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Rational design of mirror-like peptides with alanine regulation

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Abstract

To generate effective antimicrobial peptides (AMPs) with good antimicrobial activities and cell selectivity, many synthetic strategies have been implemented to facilitate the development of AMPs. However, these synthetic strategies represent only a small proportion of the methods used for the development of AMPs and are not optimal with the requirements needed for the design of AMPs. In this investigation, we designed a mirror-like structure with a lower charge and a higher number of hydrophobic amino acids. The amino acid sequence of the designed mirror-like peptides was XXYXXXYXXXYXX [X represents L (Leu) and/or A (Ala); Y represents K (Lys)]. These mirror-like peptides displayed antimicrobial activity against both Gram-positive and Gram-negative bacteria. Hemolysis activity and cytotoxicity, detected by using human red blood cells (hRBCs) and human embryonic kidney cells (HEK293), respectively, demonstrated that the frequency of Ala residues in this structure had a regulatory effect on the high hydrophobic region. In particular, KL4A6 showed a greater antimicrobial potency than the other three mirror-like peptides, folded into an α-helical structure, and displayed the highest therapeutic index, suggesting its good cell selectivity. Observations from fluorescence spectroscopy, flow cytometry, and electron microscopy experiments indicated that KL4A6 exhibited good membrane penetration potential by inducing membrane blebbing, disruption and lysis. Therefore, generating mirror-like peptides is a promising strategy for designing effective AMPs with regions of high hydrophobicity.

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Abbreviations

AMP:

Antimicrobial peptide

MIC:

Minimum inhibitory concentration

MHC:

Minimal hemolytic concentration

HRBCs:

Human red blood cells

TI:

Therapeutic index

CD:

Circular dichroism

SDS:

Sodium dodecyl sulfate

PI:

Propidium iodide

MH:

Mueller–Hinton

NPN:

N-Phenyl-1-napthylamine

FACScan:

Fluorescent activated cell scanner

ONP:

o-Nitrophenol

ONPG:

o-Nitrophenyl-β-d-galactoside

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscopy

References

  • Amiche M, Seon AA, Pierre TN, Nicolas P (1999) The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain. FEBS Lett 456(3):352–356

    Article  CAS  PubMed  Google Scholar 

  • Bell G, Gouyon PH (2003) Arming the enemy: the evolution of resistance to self-proteins. Microbiology 149(Pt 6):1367–1375. doi:10.1099/mic.0.26265-0

    Article  CAS  PubMed  Google Scholar 

  • Charpentier S, Amiche M, Mester J, Vouille V, Le Caer JP, Nicolas P, Delfour A (1998) Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics. J Biol Chem 273(24):14690–14697. doi:10.1074/jbc.273.24.14690

    Article  CAS  PubMed  Google Scholar 

  • Chen Z, Wang D, Cong Y, Wang J, Zhu J, Yang J, Hu Z, Hu X, Tan Y, Hu F, Rao X (2011) Recombinant antimicrobial peptide hPAB-beta expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol 89(2):281–291. doi:10.1007/s00253-010-2864-0

    Article  CAS  PubMed  Google Scholar 

  • Cornette JL, Cease KB, Margalit H, Spouge JL, Berzofsky JA, DeLisi C (1987) Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J Mol Biol 195(3):659–685

    Article  CAS  PubMed  Google Scholar 

  • Cornut I, Büttner K, Dasseux J-L, Dufourcq J (1994) The amphipathic α-helix concept: application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin. FEBS Lett 349(1):29–33

    Article  CAS  PubMed  Google Scholar 

  • Cronan JE (2003) Bacterial membrane lipids: where do we stand? Annu Rev Microbiol 57:203–224. doi:10.1146/annurev.micro.57.030502.090851

    Article  CAS  PubMed  Google Scholar 

  • Dathe M, Wieprecht T, Nikolenko H, Handel L, Maloy WL, MacDonald DL, Beyermann M, Bienert M (1997) Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett 403(2):208–212. doi:10.1016/s0014-5793(97)00055-0

