抗菌肽Tachyplesin Ⅰ及衍生物对大肠杆菌细胞膜作用机制的对比研究

doi:10.11843/j.issn.0366-6964.2022.09.032

摘要/Abstract

摘要： 本研究通过测定抗菌肽tachyplesin Ⅰ（TP Ⅰ）及其衍生肽TP Ⅰ-Y4对脂质体膜的作用模式，以及作用大肠杆菌后对细菌表面电位、疏水性及内、外膜渗透性和离子泄漏率的影响，对比探究了两个肽以细菌细胞膜为靶点的抑菌机理。结果表明：TP Ⅰ/TP Ⅰ-Y4作用后的大肠杆菌表面电位升高，表面疏水性增强，TP Ⅰ-Y4引起电位升高和疏水性增强稍高于TP Ⅰ。TP Ⅰ/TP Ⅰ-Y4均能在插入磷脂双分子层时扰乱脂质体膜释放出钙黄绿素，TP Ⅰ-Y4引起荧光素的泄漏率更高，但它并不完全破裂脂质体膜。TP Ⅰ/TP Ⅰ-Y4均能增加细菌内、外膜的通透性，相比之下，TP Ⅰ-Y4诱导的外膜渗透性较强，对细胞质膜的破坏作用较大，引起离子以及胞内大分子的泄漏量较多。TP Ⅰ-Y4对大肠杆菌细胞膜产生了更大的破坏作用可能是其抑菌活性相比于TP Ⅰ显著提高的主要原因。

关键词: 抗菌肽, tachyplesin Ⅰ, 衍生肽, 大肠杆菌, 膜作用机制

Abstract: The action mechanism of antimicrobial peptide tachyplesin Ⅰ(TP Ⅰ) and its analogue TP Ⅰ-Y4 targeted the cell membrane of Escherichia coli have been studied by exploring the action mode of both peptides on the liposome membrane, the effect on the bacteria surface potential, hydrophobicity, the permeability of inner and outer membrane, and ion leakage after two peptides treated E.coli. The results showed that TP Ⅰ/TP Ⅰ-Y4 induced the increase of surface potential and hydrophobicity of E.coli. TP Ⅰ-Y4 caused a slightly higher potential and hydrophobicity enhancement than TP Ⅰ. Both peptides could disturb the liposome membrane causing the calcein leakage during peptides crossing phospholipids bilayer, TP Ⅰ-Y4 caused more fluorescein leakage, but it did not completely collapse the liposome membrane. TP Ⅰ/TP Ⅰ-Y4 obviously increased the inner and outer membrane permeability of bacteria, in contrast, TP Ⅰ-Y4 induced stronger outer membrane permeability, greater damage to the plasma membrane, causing more leakage of ions and intracellular macromolecules. TP Ⅰ-Y4 had a greater destructive effect on E.coli cell membrane, which may be the main reason for the significant improvement of its antibacterial activity compared with TP Ⅰ.

Key words: antimicrobial peptide, tachyplesin Ⅰ, analogue, Escherichia coli, membrane action mechanism

中图分类号:

S859.79
R978

孙栋, 屈莎, 李璇, 唐善虎, 李思宁, 郝刚. 抗菌肽Tachyplesin Ⅰ及衍生物对大肠杆菌细胞膜作用机制的对比研究[J]. 畜牧兽医学报, 2022, 53(9): 3180-3189.

SUN Dong, QU Sha, LI Xuan, TANG Shanhu, LI Sining, HAO Gang. Comparative Study on the Membrane Action Mechanism of Tachyplesin Ⅰ and Its Analogue on Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3180-3189.

