不同疏水性氨基酸对α-螺旋抗菌肽生物学活性的影响
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摘要
天然抗菌肽具有较强的杀菌能力,但其较高的细胞毒性会使肽的细胞选择性降低。为了提高抗菌肽的选择特异性,本研究拟探讨不同疏水性氨基酸对α-螺旋抗菌肽生物学活性的影响及其抑菌机制。笔者采用α-螺旋肽GRX_2RX_3RX_2RG作为模板,分别以疏水性氨基酸色氨酸(Try,W)、苯丙氨酸(Phe,F)、缬氨酸(Val,V)、丙氨酸(Ala,A)、亮氨酸(Leu,L)和异亮氨酸(Ile,I)来替换X位置,得到了一系列富含疏水氨基酸的肽GW、GF、GV、GI、GA和GL。本研究检测了抗菌肽的二级结构、溶血活性、抑菌活性、盐离子活性,并对其抑菌的作用机制进行了研究。结果表明:通过CD光谱试验检测出GF、GI、GA和GL在细胞膜模拟环境中均表现出典型的α螺旋结构,而GV只在TFE中呈现α螺旋结构;溶血试验结果表明,GV和GA在浓度为128μmol·L~(-1)未表现出溶血活性,而其他肽均表现出较高的溶血活性;抑菌试验发现GV的最小抑菌浓度(MIC)的几何平均值为3.7μmol·L~(-1),并具有最高的治疗指数(TI);盐离子稳定性试验表明,在NH_4~+、Zn~(2+)和Fe~(3+)中GV对大肠杆菌25922(E.coli ATCC 25922)的抑菌能力较稳定。进一步通过扫描电镜和内膜通透性试验对抗菌肽GV的抑菌机制进行分析,结果观察到GV能够通过穿透E.coli ATCC 25922和金黄色葡萄球菌29213(S.aureus ATCC 29213)促使细胞内容物流出而导致细菌死亡,以及通过时间和剂量依赖的方式穿透细菌内膜。综合以上结果,富含Val的GV具有较高的细胞选择性和成为高效抗菌药物的发展潜力。
Natural antimicrobial peptides have a strong bactericidal ability, however, low cell selectivity of natural antimicrobial peptides due to their strong cytotoxicity. In order to improve the specificity of antibacterial peptides, the effects of different hydrophobic amino acids on the biological activity of α-helix antimicrobial peptides and bacteriostatic mechanisms were investigated. We employed hydrophobic amino acid tryptophan(Try, T), phenylalanine(Phe, P), valine(Val, V), alanine(Ala, A), leucine(Leu, L) and isoleucine(Ile, I) to replace X position of GRX_2RX_3RX_2RG template. Our study evaluated the secondary structure, hemolytic activity, bacteriostatic activity, salt resistance and studied bacteriostatic mechanism of peptides. The results of CD spectroscopy showed that GF, GI, GA and GL displayed typical α helix structure in the simulated environment of cell membrane, while only GV exhibited α helix structure in TFE. Hemolysis test showed that GV and GA did not exhibit hemolytic activity in the concentration of 128 μmol·L~(-1), while other peptides showed high hemolytic activity. The geometric average value of minimal inhibitory concentration(MIC) of GV which has the highest therapeutic index is 3.7 μmol·L~(-1). The stability test showed that GV had the stable antibacterial activity against E. coli ATCC 25922 in the presence of NH_4~+, Zn~(2+) and Fe~(3+). The antimicrobial mechanism of antimicrobial peptide GV was further analyzed by scanning electron microscopy and inner membrane permeability test. The results showed that GV induced outflow of bacterial content via penetrating membrane of E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 and penetrated inner membrane of E. coli ATCC 25922 by time-and dose-dependence. Collectivity, the Val-rich antimicrobial peptide GV had the highest cell selectivity, and is potential for development of highly effective antibacterial drugs.