    Article  CAS  PubMed  Google Scholar 

  • Deléage G, Combet C, Blanchet C, Geourjon C (2001) ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities. Comput Biol Med 31(4):259–267. doi:10.1016/s0010-4825(01)00008-7

    Article  PubMed  Google Scholar 

  • Dong N, Ma Q, Shan A, Lv Y, Hu W, Gu Y, Li Y (2012) Strand length-dependent antimicrobial activity and membrane-active mechanism of arginine- and valine-rich beta-hairpin-like antimicrobial peptides. Antimicrob Agents Chemother 56(6):2994–3003. doi:10.1128/AAC.06327-11

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dong N, Zhu X, Chou S, Shan A, Li W, Jiang J (2014) Antimicrobial potency and selectivity of simplified symmetric-end peptides. Biomaterials 35(27):8028–8039

    Article  CAS  PubMed  Google Scholar 

  • Fjell CD, Hiss JA, Hancock RE, Schneider G (2012) Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11(1):37–51

    CAS  Google Scholar 

  • Hancock RE (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1(3):156–164

    Article  CAS  PubMed  Google Scholar 

  • Håversen L, Kondori N, Baltzer L, Hanson L, Dolphin G, Duner K, Mattsby-Baltzer I (2010) Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial α-helix of human lactoferrin. Antimicrob Agents Chemother 54(1):418–425

    Article  PubMed Central  PubMed  Google Scholar 

  • Hirt H, Gorr SU (2013) Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa. Antimicrob Agents Chemother 57(10):4903–4910. doi:10.1128/AAC.00311-13

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Huang Y, Wiradharma N, Xu K, Ji Z, Bi S, Li L, Yang YY, Fan W (2012) Cationic amphiphilic alpha-helical peptides for the treatment of carbapenem-resistant Acinetobacter baumannii infection. Biomaterials 33(34):8841–8847. doi:10.1016/j.biomaterials.2012.08.026

    Article  CAS  PubMed  Google Scholar 

  • Idiong G, Won A, Ruscito A, Leung BO, Hitchcock AP, Ianoul A (2011) Investigating the effect of a single glycine to alanine substitution on interactions of antimicrobial peptide latarcin 2a with a lipid membrane. Eur Biophys J 40(9):1087–1100. doi:10.1007/s00249-011-0726-z

    Article  CAS  PubMed  Google Scholar 

  • Jiang Z, Vasil AI, Hale JD, Hancock RE, Vasil ML, Hodges RS (2008) Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides. Biopolymers 90(3):369–383. doi:10.1002/bip.20911

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Johansson J (1998) Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37. J Biol Chem 273(6):3718–3724. doi:10.1074/jbc.273.6.3718

    Article  CAS  PubMed  Google Scholar 

  • Kondejewski LH, Jelokhani-Niaraki M, Farmer SW, Lix B, Kay CM, Sykes BD, Hancock RE, Hodges RS (1999) Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity. J Biol Chem 274(19):13181–13192

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Xia X, Xu L, Wang Y (2013) Design of hybrid beta-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity. Biomaterials 34(1):237–250. doi:10.1016/j.biomaterials.2012.09.032

    Article  CAS  PubMed  Google Scholar 

  • Lv Y, Wang J, Gao H, Wang Z, Dong N, Ma Q, Shan A (2014) Antimicrobial properties and membrane-active mechanism of a potential alpha-helical antimicrobial derived from cathelicidin PMAP-36. PLoS One 9(1):e86364. doi:10.1371/journal.pone.0086364

    Article  PubMed Central  PubMed  Google Scholar 

  • Ma QQ, Dong N, Shan AS, Lv YF, Li YZ, Chen ZH, Cheng BJ, Li ZY (2012) Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units. Amino Acids 43(6):2527–2536. doi:10.1007/s00726-012-1334-7

