Abstract
Facing rising global antibiotics resistance, physical membrane-damaging antimicrobial peptides (AMPs) represent promising antimicrobial agents. Various strategies to design effective hybrid peptides offer many advantages in overcoming the adverse effects of natural AMPs. In this study, hybrid peptides from different species were investigated, and three hybrid antimicrobial peptides, LI, LN, and LC, were designed by combining the typical fragment of human cathelicidin-derived LL37 with either indolicidin, pig nematode cecropin P1 (CP-1) or rat neutrophil peptide-1 (NP-1). In an aqueous solution, all hybrid peptides had an unordered conformation. In simulated membrane conditions, the hybrid peptide LI displayed more β-turn and β-hairpin structures, whereas LN and LC folded into α-helix structures. The three interspecific hybrid peptides LI, LN, and LC exhibited different levels of antimicrobial activity against Gram-positive and Gram-negative bacteria. LI demonstrated the highest antimicrobial activity and cell selectivity. The results of the swimming motility indicated that LI repressed bacterial motility in a concentration-dependent method. Endotoxin binding assay demonstrated that hybrid peptide LI conserved the binding ability to LPS (polyanionic lipopolysaccharides) of its parental peptides. Fluorescence assays, flow cytometry, and SEM further revealed that hybrid peptide LI acted through different bacteriostatic mechanisms than LL37 and indolicidin and that LI killed bacterial cells via membrane damage. In summary, this study demonstrated that hybrid peptide LI produced by interspecific hybrid synthesis possessed strong cell selectivity and is a promising therapeutic candidate for drug-resistant bacteria infection.
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12 May 2018
Facing rising global antibiotics resistance, physical membrane-damaging antimicrobial peptides (AMPs) represent promising antimicrobial agents. Various strategies to design effective hybrid peptides offer many advantages in overcoming the adverse effects of natural AMPs.
Abbreviations
- AMP:
-
Antimicrobial peptide
- MIC:
-
Minimum inhibitory concentration
- MHC:
-
Minimum hemolytic concentration
- PBS:
-
Phosphate-buffered saline
- MH:
-
Mueller–Hinton
- BSA:
-
Bovine serum albumin
- CD:
-
Circular dichroism
- TFE:
-
Trifluoroethanol
- SDS:
-
Sodium dodecyl sulfate
- OM:
-
Outer membrane
- NPN:
-
N-Phenyl-1-naphthylamine
- SEM:
-
Scanning electron microscopy
- LPS:
-
Lipopolysaccharide
- PI:
-
Propidium iodide
References
Ahmad I, Perkins WR, Lupan DM et al (1995) Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. BBA Biomembr 1237:109–114. https://doi.org/10.1016/0005-2736(95)00087-J
Baek MH, Kamiya M, Kushibiki T et al (2016) Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy. J Pept Sci 22:214–221. https://doi.org/10.1002/psc.2865
Berlose JP, Convert O, Derossi D et al (1996) Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane-mimetic environments. Eur J Biochem 242:372–386. https://doi.org/10.1111/j.1432-1033.1996.0372r.x
Brogden NK, Brogden KA (2011) Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? Int J Antimicrob Agents 38:217–225
Buck M (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q Rev Biophys 31:297–355. https://doi.org/10.1017/S003358359800345X
Chou S, Shao C, Wang J et al (2016) Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance. Acta Biomater 30:78–93. https://doi.org/10.1016/j.actbio.2015.11.002
Cornette JL, Cease KB, Margalit H et al (1987) Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J Mol Biol 195:659–685. https://doi.org/10.1016/0022-2836(87)90189-6
Currie SM, Gwyer Findlay E, McFarlane AJ et al (2016) Cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans. J Immunol 196:2699–2710. https://doi.org/10.4049/jimmunol.1502478
Dashper SG, O’Brien-Simpson NM, Cross KJ et al (2005) Divalent metal cations increase the activity of the antimicrobial peptide kappacin. Antimicrob Agents Chemother 49:2322–2328. https://doi.org/10.1128/AAC.49.6.2322-2328.2005
Dong N, Ma Q, Shan A et al (2012) Strand length-dependent antimicrobial activity and membrane-active mechanism of arginine- and valine-rich β-hairpin-like antimicrobial peptides. Antimicrob Agents Chemother 56:2994–3003. https://doi.org/10.1128/AAC.06327-11
Dong N, Zhu X, Chou S et al (2014) Antimicrobial potency and selectivity of simplified symmetric-end peptides. Biomaterials 35:8028–8039. https://doi.org/10.1016/j.biomaterials.2014.06.005
Eisenhauer PB, Harwig SSL, Szklarek D et al (1989) Purification and antimicrobial properties of three defensins from rat neutrophils. Infect Immun 57:2021–2027
Ejim L, Farha MA, Falconer SB et al (2011) Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat Chem Biol 7:348–350. https://doi.org/10.1038/nchembio.559
