Mechanism of Antimicrobial Peptide Scolopin 2-NH2 Isolated from Scolopendra subspinipes mutilans

doi:10.13560/j.cnki.biotech.bull.1985.2018-0476

Abstract

Abstract: This work aims to elucidate the secondary structure,antibacterial activity and physicochemical properties of antibacterial peptide Scolopin-2（S-2）and S-2-amidated Scolopin-2-NH2（S-2-N）,and to reveal their antibacterial mechanism at the cell and molecular level. The method of minimum inhibitory concentration was used to detect the antimicrobial activity of S-2/S-2-N against 4 strains,different treatments were applied to measure their physicochemical properties,and circular dichroism spectroscopy was to determine the secondary structure S-2/S-2-N. After Escherichia coli was treated with S-2-N and S-2 respectively,flow cytometry was used to detect the efficiency of peptides penetrating cell membrane and cell membrane integrity. Flow cytometry and RT-PCR technology were together to analyze the effects of peptides on cell cycle and the genes related to intracellular DNA replication and repair. In addition to the heat resistance,acid and alkaline resistance,the resistance to ionic strength and the ability of digestive enzymes,the S-2-N also had stronger antibacterial activity than the maternal peptide S-2. The results of flow cytometry showed that in a short period of time（30 min）both peptides caused cell membrane damaged,while the efficiency of S-2-N penetrating membrane was higher. The analysis of flow cytometry and RT-PCR demonstrated that the bacterial cell cycle was blocked by S-2-N and S-2,which lowered the expressions of gene dnaA,dnaB,dnaG and SSB,but promoted the expressions of RecA and RecN. In conclusion,the antibacterial peptide S-2-N amidated from S-2 has stronger antibacterial activity than maternal peptide,and both function by the same antibacterial mechanism,i.e.,destroying the bacterial cell membrane,combining bacterial DNA and RNA,affecting the secondary structure of DNA,blocking the cell cycle of bacteria,and affecting the expressions of genes associated with DNA replication and repair in cells.

Key words: antibacterial peptide, Scolopin-2-NH2, flow cytometry, RT-PCR technique, antibacterial mechanism, genes related to the replication and repair of DNA

LU Jia, DENG Qiu-ping, REN Wen-hua. Mechanism of Antimicrobial Peptide Scolopin 2-NH2 Isolated from Scolopendra subspinipes mutilans[J]. Biotechnology Bulletin, 2018, 34(11): 179-190.

References

[1] Boman HG, Nilsson I, Rasmuson B.Inducible antibacterial defence system in drosophila[J]. Nature, 1972, 237:232-235.
[2] Jenssen H, Hamill P, Hancock REW.Peptide antimicrobial agents[J]. Clinical Microbiology Reviews, 2008, 19(3):491-511.
[3] 刘明辉, 张尚志, 马艳, 等. 抗菌肽及其分子改造研究进展[J]. 安徽农业科学, 2016, 44(16):147-150.
[4] 李冠楠, 夏雪娟, 隆耀航, 等. 抗菌肽的研究进展及其应用[J]. 动物营养学报, 2014, 26(1):17-25.
[5] Michael Z.Antimicrobial peptides of multicellular organisms[J]. Nature, 2002, 415(6870):389-395.
[6] Gudmundsson GH, Magnusson KP, Chowdhary BP, et al.Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member:comparative mapping of the locus for the human peptide antibiotic FALL-39[J]. Proc Natl Acad Sci USA, 1995, 92(15):7085-7089.
[7] Lee H, Hwang JS, Lee J, et al.Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism[J]. Biochimica et Biophysica Acta(BBA)- Biomembranes, 2015, 1848(2):634-642.
[8] Gupta R, Srivastava S.Antifungal effect of antimicrobial peptides(AMPs LR14)derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage[J]. Food Microbiology, 2014, 42(12):1-7.
[9] 燕晓翠, 杨春蕾, 姚大为, 等. 抗菌肽的国内外研究进展[J]. 天津农业科学, 2017, 23(5):35-41.
[10] 薛林贵, 马萍, 尚海, 等. 鱼类及两栖动物的抗菌肽研究进展[J]. 生物技术通报, 2017, 33(12):61-66.
[11] Bierbaum G, Sahl HG, Bierbaum G, et al.Autolytic system of Staphylococcus simulans 22:influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase[J]. Journal of Bacteriology, 1988, 169(12):5452-5458.
[12] 陈旋, 李莉蓉. 抗菌肽P7抑制大肠杆菌的非膜作用机制[J]. 微生物学报, 2016, 56(11):1737-1745.
[13] 王建忠, 徐汉江, 陈晶, 等. 抗菌肽研究进展[J]. 世界最新医学信息文摘, 2016, 16(72):77-89.
[14] Tysoe SA, Morgan RJ, Baker AD, et al.Spectroscopic investigation of differential binding modes of . DELTA. -and . LAMBDA. Ru(bpy)2(ppz)2+ with calf thymus DNA[J]. Journal of Physical Chemistry, 1993, 97(8):1707-1711.
[15] 杨平, 袁奕豪, 杨晓莉, 等. 抗菌肽高效表达及生产优化研究进展[J]. 生物技术通报, 2016, 32(3):24-30.
[16] 刘伟, 皮雄娥, 王欣. 抗菌肽与肠道健康研究新进展[J]. 微生物学报, 2016, 56(10):1537-1543.
[17] 陈亮, 李瑞静, 黄亮, 等. 微生物抗菌肽及其在动物生产中的应用[J]. 中国畜牧杂志, 2017, 53(2):15-18.
[18] Pemberton RW.Insects and other arthropods used as drugs in Korean traditional medicine[J]. J Ethnopharmacol, 1999, 65(3):207-216.
[19] You WK, et al.Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans[J]. Insect Biochemistry and Molecular Biology, 2004, 34(3):239-250.
[20] Peng K, Kong Y, et al.Two novel antimicrobial peptides from centipede venoms[J]. Toxicon, 2010, 55(2-3):274-279.
[21] Boman HG, Agerberth B, Boman A.Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine.[J]. Infecti Immun, 1993, 61(7):2978-2984.
[22] Yanmei L, Qi X, Qihao Z, et al.Overview on the recent study of antimicrobial peptides:origins, functions, relative mechanisms and application[J]. Peptides, 2012, 37(2):207-215.
[23] Junkes C, Harvey RD, Bruce KD, et al.Cyclic antimicrobial R-, W-rich peptides:the role of peptide structure and E. coli outer and inner membranes in activity and the mode of action[J]. Biophysics of Structure & Mechanism, 2011, 40(4):515-528.
[24] Gidalevitz D, Ishitsuka Y, Muresan AS, et al.Interaction of antimicrobial peptide protegrin with biomembranes[J]. Proc Natl Acad Sci USA, 2003, 100(11):6302-6307.
[25] Subbalakshmi C, Sitaram N.Mechanism of antimicrobial action of Indolicidin[J]. Fems Microbiology Letters, 1998, 160(1):91-96.
[26] Park CB, Kim HS, Kim SC.Mechanism of action of the antimicrobial peptide buforin II:buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions[J]. Biochem Biophys Res Commun, 1998, 244(1):253-257.
[27] Imura Y, Nishida M, Matsuzaki K.Action mechanism of PEGylated magainin 2 analogue peptide[J]. Biochimica Et Biophysica Acta, 2007, 1768(10):2578-2585.
[28] Anbanandam A, Albarado DD, Simons M, et al.Molecular basis for proline- and arginine-rich peptide inhibition of proteasome[J]. Journal of Molecular Biology, 2008, 384(384):219-227.