Natural antimicrobial peptides have a strong bactericidal ability, however, low cell selectivity of natural antimicrobial peptides due to their strong cytotoxicity. In order to improve the specificity of antibacterial peptides, the effects of different hydrophobic amino acids on the biological activity of α-helix antimicrobial peptides and bacteriostatic mechanisms were investigated. We employed hydrophobic amino acid tryptophan(Try, T), phenylalanine(Phe, P), valine(Val, V), alanine(Ala, A), leucine(Leu, L) and isoleucine(Ile, I) to replace X position of GRX_2RX_3RX_2RG template. Our study evaluated the secondary structure, hemolytic activity, bacteriostatic activity, salt resistance and studied bacteriostatic mechanism of peptides. The results of CD spectroscopy showed that GF, GI, GA and GL displayed typical α helix structure in the simulated environment of cell membrane, while only GV exhibited α helix structure in TFE. Hemolysis test showed that GV and GA did not exhibit hemolytic activity in the concentration of 128 μmol·L~(-1), while other peptides showed high hemolytic activity. The geometric average value of minimal inhibitory concentration(MIC) of GV which has the highest therapeutic index is 3.7 μmol·L~(-1). The stability test showed that GV had the stable antibacterial activity against E. coli ATCC 25922 in the presence of NH_4~+, Zn~(2+) and Fe~(3+). The antimicrobial mechanism of antimicrobial peptide GV was further analyzed by scanning electron microscopy and inner membrane permeability test. The results showed that GV induced outflow of bacterial content via penetrating membrane of E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 and penetrated inner membrane of E. coli ATCC 25922 by time-and dose-dependence. Collectivity, the Val-rich antimicrobial peptide GV had the highest cell selectivity, and is potential for development of highly effective antibacterial drugs.
引文
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[22] DONG N,ZHU X,CHOU S L,et al.Antimicrobial potency and selectivity of simplified symmetric-end peptides[J].Biomaterials,2014,35(27):8028-8039.
[23] BALAKRISHNAN V S,VAD B S,OTZEN D E.Novicidin’s membrane permeabilizing activity is driven by membrane partitioning but not by helicity:A biophysical study of the impact of lipid charge and cholesterol[J].Biochim Biophys Acta - Prot Proteom,2013,1834(6):996-1002.
[24] ZHU X,SHAN A S,MA Z,et al.Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa[J].Antimicrob Agents Chemother,2015,59(6):3008-3017.
[25] MAISETTA G,DI LUCA M,ESIN S,et al.Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3[J].Peptides,2008,29(1):1-6.
[26] CHEN A M,SHI Q S,OUYANG Y S,et al.Effect of Ce3+ on membrane permeability of Escherichia coli cell[J].J Rare Earths,2012,30(9):947-951.
[27] LIU Y F,XIA X,XU L,et al.Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity[J].Biomaterials,2013,34(1):237-250.
[28] GIANGASPERO A,SANDRI L,TOSSI A.Amphipathic α helical antimicrobial peptides:a systematic study of the effects of structural and physical properties on biological activity[J].FEBS J,2001,268(21):5589-5600.
[29] SONG R,WEI R B,LUO H Y,et al.Isolation and characterization of an antibacterial peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty)[J].Molecules,2012,17(3):2980-2991.
[30] WEI L,YANG J J,HE X Q,et al.Structure and function of a potent lipopolysaccharide-binding antimicrobial and anti-inflammatory peptide[J].J Med Chem,2013,56(9):3546-3556.
[31] CHEN C X,HU J,ZHANG S Z,et al.Molecular mechanisms of antibacterial and antitumor actions of designed surfactant-like peptides[J].Biomaterials,2012,33(2):592-603.
[32] ZHANG L J,ROZEK A,HANCOCK R E W.Interaction of cationic antimicrobial peptides with model membranes[J].J Biol Chem,2001,276(38):35714-35722.
[33] HUANG Y B,WANG X F,WANG H Y,et al.Studies on mechanism of action of anticancer peptides by modulation of hydrophobicity within a defined structural framework[J].Mol Cancer Ther,2011,10(3):416-426.
[34] WANG J J,CHOU S L,XU L,et al.High specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with Gly-Gly pairs[J].Sci Rep,2015,5:15963.
[35] KIM S,HYUN S,LEE Y,et al.Nonhemolytic cell-penetrating peptides:site specific introduction of glutamine and lysine residues into the α-helical peptide causes deletion of its direct membrane disrupting ability but retention of its cell penetrating ability[J].Biomacromolecules,2016,17(9):3007-3015.
[36] PATHAK N,SALAS-AUVERT R,RUCHE G,et al.Comparison of the effects of hydrophobicity,amphiphilicity,and α-helicity on the activities of antimicrobial peptides[J].Proteins,1995,22(2):182-186.
[37] KHARA J S,LIM F K,WANG Y,et al.Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis:discerning the role of hydrophobicity and helicity[J].Acta Biomater,2015,28:99-108.
[38] HUANG J F,HAO D M,CHEN Y,et al.Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria[J].Peptides,2011,32(7):1488-1495.
[39] AROURI A,DATHE M,BLUME A.The helical propensity of KLA amphipathic peptides enhances their binding to gel-state lipid membranes[J].Biophys Chem,2013,180-181:10-21.
[40] AQUILA M,BENEDUSI M,KOCH K W,et al.Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin[J].Cell Calcium,2013,53(3):180-186.
