Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

  • Kimberly D. Saint Jean
  • Karlee D. Henderson
  • Christina L. Chrom
  • Louisa E. Abiuso
  • Lindsay M. Renn
  • Gregory A. Caputo
Article
  • 109 Downloads

Abstract

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.

Keywords

Antimicrobial peptides Fluorescence Lipid binding C18G 

Supplementary material

12602_2017_9345_MOESM1_ESM.docx (3.1 mb)
ESM 1(DOCX 3.07 mb)

References

  1. 1.
    Guilhelmelli F, Vilela N, Albuquerque P, Derengowski Lda S, Silva-Pereira I, Kyaw CM (2013) Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Front Microbiol 4:353. https://doi.org/10.3389/fmicb.2013.00353 CrossRefGoogle Scholar
  2. 2.
    Tavares LS, Silva CS, de Souza VC, da Silva VL, Diniz CG, Santos MO (2013) Strategies and molecular tools to fight antimicrobial resistance: resistome, transcriptome, and antimicrobial peptides. Front Microbiol 4:412. https://doi.org/10.3389/fmicb.2013.00412 CrossRefGoogle Scholar
  3. 3.
    Aquila M, Benedusi M, Dell'Orco D (2013) Biophysical characterization of antimicrobial peptides activity: from in vitro to ex vivo techniques. Curr Protein Pept Sci 14(7):607–616CrossRefGoogle Scholar
  4. 4.
    Diehnelt CW (2013) Peptide array based discovery of synthetic antimicrobial peptides. Front Microbiol 4:402. https://doi.org/10.3389/fmicb.2013.00402 CrossRefGoogle Scholar
  5. 5.
    Tossi A, Tarantino C, Romeo D (1997) Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity. Eur J Biochem 250(2):549–558. https://doi.org/10.1111/j.1432-1033.1997.0549a.x CrossRefGoogle Scholar
  6. 6.
    Henriksen JR, Etzerodt T, Gjetting T, Andresen TL (2014) Side chain hydrophobicity modulates therapeutic activity and membrane selectivity of antimicrobial peptide mastoparan-X. PLoS One 9(3):e91007. https://doi.org/10.1371/journal.pone.0091007 CrossRefGoogle Scholar
  7. 7.
    Sharma S, Sethi S, Prasad R, Samanta P, Rajwanshi A, Malhotra S, Sharma M (2011) Characterization of low molecular weight antimicrobial peptide from human female reproductive tract. Indian J Med Res 134(5):679–687. https://doi.org/10.4103/0971-5916.90996 CrossRefGoogle Scholar
  8. 8.
    Ganz T (2003) Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 3(9):710–720. https://doi.org/10.1038/nri1180 CrossRefGoogle Scholar
  9. 9.
    Manning AJ, Kuehn MJ (2011) Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol 11:258. https://doi.org/10.1186/1471-2180-11-258 CrossRefGoogle Scholar
  10. 10.
    Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55(1):27–55. https://doi.org/10.1124/pr.55.1.2 CrossRefGoogle Scholar
  11. 11.
    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. https://doi.org/10.1016/j.plipres.2011.12.005 CrossRefGoogle Scholar
  12. 12.
    Anaya-Lopez JL, Lopez-Meza JE, Ochoa-Zarzosa A (2013) Bacterial resistance to cationic antimicrobial peptides. Crit Rev Microbiol 39(2):180–195. https://doi.org/10.3109/1040841X.2012.699025 CrossRefGoogle Scholar
  13. 13.
    Fernandez DI, Gehman JD, Separovic F (2009) Membrane interactions of antimicrobial peptides from Australian frogs. Biochim Biophys Acta 1788(8):1630–1638. https://doi.org/10.1016/j.bbamem.2008.10.007 CrossRefGoogle Scholar
  14. 14.
    Dennison SR, Wallace J, Harris F, Phoenix DA (2005) Amphiphilic alpha-helical antimicrobial peptides and their structure/function relationships. Protein Pept Lett 12(1):31–39CrossRefGoogle Scholar
  15. 15.
    Dennison SR, Harris F, Bhatt T, Singh J, Phoenix DA (2009) The effect of C-terminal amidation on the efficacy and selectivity of antimicrobial and anticancer peptides. Mol Cell Biochem 332(1–2):43–50. https://doi.org/10.1007/s11010-009-0172-8 CrossRefGoogle Scholar
  16. 16.
    Saravanan R, Li X, Lim K, Mohanram H, Peng L, Mishra B, Basu A, Lee JM, Bhattacharjya S, Leong SS (2014) Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance, and biocompatibility. Biotechnol Bioeng 111(1):37–49. https://doi.org/10.1002/bit.25003 CrossRefGoogle Scholar
