[J].Guide of China Medicine,2022,20(35):33-36.(in Chinese)
[10] 李洪宇,刘扬科,何涛,等.抗菌肽抗菌活性评价及应用研究进展[J].饲料博览,2022,359(3):18-21. LI H Y,LIU Y K,HE T,et al.Research progress on antibacterial activity evaluation and application of antimicrobial peptide[J].Feed Review,2022,359(3):18-21.(in Chinese)
[11] 张丽,武刚,祝发明,等.抗菌肽对抗生素耐药菌株的抑菌活性研究[J].畜牧与饲料科学,2020,41(2):92-95. ZHANG L,WU G,ZHU F M,et al.Evaluation of antibacterial activity of an antibacterial peptide against a clinically isolated antibiotic resistant bacterial strain[J].Animal Husbandry and Feed Science,2020,41(2):92-95.(in Chinese)
[12] ZHONG H R,XIE Z P,ZHANG S X,et al.Brevinin-GR23 from frog Hylarana guentheri with antimicrobial and antibiofilm activities against Staphylococcus aureus[J].Bioscience,Biotechnology and Biochemistry,2020,84(1):143-153.
[13] HIRT H,GORR S U.Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa[J].Antimicrobial Agents & Chemotherapy,2013,57(10):4903-4910.
[14] WANG X M,TENG D,MAO R Y,et al.Combined systems approaches reveal a multistage mode of action of a marine antimicrobial peptide against pathogenic Escherichia coli and its protective effect against bacterial peritonitis and endotoxemia[J].Antimicrobial Agents and Chemotherapy,2017,61(1):e01056-16.
[15] 田丹,杨元信.抗菌肽的抗菌活性及免疫调节功能[J].养殖与饲料,2015,5:33-35. TIAN D,YANG Y X.Antibacterial activity and immunomodulatory function of antimicrobial peptides[J].Animals Breeding and Feed,2015,5:33-35.(in Chinese)
[16] LI X,ZUO S,WANG B,et al.Antimicrobial mechanisms and clinical application prospects of antimicrobial peptides[J].Molecules, 2022,27(9):675.
[17] ZHOU L L,LIAN K Q,WANG M T,et al.The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability[J].BMC Microbiology,2022,22(1):220.
[18] 连凯琪,孟祥凤,牛维东,等.4-萜稀醇对金黄色葡萄球菌的抑菌机制[J].中国兽医学报,2022,42(7):1438-1442. LIAN K Q,MENG X F,NIU W D,et al.Antimicrobial mechanism of terpinen-4-ol against Staphylococcus aureus[J].Chinese Journal of Veterinary Science,2022,42(7):1438-1442.(in Chinese)
[19] WANG G,ZHAO G,CHAO X,et al.The characteristic of virulence,biofilm and antibiotic resistance of Klebsiella pneumoniae[J].International Journal of Environmental Research and Public Health,2020,17(17):6278.
[20] 连凯琪,周玲玲,张元臣,等.新型黏虫抗菌肽AAP-1对大肠杆菌的抗菌作用[J].中国畜牧兽医,2020,47(11):3626-3632. LIAN K Q,ZHOU L L,ZHANG Y C,et al.Antibacterial effect of novel armyworm antimicrobial peptide (AAP-1) against Escherichia coli[J].China Animal Husbandry & Veterinary Medicine,2020,47(11):3626-3632.(in Chinese)
[21] 黄璟昇,朱树馨,康伟超,等.抗菌肽BNBD5调节肺脏先天免疫抵御胸膜肺炎放线杆菌感染的研究[J].中国畜牧兽医,2023,50(10):4261-4269. HUANG J S,ZHU S X,KANG W C,et al.Study on antibacterial peptide BNBD5 regulating the innate immune response of lung against Actinobacillus pleuropneumoniae[J].China Animal Husbandry & Veterinary Medicine,2023,50(10):4261-4269.(in Chinese)
[22] 耿一鸣,李婷婷,励建荣,等.松油烯-4-醇对荧光假单胞菌抑菌能力及作用机理[J].食品科学,2022,43(1):30-36. GENG Y M,LI T T,LI J R,et al.Antibacterial activity and mechanism of terpinene-4-ol against Pseudomonas fluorescens[J].Food Science,2022,43(1):30-36.(in Chinese)
[23] HOSSEINI A,MAS J.The β-galactosidase assay in perspective:Critical thoughts for biosensor development[J].Analytical Biochemistry,2021,635:114446.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	侯宫明珠, 周海琴, 余星雨, 李娜娜, 李扬, 屈勇刚, 李彦芳, 梁晏, 严多, 杨舒涵, 邱正青, 陈斯奇. 1株羊源多重耐药肺炎克雷伯菌噬菌体的生物学特性及全基因组分析[J]. 中国畜牧兽医, 2024, 51(5): 2047-2057.