[41] KOO Y S,KIM J M,PARK I Y,et al.Structure-activity relations of parasin I,a histone H2A-derived antimicrobial peptide[J].Peptides,2008,29(7):1102-1108.
[42] SCHIBLI D J,EPAND R F,VOGEL H J,et al.Tryptophan-rich antimicrobial peptides:comparative properties and membrane interactions[J].Biochem Cell Biol,2002,80(5):667-677.
[43] MARQUETTE A,BECHINGER B.Biophysical investigations elucidating the mechanisms of action of antimicrobial peptides and their synergism[J].Biomolecules,2018,8(2):18.
[2] XIA X J,CHENG L K,ZHANG S P,et al.The role of natural antimicrobial peptides during infection and chronic inflammation[J].Antonie van Leeuwenhoek,2018,111(1):5-26.
[3] LIANG X,NONG X H,HUANG Z H,et al.Antifungal and antiviral cyclic peptides from the deep-sea-derived fungus Simplicillium obclavatum EIODSF 020[J].J Agric Food Chem,2017,65(25):5114-5121.
[4] BOTO A,DE LA LASTRA J M P,GONZáLEZ C C.The road from host-defense peptides to a new generation of antimicrobial drugs[J].Molecules,2018,23(2):E311.
[5] KUMAR P,KIZHAKKEDATHU J N,STRAUS S K.Antimicrobial peptides:diversity,mechanism of action and strategies to improve the activity and biocompatibility in vivo[J].Biomolecules,2018,8(1):E4.
[6] AGHAZADEH H,MEMARIANI H,RANJBAR R,et al.The activity and action mechanism of novel short selective LL-37-derived anticancer peptides against clinical isolates of Escherichia coli[J].Chem Biol Drug Des,2018,doi:10.1111/cbdd.13381.
[7] BROGDEN K A.Antimicrobial peptides:pore formers or metabolic inhibitors in bacteria?[J].Nat Rev Microbiol,2005,3(3):238-250.
[8] CHAN D I,PRENNER E J,VOGEL H J.Tryptophan- and arginine-rich antimicrobial peptides:structures and mechanisms of action[J].Biochim Biophys Acta - Biomembr,2006,1758(9):1184-1202.
[9] DIAS S A,FREIRE J M,PéREZ-PEINADO C,et al.New potent membrane-targeting antibacterial peptides from viral capsid proteins[J].Front Microbiol,2017,8:775.
[10] GRAF M,MARDIROSSIAN M,NGUYEN F,et al.Proline-rich antimicrobial peptides targeting protein synthesis[J].Nat Prod Rep,2017,34(7):702-711.
[11] LI J G,KOH J J,LIU S P,et al.Membrane active antimicrobial peptides:translating mechanistic insights to design[J].Front Neurosci,2017,11:73.
[12] CAO X Q,WANG Y,WU C Y,et al.Cathelicidin-OA1,a novel antioxidant peptide identified from an amphibian,accelerates skin wound healing[J].Sci Rep,2018,8(1):943.
[13] WIRADHARMA N,SNG M Y S,KHAN M,et al.Rationally designed α-helical broad-spectrum antimicrobial peptides with idealized facial amphiphilicity[J].Macromol Rapid Commun,2013,34(1):74-80.
[14] RODRíGUEZ A,VILLEGAS E,SATAKE H,et al.Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index[J].Amino Acids,2011,40(1):61-68.
[15] TAN J J,HUANG J F,HUANG Y B,et al.Effects of single amino acid substitution on the biophysical properties and biological activities of an amphipathic α-helical antibacterial peptide against Gram-negative bacteria[J].Molecules,2014,19(8):10803-10817.
[16] STRANDBERG E,ZERWECK J,HORN D,et al.Influence of hydrophobic residues on the activity of the antimicrobial peptide magainin 2 and its synergy with PGLa[J].J Pept Sci,2015,21(5):436-445.
[17] RINGSTAD L,NORDAHL E A,SCHMIDTCHEN A,et al.Composition effect on peptide interaction with lipids and bacteria:Variants of C3a peptide CNY21[J].Biophys J,2007,92(1):87-98.
[18] ZHU W L,LAN H L,PARK Y,et al.Effects of Pro → peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide[J].Biochemistry,2006,45(43):13007-13017.
[19] HU W N,JIAO W J,MA Z,et al.The influence of isoleucine and arginine on biological activity and peptide- membrane interactions of antimicrobial peptides from the bactericidal domain of AvBD4[J].Protein Pept Lett,2013,20(11):1189-1199.