    Article  CAS  PubMed  Google Scholar 

  • Ma Z, Wei D, Yan P, Zhu X, Shan A, Bi Z (2015) Characterization of cell selectivity, physiological stability and endotoxin neutralization capabilities of α-helix-based peptide amphiphiles. Biomaterials 52:517–530

    Article  CAS  PubMed  Google Scholar 

  • Mor A, Nicolas P (1994) Isolation and structure of novel defensive peptides from frog skin. Eur J Biochem 219(1–2):145–154. doi:10.1111/j.1432-1033.1994.tb19924.x

    Article  CAS  PubMed  Google Scholar 

  • Pasupuleti M, Schmidtchen A, Malmsten M (2012) Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 32(2):143–171

    Article  CAS  PubMed  Google Scholar 

  • Reisser S, Strandberg E, Steinbrecher T, Ulrich AS (2014) 3D hydrophobic moment vectors as a tool to characterize the surface polarity of amphiphilic peptides. Biophys J 106(11):2385–2394. doi:10.1016/j.bpj.2014.04.020

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schmidtchen A, Pasupuleti M, Malmsten M (2014) Effect of hydrophobic modifications in antimicrobial peptides. Adv Colloid Interface Sci 205:265–274. doi:10.1016/j.cis.2013.06.009

    Article  CAS  PubMed  Google Scholar 

  • Scorciapino MA, Pirri G, Vargiu AV, Ruggerone P, Giuliani A, Casu M, Buerck J, Wadhwani P, Ulrich AS, Rinaldi AC (2012) A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056. Biophys J 102(5):1039–1048. doi:10.1016/j.bpj.2012.01.048

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shaykhiev R, Beisswenger C, Kändler K, Senske J, Püchner A, Damm T, Behr J, Bals R (2005) Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. Am J Phys Lung Cell Mol Physiol 289(5):L842–L848

    Article  CAS  Google Scholar 

  • Shin SY, Lee SH, Yang ST, Park EJ, Lee DG, Lee MK, Eom SH, Song WK, Kim Y, Hahm KS, Kim JI (2001) Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs. J Pept Res 58(6):504–514

    Article  CAS  PubMed  Google Scholar 

  • Takahashi D, Shukla SK, Prakash O, Zhang G (2010) Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity. Biochimie 92(9):1236–1241. doi:10.1016/j.biochi.2010.02.023

    Article  CAS  PubMed  Google Scholar 

  • Taniguchi M, Takahashi N, Takayanagi T, Ikeda A, Ishiyama Y, Saitoh E, Kato T, Ochiai A, Tanaka T (2014) Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide [CL(14-25)], a partial sequence of cyanate lyase from rice. Biopolymers 102(1):58–68. doi:10.1002/bip.22399

    Article  CAS  PubMed  Google Scholar 

  • Teixeira V, Feio MJ, Bastos M (2012) Role of lipids in the interaction of antimicrobial peptides with membranes. Prog Lipid Res 51(2):149–177. doi:10.1016/j.plipres.2011.12.005

    Article  CAS  PubMed  Google Scholar 

  • Tossi A, Sandri L, Giangaspero A (2000) Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55(1):4–30. doi:10.1002/1097-0282(2000)55:1<4:AID-BIP30>3.0.CO;2-M

    Article  CAS  PubMed  Google Scholar 

  • Tripathi S, Wang G, White M, Qi L, Taubenberger J, Hartshorn KL (2015) Antiviral activity of the human cathelicidin, LL-37, and derived peptides on seasonal and pandemic influenza A viruses. PLoS One 10(4):e0124706. doi:10.1371/journal.pone.0124706

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang G (2015) Improved methods for classification, prediction, and design of antimicrobial peptides. Methods Mol Biol 1268:43–66. doi:10.1007/978-1-4939-2285-7_3

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang K-r, Zhang B-z, Zhang W, Yan J-x, Li J, Wang R (2008) Antitumor effects, cell selectivity and structure–activity relationship of a novel antimicrobial peptide polybia-MPI. Peptides 29(6):963–968