Fox MA, Thwaite JE, Ulaeto DO et al (2012) Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II. Peptides 33:197–205. https://doi.org/10.1016/j.peptides.2012.01.013
Hagar JA, Powell DA, Aachoui Y et al (2013) Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science (80-) 341:1250–1253. https://doi.org/10.1126/science.1240988
Haisma EM, De Breij A, Chan H et al (2014) LL-37-derived peptides eradicate multidrug-resistant Staphylococcus aureus from thermally wounded human skin equivalents. Antimicrob Agents Chemother 58:4411–4419. https://doi.org/10.1128/AAC.02554-14
Herce HD, Garcia AE (2007) Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes. Proc Natl Acad Sci USA 104:20805–20810. https://doi.org/10.1073/pnas.0706574105
Imamura Y, Higashiyama Y, Tomono K et al (2005) Azithromycin exhibits bactericidal effects on Pseudomonas aeruginosa through interaction with the outer membrane. Antimicrob Agents Chemother 49:1377–1380. https://doi.org/10.1128/AAC.49.4.1377-1380.2005
Javadpour MM, Juban MM, Lo WCJ et al (1996) De novo antimicrobial peptides with low mammalian cell toxicity. J Med Chem 39:3107–3113. https://doi.org/10.1021/jm9509410
Jing W, Demcoe AR, Vogel HJ (2003) Conformation of a bactericidal domain of puroindoline a: structure and mechanism of action of a 13-residue antimicrobial peptide. J Bacteriol 185:4938–4947. https://doi.org/10.1128/JB.185.16.4938-4947.2003
Jing W, Svendsen JS, Vogel HJ (2006) Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin. Biochem Cell Biol 84:312–326. https://doi.org/10.1139/o06-052
Kawashima H, Katayama M, Yoshida R et al (2016) A dimer model of human calcitonin13–32 forms an α-helical structure and robustly aggregates in 50% aqueous 2,2,2-trifluoroethanol solution. J Pept Sci 480–484. https://doi.org/10.1002/psc.2891
Le C-F, Yusof MYM, Hassan H et al (2015) In vitro properties of designed antimicrobial peptides that exhibit potent antipneumococcal activity and produces synergism in combination with penicillin. Sci Rep 5:9761. https://doi.org/10.1038/srep09761
Li J, Koh JJ, Liu S et al (2017) Membrane active antimicrobial peptides: translating mechanistic insights to design. Front Neurosci 11:73
Liu YF, Xia X, Xu L, Wang YZ (2013) Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity. Biomaterials 34:237–250. https://doi.org/10.1016/j.biomaterials.2012.09.032
Lv Y, Wang J, Gao H et al (2014) Antimicrobial properties and membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin PMAP-36. PLoS ONE 9:1–12. https://doi.org/10.1371/journal.pone.0086364
Lyu Y, Yang X, Goswami S et al (2017) Amphiphilic tobramycin-lysine conjugates sensitize multidrug resistant Gram-negative bacteria to rifampicin and minocycline. J Med Chem 60:3684–3702. https://doi.org/10.1021/acs.jmedchem.6b01742
Ma Z, Wei D, Yan P et al (2015) Characterization of cell selectivity, physiological stability and endotoxin neutralization capabilities of α-helix-based peptide amphiphiles. Biomaterials 52:517–530. https://doi.org/10.1016/j.biomaterials.2015.02.063
Ma L, Wang Y, Wang M et al (2016) Effective antimicrobial activity of Cbf-14, derived from a cathelin-like domain, against penicillin-resistant bacteria. Biomaterials 87:32–45. https://doi.org/10.1016/j.biomaterials.2016.02.011
Malanovic N, Lohner K (2016) Gram-positive bacterial cell envelopes: the impact on the activity of antimicrobial peptides. Biochim Biophys Acta Biomembr 1858:936–946. https://doi.org/10.1016/j.bbamem.2015.11.004
Memariani H, Shahbazzadeh D, Ranjbar R et al (2017) Design and characterization of short hybrid antimicrobial peptides from pEM-2, mastoparan-VT1, and mastoparan-B. Chem Biol Drug Des 89:327–338. https://doi.org/10.1111/cbdd.12864
Mikut R, Ruden S, Reischl M et al (2016) Improving short antimicrobial peptides despite elusive rules for activity. Biochim Biophys Acta Biomembr 1858:1024–1033. https://doi.org/10.1016/j.bbamem.2015.12.013
Mura M, Wang J, Zhou Y et al (2016) The effect of amidation on the behaviour of antimicrobial peptides. Eur Biophys J 45:195–207
Neelabh Singh K, Rani J (2016) Sequential and structural aspects of antifungal peptides from animals, bacteria and fungi based on bioinformatics tools. Probiotics Antimicrob Proteins 8:85–101
Nguyen VS, Tan KW, Ramesh K et al (2017) Structural basis for the bacterial membrane insertion of dermcidin peptide, DCD-1L. Sci Rep 7:13923. https://doi.org/10.1038/s41598-017-13600-z
Niyonsaba F, Iwabuchi K, Someya A et al (2002) A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis. Immunology 106:20–26. https://doi.org/10.1046/j.1365-2567.2002.01398.x
Oren Z, Lerman JC, Gudmundsson GH et al (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 341:501–513
