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Macromolecule Biosynthesis Assay and Fluorescence Spectroscopy Methods to Explore Antimicrobial Peptide Mode(s) of Action

  • Bimal Jana
  • Kristin Renee Baker
  • Luca Guardabassi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1548)

Abstract

Antimicrobial peptides (AMPs) are viable alternatives to the currently available antimicrobials, and numerous studies have investigated their possible use as therapeutic agents for specific clinical applications. AMPs are a diverse class of antimicrobials that often act upon the bacterial cell membrane but may exhibit additional modes of action. Identification of the multiple modes of action requires a comprehensive study at subinhibitory concentrations and careful data analysis since additional modes of action can be eclipsed by AMP action on the cell membrane.

Techniques that measure the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods to identify AMP mode(s) of action. Detailed protocols designed to measure inhibition of DNA, RNA, protein, and cell wall synthesis or membrane de-energization are presented and discussed for optimal application of these two techniques as well as to enable accurate interpretation of the experimental findings.

Key words

Antimicrobial peptide Mode(s) of action Macromolecule biosynthesis Fluorescence spectroscopy Proton motive force Membrane de-energization/depolarization 

Notes

Acknowledgments

The work reported here has been supported by grants from the University of Copenhagen Centre for Control of Antimicrobial Resistance (UC-Care) and Zoetis.

References

  1. 1.
    European Centre for Disease Prevention and Control (2015) Antimicrobial resistance surveillance in Europe 2014. Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). ECDC, Stockholm. doi: 10.2900/23549 Google Scholar
  2. 2.
    Tavares LS, Silva CSF, 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 4:1–11CrossRefGoogle Scholar
  3. 3.
    Nizet V (2006) Antimicrobial peptide resistance mechanisms of human bacterial pathogens. Curr Issues Mol Biol 8:11–26PubMedGoogle Scholar
  4. 4.
    Guilhelmelli F, Vilela N, Albuquerque P, Derengowski L d S, Silva-Pereira I, Kyaw CM (2013) Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Front Microbiol 4:1–12CrossRefGoogle Scholar
  5. 5.
    Marcellini L, Giammatteo M, Aimola P, Mangoni ML (2010) Fluorescence and electron microscopy methods for exploring antimicrobial peptides mode(s) of action. Methods Mol Biol 618:249–266CrossRefPubMedGoogle Scholar
  6. 6.
    Wimley WC (2015) Determining the effects of membrane-interacting peptides on membrane integrity. In: Cell-penetrating peptides: methods and protocols. Humana Press, New York, NY, pp 89–106CrossRefGoogle Scholar
  7. 7.
    Hancock REW, Rozek A (2002) Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett 206:143–149CrossRefPubMedGoogle Scholar
  8. 8.
    Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman V, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K (2015) A new antibiotic kills pathogens without detectable resistance. Nature 517:455–459CrossRefPubMedGoogle Scholar
  9. 9.
    Weigel PH, Englund PT (1976) Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers. J Biol Chem 252:1148–1155Google Scholar
  10. 10.
    Farha MA, Verschoor CP, Bowdish D, Brown ED (2013) Collapsing the proton motive force to identify synergistic combinations against Staphylococcus aureus. Chem Biol 20:1168–1178CrossRefPubMedGoogle Scholar
  11. 11.
    Münch D, Müller A, Schneider T, Kohl B, Wenzel M, Bandow JE, Maffioli S, Sosio M, Donadio S, Wimmer R, Sahl HG (2014) The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions. J Biol Chem 289:12063–12076CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bulathsinghala CM, Jana B, Baker KR, Postle K (2013) ExbB cytoplasmic loop deletions cause immediate, proton motive force-independent growth arrest. J Bacteriol 195:4580–4591CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Bimal Jana
    • 1
  • Kristin Renee Baker
    • 1
  • Luca Guardabassi
    • 1
    • 2
  1. 1.Department of Biomedical SciencesRoss University School of Veterinary MedicineBasseterreSt Kitts and Nevis
  2. 2.Department of Veterinary Disease Biology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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