参考文献 31

[1]	BROWN E D, WRIGHT G D. Antibacterial drug discovery in the resistance era[J]. Nature, 2016, 529(7586):336-343.
[2]	RAO L L, LV L C, ZENG Z L, et al. Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003-2012[J]. Vet Microbiol, 2014, 172(3-4):534-541.
[3]	DA SILVA A C, RODRIGUES M X, SILVA N C C. Methicillin-resistant Staphylococcus aureus in food and the prevalence in Brazil:a review[J]. Braz J Microbiol, 2020, 51(1):347-356.
[4]	DA COSTA J P, COVA M, FERREIRA R, et al. Antimicrobial peptides:an alternative for innovative medicines?[J]. Appl Microbiol Biotechnol, 2015, 99(5):2023-2040.
[5]	刘志新, 沈腾飞, 蔡开蕊, 等. 猪源抗菌肽PMAP-23的改造及其活性测定[J]. 中国预防兽医学报, 2020, 42(8):809-815.LIU Z X, SHEN T F, CAI K R, et al. Molecular design and antibacterial activity of antimicrobial peptide PMAP-23[J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(8):809-815. (in Chinese)
[6]	CHOU S L, SHAO C X, WANG J J, et al. Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance[J]. Acta Biomater, 2016, 30:78-93.
[7]	NAKAMURA T, FURUNAKA H, MIYATA T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure[J]. J Biol Chem, 1988, 263(32):16709-16713.
[8]	李寒梅, 唐勇军, 王顺启, 等. 抗菌肽鲎素的研究进展[J]. 生命科学研究, 2018, 22(4):338-344.LI H M, TANG Y J, WANG S Q, et al. Research progresses on antimicrobial peptide tachyplesins[J]. Life Science Research, 2018, 22(4):338-344. (in Chinese)
[9]	YU R L, WANG J Y, SO L Y, et al. Enhanced activity against multidrug-resistant bacteria through coapplication of an analogue of tachyplesin I and an inhibitor of the QseC/B signaling pathway[J]. J Med Chem, 2020, 63(7):3475-3484.
[10]	DING H, JIN G, ZHANG L J, et al. Effects of tachyplesin I on human U251 glioma stem cells[J]. Mol Med Rep, 2015, 11(4):2953-2958.
[11]	MORVAN A, IWANAGA S, COMPS M, et al. In vitro activity of the limulus antimicrobial peptide tachyplesin I on marine bivalve pathogens[J]. J Invertebr Pathol, 1997, 69(2):177-182.
[12]	LI H M, LIU B, WU J, et al. The inhibitory effect of tachyplesin I on thrombosis and its mechanisms[J]. Chem Biol Drug Design, 2019, 94(3):1672-1679.
[13]	陈飞龙. Lactobacillus paracasei FX-6产抗菌肽对金黄色葡萄球菌作用机制及其应用研究[D]. 广州:华南农业大学, 2016.CHEN F L. The action mechanism on Staphylococcus aureus and application of antimicrobial peptide produced by Lactobacillus paracasei FX-6[D]. Guangzhou:South China Agricultural University, 2016. (in Chinese)
[14]	郝刚. BuforinⅡ抗菌肽的分子设计及对DNA作用抑菌机理研究[D]. 无锡:江南大学, 2009.HAO G. Studies on the molecular design and antimicrobial mechanism of DNA action of antimicrobial peptide BuforinⅡ[D]. Wuxi:Jiangnan University, 2009. (in Chinese)
[15]	赵多艳, 卫延安. 改性大豆磷脂在脂质体制备中的应用研究[J]. 中国油脂, 2017, 42(12):149-153.ZHAO D Y, WEI Y A. Application of modified soybean phospholipid in preparation of liposomes[J]. China Oils and Fats, 2017, 42(12):149-153. (in Chinese)
[16]	冯建岭. ε-聚赖氨酸的防腐效果及对大肠杆菌抑制机理的研究[D]. 济南:齐鲁工业大学, 2014.FENG J L. Preservative effect of ε-polylysine and its antibacterial mechanism against Escherichia coli[D]. Ji'nan:Qilu University of Technology, 2014. (in Chinese)
