抗菌肽Met-Ala-Lys对铜绿假单胞菌细胞膜的作用机制
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摘要
以抗菌肽Met-Ala-Lys(MAK)和铜绿假单胞菌为研究对象,采用NPN荧光探针法,圆二色谱法以及原子吸收光谱测定测定铜绿假单胞菌细胞膜结构的变化,探讨MAK对目标菌生长的影响作用机制。结果表明:随着MAK对铜绿假单胞菌作用时间的增加,MAK的二级结构尤其是无规则卷曲结构发生变化明显,细胞内膜(吸附速率从54.03%降至27.33%)和外膜(荧光强度在前30 min持续增强,30 min后基本稳定在45左右)受到影响,通透性明显增强,导致钾离子(从处理10 min时迅速增加)和蛋白质泄漏量(从4.65μg/m L到10.25μg/m L)以及细胞膜的疏水性随之增加,细胞表面膜电位发生变化。表明MAK能改变铜绿假单胞菌的表面特性,抑制其生长,进而起到抗菌作用。本研究为抗菌肽MAK的抗菌机理制研究奠定一定的理论基础。
The present study investigated the antibacterial effected of the peptide Met-Ala-Lys(MAK) on the structure of Pseudomonas aeruginosa based on the NPN fluorescence method, the circular dichroism spectroscopy analysis, and the level of potassium ions and protein were employed respectively to characterize the extracellular membrane permeability, the variation of secondary structure, and the cell surface hydrophobicity and its endometrial permeability induced by the MAK. The results showed that with the increase of time, the secondary-structure,specially the Random coil, of MAK changed, resulting in the leakage of potassium(getting rapidly from the first 10 min) and protein were significantly high(from 4.65 μg/m L to 10.25 μg/m L), indicating that the inner(from 54.03% to 27.33%) and outer(increase rapidly in the first30 min and basically stable at around 45 after 30 min) membrane permeability were changed, thus, MAK can change the surface characteristics of Pseudomonas aeruginosa and contribute to the inhibition of bacterial growth. These results may provide some information for further study of antibacterial peptide, especially in the aspect of the mechanism of antibacterial peptide Met-Ala-Lys.
The present study investigated the antibacterial effected of the peptide Met-Ala-Lys(MAK) on the structure of Pseudomonas aeruginosa based on the NPN fluorescence method, the circular dichroism spectroscopy analysis, and the level of potassium ions and protein were employed respectively to characterize the extracellular membrane permeability, the variation of secondary structure, and the cell surface hydrophobicity and its endometrial permeability induced by the MAK. The results showed that with the increase of time, the secondary-structure,specially the Random coil, of MAK changed, resulting in the leakage of potassium(getting rapidly from the first 10 min) and protein were significantly high(from 4.65 μg/m L to 10.25 μg/m L), indicating that the inner(from 54.03% to 27.33%) and outer(increase rapidly in the first30 min and basically stable at around 45 after 30 min) membrane permeability were changed, thus, MAK can change the surface characteristics of Pseudomonas aeruginosa and contribute to the inhibition of bacterial growth. These results may provide some information for further study of antibacterial peptide, especially in the aspect of the mechanism of antibacterial peptide Met-Ala-Lys.