Park IY, Cho JH, Kim KS et al (2004) Helix stability confers salt resistance upon helical antimicrobial peptides. J Biol Chem 279:13896–13901. https://doi.org/10.1074/jbc.M311418200
Park KH, Nan YH, Park Y et al (2009) Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides. Biochim Biophys Acta Biomembr 1788:1193–1203. https://doi.org/10.1016/j.bbamem.2009.02.020
Paul K, Erhardt M, Hirano T et al (2008) Energy source of flagellar type III secretion. Nature 451:489–492. https://doi.org/10.1038/nature06497
Paulmann M, Arnold T, Linke D et al (2012) Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat. J Biol Chem 287:8434–8443. https://doi.org/10.1074/jbc.M111.332270
Paulsen VS, Blencke HM, Benincasa M et al (2013) Structure-activity relationships of the antimicrobial peptide arasin 1—and mode of action studies of the N-terminal, proline-rich region. PLoS ONE. https://doi.org/10.1371/journal.pone.0053326
Rokitskaya TI, Kolodkin NI, Kotova EA, Antonenko YN (2011) Indolicidin action on membrane permeability: carrier mechanism versus pore formation. Biochim Biophys Acta Biomembr 1808:91–97. https://doi.org/10.1016/j.bbamem.2010.09.005
Saugar JM, Rodriguez-Hernandez MJ, de la Torre BG et al (2006) Activity of cecropin A-melittin hybrid peptides against colistin-resistant clinical strains of Acinetobacter baumannii: molecular basis for the differential mechanisms of action. Antimicrob Agents Chemother 50:1251–1256. https://doi.org/10.1128/AAC.50.4.1251
Selsted ME, Novotny MJ, Morris WL et al (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 267:4292–4295
Shang D, Zhang Q, Dong W et al (2016) The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization. Acta Biomater 33:153–165. https://doi.org/10.1016/j.actbio.2016.01.019
Shin A, Lee E, Jeon D et al (2015) Peptoid-substituted hybrid antimicrobial peptide derived from papiliocin and magainin 2 with enhanced bacterial selectivity and anti-inflammatory activity. Biochemistry 54:3921–3931. https://doi.org/10.1021/acs.biochem.5b00392
Tsai PW, Cheng YL, Hsieh WP, Lan CY (2014) Responses of Candida albicans to the human antimicrobial peptide LL-37. J Microbiol 52:581–589. https://doi.org/10.1007/s12275-014-3630-2
Uppu DSSM, Haldar J (2016) Lipopolysaccharide neutralization by cationic-amphiphilic polymers through pseudoaggregate formation. Biomacromolecules 17:862–873. https://doi.org/10.1021/acs.biomac.5b01567
Wang P, Bang JK, Kim HJ et al (2009) Antimicrobial specificity and mechanism of action of disulfide-removed linear analogs of the plant-derived Cys-rich antimicrobial peptide Ib-AMP1. Peptides 30:2144–2149. https://doi.org/10.1016/j.peptides.2009.09.020
Wei X-B, Wu R-J, Si D-Y et al (2016) Novel hybrid peptide cecropin A (1–8)-LL37 (17–30) with potential antibacterial activity. Int J Mol Sci 17:983. https://doi.org/10.3390/ijms17070983
Wimley WC, White SH (1996) Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nature 3:842–848. https://doi.org/10.1038/nsb1096-842
Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395. https://doi.org/10.1038/415389a
Zelezetsky I, Tossi A (2006) Alpha-helical antimicrobial peptides—using a sequence template to guide structure-activity relationship studies. Biochim Biophys Acta Biomembr 1758:1436–1449
Zhu X, Dong N, Wang Z et al (2014a) Design of imperfectly amphipathic α-helical antimicrobial peptides with enhanced cell selectivity. Acta Biomater 10:244–257. https://doi.org/10.1016/j.actbio.2013.08.043
Zhu X, Ma Z, Wang J et al (2014b) Importance of tryptophan in transforming an amphipathic peptide into a Pseudomonas aeruginosa-targeted antimicrobial peptide. PLoS ONE. https://doi.org/10.1371/journal.pone.0114605
Zhu X, Zhang L, Wang J et al (2015) Characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity. Acta Biomater 18:155–167. https://doi.org/10.1016/j.actbio.2015.02.023
Funding
We gratefully acknowledge the financial support from the Natural Science Foundation of Heilongjiang Province, China (41400172-4-15322), the National Natural Science Foundation of China (31472104, 31501914, and 31672434), the China Postdoctoral Science Foundation (2015M571385), the Postdoctoral Science Foundation of Heilongjiang Province, China (LBH-Z14026), the Special Financial Grant from the China Postdoctoral Science Foundation (2016T90267), and the Academic Backbone Project of Northeast Agricultural University (16XG14).
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Dong, N., Li, X.R., Xu, X.Y. et al. Characterization of bactericidal efficiency, cell selectivity, and mechanism of short interspecific hybrid peptides. Amino Acids 50, 453–468 (2018). https://doi.org/10.1007/s00726-017-2531-1
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DOI: https://doi.org/10.1007/s00726-017-2531-1