  17. 17.
    Wimley WC, Hristova K (2011) Antimicrobial peptides: successes, challenges and unanswered questions. J Membr Biol 239(1–2):27–34. https://doi.org/10.1007/s00232-011-9343-0 CrossRefGoogle Scholar
  18. 18.
    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. https://doi.org/10.1002/bip.22399 CrossRefGoogle Scholar
  19. 19.
    Almeida PF, Ladokhin AS, White SH (2012) Hydrogen-bond energetics drive helix formation in membrane interfaces. Biochim Biophys Acta 1818(2):178–182. https://doi.org/10.1016/j.bbamem.2011.07.019 CrossRefGoogle Scholar
  20. 20.
    Harris F, Dennison S, Phoenix DA (2006) The prediction of hydrophobicity gradients within membrane interactive protein alpha-helices using a novel graphical technique. Protein Pept Lett 13(6):595–600CrossRefGoogle Scholar
  21. 21.
    Liu LP, Deber CM (1999) Combining hydrophobicity and helicity: a novel approach to membrane protein structure prediction. Bioorg Med Chem 7(1):1–7CrossRefGoogle Scholar
  22. 22.
    Krishnakumar SS, London E (2007) Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes. J Mol Biol 374(3):671–687. https://doi.org/10.1016/j.jmb.2007.09.037 CrossRefGoogle Scholar
  23. 23.
    Dennison SR, Morton LH, Harris F, Phoenix DA (2008) The impact of membrane lipid composition on antimicrobial function of an alpha-helical peptide. Chem Phys Lipids 151(2):92–102. https://doi.org/10.1016/j.chemphyslip.2007.10.007 CrossRefGoogle Scholar
  24. 24.
    Shaw JE, Alattia JR, Verity JE, Prive GG, Yip CM (2006) Mechanisms of antimicrobial peptide action: studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy. J Struct Biol 154(1):42–58. https://doi.org/10.1016/j.jsb.2005.11.016 CrossRefGoogle Scholar
  25. 25.
    Li Y, Xiang Q, Zhang Q, Huang Y, Su Z (2012) Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides 37(2):207–215. https://doi.org/10.1016/j.peptides.2012.07.001 CrossRefGoogle Scholar
  26. 26.
    Chen B, Fan DQ, Zhu KX, Shan ZG, Chen FY, Hou L, Cai L, Wang KJ (2015) Mechanism study on a new antimicrobial peptide Sphistin derived from the N-terminus of crab histone H2A identified in haemolymphs of Scylla paramamosain. Fish Shellfish Immunol 47(2):833–846. https://doi.org/10.1016/j.fsi.2015.10.010 CrossRefGoogle Scholar
  27. 27.
    Bocchinfuso G, Bobone S, Mazzuca C, Palleschi A, Stella L (2011) Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides. Cell Mol Life Sci 68(13):2281–2301. https://doi.org/10.1007/s00018-011-0719-1 CrossRefGoogle Scholar
  28. 28.
    JX L, Damodaran K, Blazyk J, Lorigan GA (2005) Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes. Biochemistry 44(30):10208–10217. https://doi.org/10.1021/bi050730p CrossRefGoogle Scholar
  29. 29.
    Han F, Liu Y, Xie Y, Gao Y, Luan C, Wang Y (2011) Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties, cytotoxicity and mechanism of action. World J Microbiol Biotechnol 27(8):1847–1857. https://doi.org/10.1007/s11274-010-0643-9 CrossRefGoogle Scholar
  30. 30.
    Lohner K, Blondelle SE (2005) Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics. Comb Chem High Throughput Screen 8(3):241–256CrossRefGoogle Scholar
  31. 31.
    Darveau RP, Blake J, Seachord CL, Cosand WL, Cunningham MD, Cassiano-Clough L, Maloney G (1992) Peptides related to the carboxyl terminus of human platelet factor IV with antibacterial activity. J Clin Invest 90(2):447–455. https://doi.org/10.1172/JCI115880 CrossRefGoogle Scholar
  32. 32.
    Bader MW, Navarre WW, Shiau W, Nikaido H, Frye JG, McClelland M, Fang FC, Miller SI (2003) Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides. Mol Microbiol 50(1):219–230CrossRefGoogle Scholar
  33. 33.
    Peck-Miller KA, Darveau RP, Fell HP (1993) Identification of serum components that inhibit the tumoricidal activity of amphiphilic alpha helical peptides. Cancer Chemother Pharmacol 32(2):109–115CrossRefGoogle Scholar