引文
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[15]Huifan Liu,Ye Hongji,Sun Chongzhen et al.Antioxidant activity in HepG2 cells,immunomodulatory effects in RAW264.7 cells and absorption characteristics in Caco-2 cells of the peptide fraction isolated from Dendrobium aphyllum[J].International Journal of Food Science&Technology,2018,53(9):2027-2036
[16]徐灿.粒毛盘菌黑色素及其衍生物结构、生物活性与作用机制[D].合肥:合肥工业大学,2017XU Can.Structure biological activity and mechanism of melanin and its derivatives[D].Hefei:Hefei University of Technology,2017
[17]陈玉洁.酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对致病性大肠杆菌的抑菌和细胞表面特性的影响[J].微生物学通报,2015,42(4):683-689CHEN Yu-jie.Effect of Kluyveromyces marxianus metabolic antibacterial complex extracted from acid mare milk on bacteriostasis and cell surface characteristics of pathogenic Escherichia coli[J].Journal of Microbiology,2015,42(4):683-689
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[19]陶凤云,张新妙,俞军,等.茶树油抗菌作用机理研究进展[J].中国抗生素杂志,2006,31(5):261-266TAO Feng-yun,ZHANG Xin-miao,YU Jun,et al.Research progress on antibacterial mechanism of tea tree oil[J].Chinese Journal of Antibiotics,2006,31(5):261-266
[20]刘辉.抑制耐冷菌的乳酸菌抗菌肽筛选及其结构和抗菌机理研究[D].哈尔滨:哈尔滨工业大学,2016LIU Hui.Screening structure and antibacterial mechanism of antimicrobial peptides from Lactic acid bacteria inhibiting cold-resistant bacteria[D].Harbin:Harbin Institute of Technology,2016
[21]李宁宁,吴惊香,丁典,等.胰岛细胞ATP敏感的钾离子通道的结构[J].科学新闻,2018,546(4):34LI Ning-ning,WU Jing-xiang,DING Dian,et al.Structure of atp-sensitive potassium channels in islet cells[J].Science News,2018,546(4):34
[22]孙宜君.螺旋藻抗菌肽的纯化鉴定及其抑菌机理的研究[D].北京:北京林业大学,2016SUN Yi-jun.Purification characterization and mechanism of action of an antibacterial peptide from Spirulina platensis[D].Beijing:Beijing Forestry University,2016
[23]林凯蕾.圆二色光谱预测蛋白质二级结构组成方法评析[D].上海:复旦大学,2012LIN Kai-lei.Evaluation and analysis on the method of circular dichroism to predict protein secondary structure composition[D].Shanghai:Fudan University,2012
[24]陶立国.人工设计多肽的结构及其抗菌机理研究[D].无锡:江苏大学,2016TAO Li-guo.Structure and antibacterial mechanism of artificially designed polypeptides[D].Wuxi:Jiangsu University,2016
[25]韩宇鹏,杨静静,汪薛良,等.采用圆二色谱和傅立叶变换红外光谱法研究糖化与煮沸过程中蛋白质Z的二级结构变化[J].啤酒科技,2015(11):56-61HAN Yu-peng,YANG Jing-jing,WANG Xue-liang,et al.Study on secondary structure changes of protein Z during saccharification and boiling by circular dichrography and Fourier transform infrared spectroscopy[J].Beer Science and Technology,2015,11:56-61
[26]舒展,张毅,谢虹忆,等.硅酸盐黏土矿物在抗菌方面研究进展[J].材料工程,2018,46(4):23-30SHU Zhan,ZHANG Yi,XIE Hong-yi,et al.Advances in research on antibacterial properties of Silicate clay Minerals[J].Material Engineering,2018,46(4):23-30
[27]孙杰.Ag/Ti O2纳米材料的制备及抗菌、抗螨性能和机理研究[D].深圳:深圳大学,2017SUN Jie.Preparation of Ag/Ti O2 nanocomposites and its antibacterial and antimite performance and mechanism[D].Shenzhen:Shenzhen University,2016
[28]曾嵘,夏其昌.蛋白质组学研究进展与趋势[J].中国科学院院刊,2002,3(3):17-25ZENG Rong,XIA Qi-chang.Advances and trends in proteomics research[J].Bulletin of Chinese Academy of Sciences,2002,3(3):17-25
[29]常丽.季鏻盐改性聚丙烯腈纤维及其抗菌性能与机理研究[D].长沙:湖南大学,2016CHANG Li.Study on The synthesis,antimicrobial activities and mechanism of polyacrylonitrile fibers modified with quaternary phosphonium salts[D].Changsha:Hunan:Hunan University,2016