    Article  CAS  PubMed  Google Scholar 

  • Wang G, Li X, Wang Z (2009) APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 37(suppl 1):D933–D937

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3(2):163–175

    Article  CAS  PubMed  Google Scholar 

  • Wieprecht T, Dathe M, Beyermann M, Krause E, Maloy WL, MacDonald DL, Bienert M (1997) Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes. Biochemistry 36(20):6124–6132

    Article  CAS  PubMed  Google Scholar 

  • Wiradharma N, Liu SQ, Yang YY (2012) Branched and 4-arm starlike alpha-helical peptide structures with enhanced antimicrobial potency and selectivity. Small 8(3):362–366. doi:10.1002/smll.201101672

    Article  CAS  PubMed  Google Scholar 

  • Wu C-SC, Ikeda K, Yang JT (1981) Ordered conformation of polypeptides and proteins in acidic dodecyl sulfate solution. Biochemistry 20(3):566–570

    Article  CAS  PubMed  Google Scholar 

  • Xi D, Teng D, Wang X, Mao R, Yang Y, Xiang W, Wang J (2013) Design, expression and characterization of the hybrid antimicrobial peptide LHP7, connected by a flexible linker, against Staphylococcus and Streptococcus. Process Biochem 48(3):453–461. doi:10.1016/j.procbio.2013.01.008

    Article  CAS  Google Scholar 

  • Xu W, Zhu X, Tan T, Li W, Shan A (2014) Design of embedded-hybrid antimicrobial peptides with enhanced cell selectivity and anti-biofilm activity. PLoS One 9(6):e98935. doi:10.1371/journal.pone.0098935

    Article  PubMed Central  PubMed  Google Scholar 

  • Xu L, Chou S, Wang J, Shao C, Li W, Zhu X, Shan A (2015) Antimicrobial activity and membrane-active mechanism of tryptophan zipper-like beta-hairpin antimicrobial peptides. Amino Acids. doi:10.1007/s00726-015-2029-7

    Google Scholar 

  • Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12(1):7–8. doi:10.1038/nmeth.3213

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55(1):27–55. doi:10.1124/pr.55.1.2

    Article  CAS  PubMed  Google Scholar 

  • Zhu X, Dong N, Wang Z, Ma Z, Zhang L, Ma Q, Shan A (2014) Design of imperfectly amphipathic α-helical antimicrobial peptides with enhanced cell selectivity. Acta Biomater 10(1):244–257

    Article  CAS  PubMed  Google Scholar 

  • Zhu X, Shan A, Ma Z, Xu W, Wang J, Chou S, Cheng B (2015) Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa. Antimicrob Agents Chemother 59(6):3008–3017. doi:10.1128/AAC.04830-14

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

We gratefully acknowledge the financial support from the National Science and Technology Support Program (BAD10B03), the National Natural Research Foundation of China (31272453, 31472104), the National Basic Research Program (Grant no. 2012CB124703), the China Agriculture Research System (CARS-36), the Program of Ministry of Education of China (20092325110009), and the Key Research Program of Heilongjiang Education Bureau (1254CGZH22, 11551z003).

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Authors and Affiliations

  1. Institute of Animal Nutrition, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin, 150030, People’s Republic of China

    Weizhong Li, Tingting Tan, Wei Xu, Lin Xu, Na Dong, Deying Ma & Anshan Shan

  2. Weifang University, Weifang, 261061, People’s Republic of China

    Weizhong Li

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  1. Weizhong Li
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  2. Tingting Tan
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  3. Wei Xu
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  4. Lin Xu
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  5. Na Dong
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  6. Deying Ma
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  7. Anshan Shan
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Correspondence to Deying Ma or Anshan Shan.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

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Li, W., Tan, T., Xu, W. et al. Rational design of mirror-like peptides with alanine regulation. Amino Acids 48, 403–417 (2016). https://doi.org/10.1007/s00726-015-2094-y

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