[20] CHEN Y X,GUARNIERI M T,VASIL A I,et al.Role of peptide hydrophobicity in the mechanism of action of α-helical antimicrobial peptides[J].Antimicrob Agents Chemother,2007,51(4):1398-1406.
[21] LEE E,SHIN A,JEONG K W,et al.Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1[J].PLoS One,2014,9(12):e114453.
[22] DONG N,ZHU X,CHOU S L,et al.Antimicrobial potency and selectivity of simplified symmetric-end peptides[J].Biomaterials,2014,35(27):8028-8039.
[23] BALAKRISHNAN V S,VAD B S,OTZEN D E.Novicidin’s membrane permeabilizing activity is driven by membrane partitioning but not by helicity:A biophysical study of the impact of lipid charge and cholesterol[J].Biochim Biophys Acta - Prot Proteom,2013,1834(6):996-1002.
[24] ZHU X,SHAN A S,MA Z,et al.Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa[J].Antimicrob Agents Chemother,2015,59(6):3008-3017.
[25] MAISETTA G,DI LUCA M,ESIN S,et al.Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3[J].Peptides,2008,29(1):1-6.
[26] CHEN A M,SHI Q S,OUYANG Y S,et al.Effect of Ce3+ on membrane permeability of Escherichia coli cell[J].J Rare Earths,2012,30(9):947-951.
[27] LIU Y F,XIA X,XU L,et al.Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity[J].Biomaterials,2013,34(1):237-250.
[28] GIANGASPERO A,SANDRI L,TOSSI A.Amphipathic α helical antimicrobial peptides:a systematic study of the effects of structural and physical properties on biological activity[J].FEBS J,2001,268(21):5589-5600.
[29] SONG R,WEI R B,LUO H Y,et al.Isolation and characterization of an antibacterial peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty)[J].Molecules,2012,17(3):2980-2991.
[30] WEI L,YANG J J,HE X Q,et al.Structure and function of a potent lipopolysaccharide-binding antimicrobial and anti-inflammatory peptide[J].J Med Chem,2013,56(9):3546-3556.
[31] CHEN C X,HU J,ZHANG S Z,et al.Molecular mechanisms of antibacterial and antitumor actions of designed surfactant-like peptides[J].Biomaterials,2012,33(2):592-603.
[32] ZHANG L J,ROZEK A,HANCOCK R E W.Interaction of cationic antimicrobial peptides with model membranes[J].J Biol Chem,2001,276(38):35714-35722.
[33] HUANG Y B,WANG X F,WANG H Y,et al.Studies on mechanism of action of anticancer peptides by modulation of hydrophobicity within a defined structural framework[J].Mol Cancer Ther,2011,10(3):416-426.
[34] WANG J J,CHOU S L,XU L,et al.High specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with Gly-Gly pairs[J].Sci Rep,2015,5:15963.
[35] KIM S,HYUN S,LEE Y,et al.Nonhemolytic cell-penetrating peptides:site specific introduction of glutamine and lysine residues into the α-helical peptide causes deletion of its direct membrane disrupting ability but retention of its cell penetrating ability[J].Biomacromolecules,2016,17(9):3007-3015.
[36] PATHAK N,SALAS-AUVERT R,RUCHE G,et al.Comparison of the effects of hydrophobicity,amphiphilicity,and α-helicity on the activities of antimicrobial peptides[J].Proteins,1995,22(2):182-186.
[37] KHARA J S,LIM F K,WANG Y,et al.Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis:discerning the role of hydrophobicity and helicity[J].Acta Biomater,2015,28:99-108.
[38] HUANG J F,HAO D M,CHEN Y,et al.Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria[J].Peptides,2011,32(7):1488-1495.
[39] AROURI A,DATHE M,BLUME A.The helical propensity of KLA amphipathic peptides enhances their binding to gel-state lipid membranes[J].Biophys Chem,2013,180-181:10-21.
[40] AQUILA M,BENEDUSI M,KOCH K W,et al.Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin[J].Cell Calcium,2013,53(3):180-186.
[41] KOO Y S,KIM J M,PARK I Y,et al.Structure-activity relations of parasin I,a histone H2A-derived antimicrobial peptide[J].Peptides,2008,29(7):1102-1108.
[42] SCHIBLI D J,EPAND R F,VOGEL H J,et al.Tryptophan-rich antimicrobial peptides:comparative properties and membrane interactions[J].Biochem Cell Biol,2002,80(5):667-677.
[43] MARQUETTE A,BECHINGER B.Biophysical investigations elucidating the mechanisms of action of antimicrobial peptides and their synergism[J].Biomolecules,2018,8(2):18.
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