[2]宋宏霞,曾名勇,刘尊英,等.抗菌肽的生物活性及其作用机理[J].食品工业科技,2006,27(9):185-197SONG Hong-xia,ZENG Ming-yong,LIU Zun-ying,et al.Biological activity and mechanism of antimicrobial peptides[J].Science and Technology of Food Industry,2006,27(9):185-19
[3]Ng T B,Jingyi Liu,Jack Ho Wong,et al.Review of research on Dendrobium,a prized folk medicine[J].Appl Microbiol Biotechnol,2012,93(5):1795-1803
[4]李桂琳,白燕冰,白学慧,等.兜唇石斛扦插繁殖试验[J].热带农业科技,2011,34(2):32-34LI Gui-lin,BAI Yan-bing,BAI Xue-hui,et al.Experiment of Dendrobium aphyllumin cuttage propagation[J].Science and Technology of Tropical Agriculture,2011,34(2):32-34
[5]Liu Huifan,Ma Juanjuan,Wu Hui.Detoxifying effects of ultrafiltration fractions of Dendrobium aphyllum peptides on chemical and AAPH-induced oxidative stress[J].RSC Adv,2017,7:48913-48924
[6]刘袆帆.兜唇石斛免疫活性多糖及抗氧化肽的结构鉴定及功能表征[D].广州:华南理工大学,2018LIU Hui-fan.The Identification of structure and characterization of immunoregulatory polysaccharide and antioxidative peptides isolated from Dendrobium aphyllumin[D].Guangzhou:South China University of Technology,2018
[7]宋丽雅,倪正,樊琳娜,等.花椒抑菌成分提取方法及抑菌机理研究[J].中国食品学报,2016,16(3):125-130SONG Li-ya,NI Zheng,FAN Nin-na,et al.Study on the extraction method and antibacterial mechanism of Chinese prickly ash[J].Chinese Journal of Food Science,2016,16(3):125-130
[8]汪陈洁.乳酸对常见食源性致病菌的抗菌活性与作用机理[D].武汉:华中农业大学,2014WANG Chen-jie.Antimicrobial activity and mode of action of lactic acid against common food-borne pathogens[D].Wuhan:Huazhong Agricultural University,2014
[9]郑少萌.四种多肽的合成和分离纯化的基础研究[D].北京:首都师范大学,2014ZHENG Shao-meng.Basic research on synthesis isolation and purification of four polypeptides[D].Beijing:Capital Normal University,2014
[10]彭博,郭中敏,陆家海.人工合成抗菌肽的常用方法及应用前景[J].中国抗生素杂志,2012,37(3):176-183PENG Bo,GUO Zhong-min,LU Jia-hai,et al.Common methods and application prospect of synthetic antimicrobial peptides[J].Chinese Journal of Antibiotics,2012,37(3):176-183
[11]赵晴,张甲耀,陈兰洲,等.疏水性石油烃降解菌细胞表面疏水性及降解特性[J].环境科学学报,2005,26(5):132-136ZHAO Qing,ZHANG Jia-yao,CHEN Lan-zhou,et al.Hydrophobicity and degradation characteristics of cell surface of hydrophobic oil-hydrocarbon degrading bacteria[J].Journal of Environmental Sciences,2005,26(5):132-136
[12]孙宜君.螺旋藻抗菌肽的纯化鉴定及其抑菌机理的研究[D].北京:北京林业大学,2016SUN Yi-jun.Purification characterization and mechanism of action of an antibacterial peptidie from Spirulina platensis[D].Beijing:Beijing Forestry University,2016
[13]候利霞,翟培,施用晖,等.家蝇抗菌肽对细菌细胞表面特性影响及其作用机理的研究[J].微生物学通报,2007,34(3):434-437HOU Li-xia,ZHAI Pei,SHI Yong-hui,et al.Effects of Housefly antimicrobial peptides on bacterial cell surface characteristics and their mechanism of action[J].Journal of Microbiology,2007,34(3):434-437
[14]丁闰蓉.培养基添加剂对重组大肠杆菌胞外生产重组α-环糊精葡萄糖基转移酶的影响[D].无锡:江南大学,2009DING Run-rong.Effect of medium additives on extracellular production of recombinant-cyclodextrin glucose transferase in E.coli[D].Wuxi:Southern Yangtze University,2009
[15]Huifan Liu,Ye Hongji,Sun Chongzhen et al.Antioxidant activity in HepG2 cells,immunomodulatory effects in RAW264.7 cells and absorption characteristics in Caco-2 cells of the peptide fraction isolated from Dendrobium aphyllum[J].International Journal of Food Science&Technology,2018,53(9):2027-2036