  34. 34.
    Bader MW, Sanowar S, Daley ME, Schneider AR, Cho U, Xu W, Klevit RE, Le Moual H, Miller SI (2005) Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122(3):461–472. https://doi.org/10.1016/j.cell.2005.05.030 CrossRefGoogle Scholar
  35. 35.
    Peck-Miller KA, Blake J, Cosand WL, Darveau RP, Fell HP (1994) Structure-activity analysis of the antitumor and hemolytic properties of the amphiphilic alpha-helical peptide, C18G. Int J Pept Protein Res 44(2):143–151CrossRefGoogle Scholar
  36. 36.
    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. https://doi.org/10.1038/nprot.2007.521 CrossRefGoogle Scholar
  37. 37.
    Burman LG, Nordstrom K, Boman HG (1968) Resistance of Escherichia coli to penicillins. V. Physiological comparison of two isogenic strains, one with chromosomally and one with episomally mediated ampicillin resistance. J Bacteriol 96(2):438–446Google Scholar
  38. 38.
    Ridgway Z, Picciano AL, Gosavi PM, Moroz YS, Angevine CE, Chavis AE, Reiner JE, Korendovych IV, Caputo GA (2015) Functional characterization of a melittin analog containing a non-natural tryptophan analog. Biopolymers 104(4):384–394. https://doi.org/10.1002/bip.22624 CrossRefGoogle Scholar
  39. 39.
    Caputo GA, London E (2013) Analyzing transmembrane protein and hydrophobic helix topography by dual fluorescence quenching. Methods Mol Biol 974:279–295. https://doi.org/10.1007/978-1-62703-275-9_13 CrossRefGoogle Scholar
  40. 40.
    Caputo GA, London E (2003) Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes. Biochemistry 42(11):3265–3274. https://doi.org/10.1021/bi026696l CrossRefGoogle Scholar
  41. 41.
    D. Armstrong and R. Zidovetski, Helical Wheel Projections, http://rzlab.ucr.edu/scripts/wheel/wheel.cgi; Version: Id: wheel.pl,v 1.4 2009–10-20 21:23:36 don Exp
  42. 42.
    Choi J, Groisman EA (2016) Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence. Mol Microbiol 101(6):1024–1038. https://doi.org/10.1111/mmi.13439 CrossRefGoogle Scholar
  43. 43.
    Guina T, Yi EC, Wang H, Hackett M, Miller SI (2000) A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides. J Bacteriol 182(14):4077–4086CrossRefGoogle Scholar
  44. 44.
    Hovakeemian SG, Liu R, Gellman SH, Heerklotz H (2015) Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents. Soft Matter 11(34):6840–6851. https://doi.org/10.1039/c5sm01521a CrossRefGoogle Scholar
  45. 45.
    Fahie M, Romano FB, Chisholm C, Heuck AP, Zbinden M, Chen M (2013) A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A. J Biol Chem 288(43):31042–31051. https://doi.org/10.1074/jbc.M113.475350 CrossRefGoogle Scholar
  46. 46.
    Hanna SL, Huang JL, Swinton AJ, Caputo GA, Vaden TD (2017) Synergistic effects of polymyxin and ionic liquids on lipid vesicle membrane stability and aggregation. Biophys Chem 227:1–7. https://doi.org/10.1016/j.bpc.2017.05.002 CrossRefGoogle Scholar
  47. 47.
    Zelezetsky I, Tossi A (2006) Alpha-helical antimicrobial peptides—using a sequence template to guide structure-activity relationship studies. Biochim Biophys Acta 1758(9):1436–1449. https://doi.org/10.1016/j.bbamem.2006.03.021 CrossRefGoogle Scholar
  48. 48.
    Feder R, Dagan A, Mor A (2000) Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity. J Biol Chem 275(6):4230–4238CrossRefGoogle Scholar
  49. 49.
    Powers JP, Hancock RE (2003) The relationship between peptide structure and antibacterial activity. Peptides 24(11):1681–1691. https://doi.org/10.1016/j.peptides.2003.08.023 CrossRefGoogle Scholar
  50. 50.
    Kuroda K, Caputo GA, DeGrado WF (2009) The role of hydrophobicity in the antimicrobial and hemolytic activities of polymethacrylate derivatives. Chemistry 15(5):1123–1133. https://doi.org/10.1002/chem.200801523 CrossRefGoogle Scholar
  51. 51.
    Takahashi H, Caputo GA, Vemparala S, Kuroda K (2017) Synthetic random copolymers as a molecular platform to mimic host-defense antimicrobial peptides. Bioconjug Chem 28(5):1340–1350. https://doi.org/10.1021/acs.bioconjchem.7b00114 CrossRefGoogle Scholar
