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Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides

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Antimicrobial Peptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1548))

Abstract

Antimicrobial peptides (AMPs) are currently intensely studied because of their potential as new bactericidal and bacteriostatic agents. The mechanism of action of numerous AMPs involves the permeabilization of bacterial membranes. Several methods have been developed to study peptide–membrane interactions; in particular optical spectroscopy methods are widely used. The intrinsic fluorescence properties of the Trp indole ring in Trp-containing AMPs can be exploited by measuring the fluorescence blue shift and acrylamide-induced fluorescence quenching. One important aspect of such studies is the use of distinct models of the bacterial membrane, in most cases large unilamellar vesicles (LUVs) with different, yet well-defined, phospholipid compositions. Deploying LUVs that are preloaded with fluorescent dyes, such as calcein, also allows for the study of vesicle permeabilization by AMPs. In addition, experiments using genetically engineered live Escherichia coli cells can be used to distinguish between the effects of AMPs on the outer and inner membranes of gram-negative bacteria. In combination, these methods can provide a detailed insight into the mode of action of AMPs.

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References

  1. Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29:464–472

    Article  CAS  PubMed  Google Scholar 

  2. Matsuzaki K (2009) Control of cell selectivity of antimicrobial peptides. Biochim Biophys Acta 1788:1687–1692

    Article  CAS  PubMed  Google Scholar 

  3. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55

    Article  CAS  PubMed  Google Scholar 

  4. Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28

    Article  CAS  PubMed  Google Scholar 

  5. Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19:491–511

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66:236–248

    Article  CAS  PubMed  Google Scholar 

  7. Lohner K, Sevcsik E, Pabst G (2008) Liposome-based biomembrane mimetic systems: implications for lipid-peptide interactions. In: Advances in planar lipid bilayers and liposomes. Elsevier, Amsterdam, pp 103–132

    Google Scholar 

  8. Riedl S, Rinner B, Asslaber M et al (2011) In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim Biophys Acta 1808:2638–2645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Abraham T, Lewis RN, Hodges RS et al (2005) Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes. Biochemistry 44:11279–11285

    Article  CAS  PubMed  Google Scholar 

  10. Lohner K, Prenner EJ (1999) Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems. Biochim Biophys Acta 1462:141–156

    Article  CAS  PubMed  Google Scholar 

  11. Andrushchenko VV, Aarabi MH, Nguyen LT et al (2008) Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes. Biochim Biophys Acta 1778:1004–1014

    Article  CAS  PubMed  Google Scholar 

  12. Andrushchenko VV, Vogel HJ, Prenner EJ (2006) Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy. Biochim Biophys Acta 1758:1596–1608

    Article  CAS  PubMed  Google Scholar 

  13. Andrushchenko VV, Vogel HJ, Prenner EJ (2007) Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry. Biochim Biophys Acta 1768:2447–2458

    Article  CAS  PubMed  Google Scholar 

  14. Pate M, Blazyk J (2008) Methods for assessing the structure and function of cationic antimicrobial peptides. In: Champney WS (ed) New antibiotics targets. Humana Press, Totowa, NJ, pp 155–173

    Chapter  Google Scholar 

  15. Lakowicz JR (2006) Protein fluorescence. In: Lakowicz JR (ed) Principles of fluorescence spectroscopy. Springer, New York, NY, pp 529–575

    Chapter  Google Scholar 

  16. Lakowicz JR (2006) Quenching of fluorescence. In: Lakowicz JR (ed) Principles of fluorescence spectroscopy. Springer, New York, NY, pp 277–330

    Chapter  Google Scholar 

  17. Eftink MR, Ghiron CA (1981) Fluorescence quenching studies with proteins. Anal Biochem 114:199–227

    Article  CAS  PubMed  Google Scholar 

  18. Shashidhara KS, Gaikwad SM (2007) Fluorescence quenching and time-resolved fluorescence studies of alpha-mannosidase from Aspergillus fischeri (NCIM 508). J Fluoresc 17:599–605

    Article  CAS  PubMed  Google Scholar 

  19. Hristova K, Selsted ME, White SH (1997) Critical role of lipid composition in membrane permeabilization by rabbit neutrophil defensins. J Biol Chem 272:24224–24233

    Article  CAS  PubMed  Google Scholar 

  20. Wimley WC, Selsted ME, White SH (1994) Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores. Protein Sci 3:1362–1373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Stella L, Mazzuca C, Venanzi M et al (2004) Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV. Biophys J 86:936–945

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ambroggio EE, Separovic F, Bowie JH et al (2005) Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein. Biophys J 89:1874–1881

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Belokoneva OS, Satake H, Mal’tseva EL et al (2004) Pore formation of phospholipid membranes by the action of two hemolytic arachnid peptides of different size. Biochim Biophys Acta 1664:182–188

    Article  CAS  PubMed  Google Scholar 

  24. Lehrer RI, Barton A, Ganz T (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol Methods 108:153–158

    Article  CAS  PubMed  Google Scholar 

  25. Chongsiriwatana NP, Barron AE (2010) Comparing bacterial membrane interactions of antimicrobial peptides. In: Giuliani A, Rinaldi AC (eds) Antimicrobial peptides methods and protocols. Humana Press, Totowa, NJ, pp 171–182

    Chapter  Google Scholar 

  26. Epand RF, Pollard JE, Wright JO et al (2010) Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins. Antimicrob Agents Chemother 54:3708–3713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Eriksson M, Nielsen PE, Good L (2002) Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli. J Biol Chem 277:7144–7147

    Article  CAS  PubMed  Google Scholar 

  28. Kaback HR, Sahin-tóth M, Weinglass AB (2001) The kamikaze approach to membrane transport. Nat Rev Mol Cell Biol 2:610–620

    Article  CAS  PubMed  Google Scholar 

  29. Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Avanti Polar Lipids Inc. (2015) Preparation of liposomes

    Google Scholar 

  31. Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168

    Article  CAS  PubMed  Google Scholar 

  32. Ames BN, Neufeld EF, Ginsberg V (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8:115–118

    Article  CAS  Google Scholar 

  33. Lehrer R, Barton A, Daher KA et al (1989) Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest 84:553–561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Andrushchenko VV, Vogel HJ, Prenner EJ (2007) Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides. J Pept Sci 13:37–43

    Article  CAS  PubMed  Google Scholar 

  35. De Kroon AIPM, Soekarjo MW, De Gier J et al (1990) The role of charge and hydrophobicity in peptide-lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers. Biochemistry 29:8229–8240

    Article  PubMed  Google Scholar 

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Acknowledgments

Research on antimicrobial peptides has been funded by a grant from the “Novel alternatives to antibiotics” program from the Canadian Institutes of Health Research.

E. coli ML35p was kindly provided by Dr. Robert Lehrer from the David Geffen School of Medicine at UCLA, Los Angeles, CA.

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Authors and Affiliations

  1. Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada, T2N 1N4

    Mauricio Arias & Hans J. Vogel

Authors
  1. Mauricio Arias
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  2. Hans J. Vogel
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Corresponding author

Correspondence to Hans J. Vogel .

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Editors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Paul R. Hansen

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Arias, M., Vogel, H.J. (2017). Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides. In: Hansen, P. (eds) Antimicrobial Peptides. Methods in Molecular Biology, vol 1548. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6737-7_10

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  • DOI: https://doi.org/10.1007/978-1-4939-6737-7_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6735-3

  • Online ISBN: 978-1-4939-6737-7

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