[16]徐灿.粒毛盘菌黑色素及其衍生物结构、生物活性与作用机制[D].合肥:合肥工业大学,2017XU Can.Structure biological activity and mechanism of melanin and its derivatives[D].Hefei:Hefei University of Technology,2017
[17]陈玉洁.酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对致病性大肠杆菌的抑菌和细胞表面特性的影响[J].微生物学通报,2015,42(4):683-689CHEN Yu-jie.Effect of Kluyveromyces marxianus metabolic antibacterial complex extracted from acid mare milk on bacteriostasis and cell surface characteristics of pathogenic Escherichia coli[J].Journal of Microbiology,2015,42(4):683-689
[18]赵广旭.基于疏水性和螺旋性对膜活性多肽的设计与活性研究[D].长春:吉林大学,2017ZHAO Guang-xu.Design and activity study of membrane active polypeptides based on hydrophobicity and helicity[D].Changchun:Jilin University,2017
[19]陶凤云,张新妙,俞军,等.茶树油抗菌作用机理研究进展[J].中国抗生素杂志,2006,31(5):261-266TAO Feng-yun,ZHANG Xin-miao,YU Jun,et al.Research progress on antibacterial mechanism of tea tree oil[J].Chinese Journal of Antibiotics,2006,31(5):261-266
[20]刘辉.抑制耐冷菌的乳酸菌抗菌肽筛选及其结构和抗菌机理研究[D].哈尔滨:哈尔滨工业大学,2016LIU Hui.Screening structure and antibacterial mechanism of antimicrobial peptides from Lactic acid bacteria inhibiting cold-resistant bacteria[D].Harbin:Harbin Institute of Technology,2016
[21]李宁宁,吴惊香,丁典,等.胰岛细胞ATP敏感的钾离子通道的结构[J].科学新闻,2018,546(4):34LI Ning-ning,WU Jing-xiang,DING Dian,et al.Structure of atp-sensitive potassium channels in islet cells[J].Science News,2018,546(4):34
[22]孙宜君.螺旋藻抗菌肽的纯化鉴定及其抑菌机理的研究[D].北京:北京林业大学,2016SUN Yi-jun.Purification characterization and mechanism of action of an antibacterial peptide from Spirulina platensis[D].Beijing:Beijing Forestry University,2016
[23]林凯蕾.圆二色光谱预测蛋白质二级结构组成方法评析[D].上海:复旦大学,2012LIN Kai-lei.Evaluation and analysis on the method of circular dichroism to predict protein secondary structure composition[D].Shanghai:Fudan University,2012
[24]陶立国.人工设计多肽的结构及其抗菌机理研究[D].无锡:江苏大学,2016TAO Li-guo.Structure and antibacterial mechanism of artificially designed polypeptides[D].Wuxi:Jiangsu University,2016
[25]韩宇鹏,杨静静,汪薛良,等.采用圆二色谱和傅立叶变换红外光谱法研究糖化与煮沸过程中蛋白质Z的二级结构变化[J].啤酒科技,2015(11):56-61HAN Yu-peng,YANG Jing-jing,WANG Xue-liang,et al.Study on secondary structure changes of protein Z during saccharification and boiling by circular dichrography and Fourier transform infrared spectroscopy[J].Beer Science and Technology,2015,11:56-61
[26]舒展,张毅,谢虹忆,等.硅酸盐黏土矿物在抗菌方面研究进展[J].材料工程,2018,46(4):23-30SHU Zhan,ZHANG Yi,XIE Hong-yi,et al.Advances in research on antibacterial properties of Silicate clay Minerals[J].Material Engineering,2018,46(4):23-30
[27]孙杰.Ag/Ti O2纳米材料的制备及抗菌、抗螨性能和机理研究[D].深圳:深圳大学,2017SUN Jie.Preparation of Ag/Ti O2 nanocomposites and its antibacterial and antimite performance and mechanism[D].Shenzhen:Shenzhen University,2016
[28]曾嵘,夏其昌.蛋白质组学研究进展与趋势[J].中国科学院院刊,2002,3(3):17-25ZENG Rong,XIA Qi-chang.Advances and trends in proteomics research[J].Bulletin of Chinese Academy of Sciences,2002,3(3):17-25
[29]常丽.季鏻盐改性聚丙烯腈纤维及其抗菌性能与机理研究[D].长沙:湖南大学,2016CHANG Li.Study on The synthesis,antimicrobial activities and mechanism of polyacrylonitrile fibers modified with quaternary phosphonium salts[D].Changsha:Hunan:Hunan University,2016