[2]	詹同彤, 王平峰, 郑晓美, 文琦琦, 叶刚, 邓林华, 由玉岩, 何鸣, 王茜, 李才武, 凌珊珊. 大熊猫源肺炎克雷伯菌研究进展[J]. 中国畜牧兽医, 2024, 51(2): 770-778.
[3]	李婷婷, 刘子超, 周永顺, 何营, 耿方豪, 陈洁然, 高剑峰. 两种处理方法对小鼠骨髓源性树突状细胞体外递呈布鲁氏菌免疫多肽组分析的影响[J]. 中国畜牧兽医, 2023, 50(9): 3730-3739.
[4]	盛席静, 张凡, 刘方佳, 刘泉, 曹征征, 胡天宇, 李东华, 吴梦慧, 戴梦红. 动物源肺炎克雷伯菌致病性与耐药性研究进展[J]. 中国畜牧兽医, 2023, 50(7): 2966-2973.
[5]	布阿依夏木·库尔班, 王义鑫, 杨铖熹, 耿子健, 徐滢清, 罗刚. 天蚕素A和溶菌酶杂合肽在毕赤酵母中重组表达及其活性分析[J]. 中国畜牧兽医, 2023, 50(12): 5032-5042.
[6]	冯亚楠, 杨娜, 毛若雨, 郝娅, 马炫炫, 滕达, 王建华. 高效液相色谱法检测鼠饲料中抗菌肽NZ2114[J]. 中国畜牧兽医, 2023, 50(11): 4463-4472.
[7]	黄璟昇, 朱树馨, 康伟超, 邓志杰, 杨云梅, 刘城志, 易丹丹, 何家康, 梁正敏. 抗菌肽BNBD5调节肺脏先天免疫抵御胸膜肺炎放线杆菌感染的研究[J]. 中国畜牧兽医, 2023, 50(10): 4261-4269.
[8]	邓丽, 李杰, 刘保国, 杭柏林. 牛血红蛋白β亚基片段衍生抗菌肽的分子设计及其抗菌活性分析[J]. 中国畜牧兽医, 2022, 49(9): 3589-3598.
[9]	宇晓军, 李丹丹, 厉学武, 王群, 王光, 王利华. 抗菌肽对育成期母貂免疫功能及肠组织形态的影响[J]. 中国畜牧兽医, 2022, 49(2): 443-453.
[10]	陈强, 程悦宁, 冯秋菊, 郭利, 张淑琴, 谭斌, 易立, 赵权, 程世鹏, 孙娜. 水貂肺炎克雷伯菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2022, 49(2): 700-708.
[11]	王哲红, 吴桐忠, 赵玉宾, 张星星, 韩猛立, 钟发刚, 胡建军, 黄新. 石河子地区某规模化奶牛场肺炎克雷伯菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2021, 48(9): 3464-3472.
[12]	于佳民, 赵倩, 张志焱, 徐海燕, 谷巍. 多黏类芽孢杆菌抗菌肽的分离纯化及特性研究[J]. 中国畜牧兽医, 2021, 48(8): 2830-2837.
[13]	张欣, 潘晨浩, 赵瑞利, 姜轩, 金天明, 赵洋, 马吉飞, 于恩远, 李留安, 张振洲, 李桂霞, 李瑞忠. 抗菌肽Temprine-La(FS)抑制2型猪链球菌生物被膜形成的研究[J]. 中国畜牧兽医, 2021, 48(1): 324-337.
[14]	姜晓文, 燕鸽, 李叔洪, 于文会, 魏佳宇, 艾莉雯, 白文. 板蓝根微粉水提物抗大肠杆菌活性及其机制的探究[J]. 中国畜牧兽医, 2020, 47(9): 2968-2978.
[15]	翟培, 韩晋辉, 潘晓瑜. 基于天蚕素A和胡蜂毒素的杂合肽设计及活性鉴定[J]. 中国畜牧兽医, 2020, 47(9): 3006-3013.