[17]	苏战强, 陈阳, 夏利宁, 等. 乳糖诱导大肠杆菌β-D-半乳糖苷酶试验[J]. 中国兽医杂志, 2016, 52(9):24-27.SU Z Q, CHEN Y, XIA L N, et al. Lactose-induced β-D-galactosidase test of Escherichia coli[J]. Chinese Journal of Veterinary Medicine, 2016, 52(9):24-27. (in Chinese)
[18]	FU J Y, YANG H, WANG H L, et al.[ARTICLE WITHDRAWN] Antimicrobial peptide bsn-37 inhibits the growth of Escherichia coli by targeting its cell wall and membrane[J]. Mater Express, 2020, 10(8):1260-1264.
[19]	夏锦尧. 实用荧光分析法[M]. 北京:中国人民公安大学出版社, 1992:442.XIA J Y. Practical fluorescence analysis[M]. Beijing:China People's Public Security University Press, 1992:442. (in Chinese)
[20]	侯温甫, 欧阳何一, 吴忌, 等. 曲酸对冷鲜鸭肉中优势腐败菌的抑制作用及其抑菌机理[J]. 食品科学, 2019, 40(1):278-285.HOU W F, OUYANG H Y, WU J, et al. Inhibitory effect of kojic acid on dominant spoilage bacteria in chilled duck meat and its mechanism[J]. Food Science, 2019, 40(1):278-285. (in Chinese)
[21]	SITOHY M Z, MAHGOUB S A, OSMAN A O. In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit[J]. Int J Food Microbiol, 2012, 154(1-2):19-29.
[22]	EPAND R F, POLLARD J E, WRIGHT J O, et al. Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins[J]. Antimicrob Agents Chemother, 2010, 54(9):3708-3713.
[23]	王家俊. 非完美两亲性α螺旋肽的构效关系及其抗酶解活性的研究[D]. 哈尔滨:东北农业大学, 2019.WANG J J. Studies on structure-function relationship and protease-resistant activity of imperfectly amphiphilic α-helical peptides[D]. Harbin:Northeast Agricultural University, 2019. (in Chinese)
[24]	王青, 徐彦召, 杨磊, 等. 一种广谱、低毒的抗菌肽类似物HJH-3的生物学活性和膜活性的测定[J]. 中国预防兽医学报, 2019, 41(8):800-806.WANG Q, XU Y Z, YANG L, et al. Determination of bioactivity and membrane activity of bovine antimicrobial peptide HJH-3[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(8):800-806. (in Chinese)
[25]	DONG Z, LUO W J, ZHONG H R, et al. Molecular cloning and characterization of antimicrobial peptides from skin of Hylarana guentheri[J]. Acta Biochim Biophys Sin (Shanghai), 2017, 49(5):450-457.
[26]	孙栋, 蔡寅川, 蒋思雨, 等.抗菌肽Tachyplesin Ⅰ的分子设计、结构与活性分析[J].畜牧兽医学报, 2022, 53(6):1905-1913.SUN D, CAI Y C, JIANG S Y, et al. Molecular design, structure and activity analysis of antimicrobial peptide tachyplesin Ⅰ[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6):1905-1913.(in Chinese)
[27]	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)
[28]	DOHERTY T, WARING A J, HONG M. Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR[J]. Biochim Biophys Acta, 2006, 1758(9):1285-1291.
[29]	KUSHIBIKI T, KAMIYA M, AIZAWA T, et al. Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide[J]. Biochim Biophys Acta (BBA)-Proteins Proteomics, 2014, 1844(3):527-534.
[30]	HONG J, GUAN W T, JIN G, et al. Mechanism of tachyplesin Ⅰ injury to bacterial membranes and intracellular enzymes, determined by laser confocal scanning microscopy and flow cytometry[J]. Microbiol Res, 2015, 170:69-77.
[31]	谢海伟, 杨贤松, 柯春林, 等. 化学合成抗菌肽抑制致病性大肠杆菌F41的分子作用机理[J]. 现代食品科技, 2016, 32(1):44-51.XIE H W, YANG X S, KE C L, et al. Molecular mechanisms of novel synthetic analogs of tachyplesin against pathogenic Escherichia coli F41[J]. Modern Food Science and Technology, 2016, 32(1):44-51. (in Chinese)