  52. 52.
    Kuroda K, Caputo GA (2013) Antimicrobial polymers as synthetic mimics of host-defense peptides. Wiley Interdiscip Rev Nanomed Nanobiotechnol 5(1):49–66. https://doi.org/10.1002/wnan.1199 CrossRefGoogle Scholar
  53. 53.
    Zhao G, London E (2006) An amino acid “transmembrane tendency” scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 15(8):1987–2001. https://doi.org/10.1110/ps.062286306 CrossRefGoogle Scholar
  54. 54.
    Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179(1):125–142CrossRefGoogle Scholar
  55. 55.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132CrossRefGoogle Scholar
  56. 56.
    Ojemalm K, Higuchi T, Lara P, Lindahl E, Suga H, von Heijne G (2016) Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids. Proc Natl Acad Sci U S A 113(38):10559–10564. https://doi.org/10.1073/pnas.1606776113 CrossRefGoogle Scholar
  57. 57.
    Gebhardt M, Henkes LM, Tayefeh S, Hertel B, Greiner T, Van Etten JL, Baumeister D, Cosentino C, Moroni A, Kast SM, Thiel G (2012) Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels. Biochemistry 51(28):5571–5579. https://doi.org/10.1021/bi3006016 CrossRefGoogle Scholar
  58. 58.
    Chamberlain AK, Lee Y, Kim S, Bowie JU (2004) Snorkeling preferences foster an amino acid composition bias in transmembrane helices. J Mol Biol 339(2):471–479. https://doi.org/10.1016/j.jmb.2004.03.072 CrossRefGoogle Scholar
  59. 59.
    Caputo GA, London E (2003) Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices. Biochemistry 42(11):3275–3285. https://doi.org/10.1021/bi026697d CrossRefGoogle Scholar
  60. 60.
    Palermo EF, Vemparala S, Kuroda K (2012) Cationic spacer arm design strategy for control of antimicrobial activity and conformation of amphiphilic methacrylate random copolymers. Biomacromolecules 13(5):1632–1641. https://doi.org/10.1021/bm300342u CrossRefGoogle Scholar
  61. 61.
    Morton LA, Yang H, Saludes JP, Fiorini Z, Beninson L, Chapman ER, Fleshner M, Xue D, Yin H (2013) MARCKS-ED peptide as a curvature and lipid sensor. ACS Chem Biol 8(1):218–225. https://doi.org/10.1021/cb300429e CrossRefGoogle Scholar
  62. 62.
    Vanni S, Vamparys L, Gautier R, Drin G, Etchebest C, Fuchs PF, Antonny B (2013) Amphipathic lipid packing sensor motifs: probing bilayer defects with hydrophobic residues. Biophys J 104(3):575–584. https://doi.org/10.1016/j.bpj.2012.11.3837 CrossRefGoogle Scholar
  63. 63.
    de Jesus AJ, White OR, Flynn AD, Yin H (2016) Determinants of curvature-sensing behavior for MARCKS-fragment peptides. Biophys J 110(9):1980–1992. https://doi.org/10.1016/j.bpj.2016.04.007 CrossRefGoogle Scholar
  64. 64.
    Mensa B, Kim YH, Choi S, Scott R, Caputo GA, DeGrado WF (2011) Antibacterial mechanism of action of arylamide foldamers. Antimicrob Agents Chemother 55(11):5043–5053. https://doi.org/10.1128/AAC.05009-11 CrossRefGoogle Scholar
  65. 65.
    Choi S, Isaacs A, Clements D, Liu D, Kim H, Scott RW, Winkler JD, DeGrado WF (2009) De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers. Proc Natl Acad Sci U S A 106(17):6968–6973. https://doi.org/10.1073/pnas.0811818106 CrossRefGoogle Scholar
  66. 66.
    Su Y, DeGrado WF, Hong M (2010) Orientation, dynamics, and lipid interaction of an antimicrobial arylamide investigated by 19F and 31P solid-state NMR spectroscopy. J Am Chem Soc 132(26):9197–9205. https://doi.org/10.1021/ja103658h CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Kimberly D. Saint Jean
    • 1
  • Karlee D. Henderson
    • 1
  • Christina L. Chrom
    • 1
  • Louisa E. Abiuso
    • 1
  • Lindsay M. Renn
    • 1
  • Gregory A. Caputo
    • 1
    • 2
  1. 1.Department of Chemistry and BiochemistryRowan UniversityGlassboroUSA
  2. 2.Department of Molecular and Cellular BiosciencesRowan UniversityGlassboroUSA

Personalised recommendations