Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

High-throughput generation of small antibacterial peptides with improved activity

Abstract

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12-amino-acid peptides based on a linearized variant (RLARIVVIRVAR-NH2) of the bovine peptide bactenecin was screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, we designed optimized 12-mer peptides showing broad spectrum activities with minimal inhibitory concentrations (MIC) as low as 0.5 μg/ml against Escherichia coli. Similarly, we generated an 8-mer substituted peptide that showed broad spectrum activity, with an MIC of 2 μg/ml, against E. coli and Staphylococcus aureus.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Comparison of a 'classical' killing curve and a lux killing assay of Pseudomonas aeruginosa Bac2A.
Figure 2: Complete substitution analysis for Bac2A.

Similar content being viewed by others

References

  1. Hancock, R.E.W. Cationic peptides: effectors in innate immunity & novel antimicrobials. Lancet Infect. Dis. 1, 156–164 (2001).

    Article  CAS  Google Scholar 

  2. Gough, M., Hancock, R.E.W. & Kelly, N.M. Anti-endotoxic potential of cationic peptide antimicrobials. Infect. Immun. 64, 4922–4927 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Hancock, R.E.W. & Rozek, A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol. Lett. 206, 143–149 (2002).

    Article  CAS  Google Scholar 

  4. Romeo, D., Skerlavaj, B., Bolognesi, M. & Gennaro, R. Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils. J. Biol. Chem. 263, 9573–9575 (1988).

    CAS  PubMed  Google Scholar 

  5. Wu, M. & Hancock, R.E.W. Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide. Antimicrob. Agents Chemother. 43, 1274–1276 (1999).

    Article  CAS  Google Scholar 

  6. Frank, R. Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217–9232 (1992).

    Article  CAS  Google Scholar 

  7. Kramer, A. et al. Molecular basis for the binding promiscuity of an anti-p24 (HIV-1) monoclonal antibody. Cell 91, 799–809 (1997).

    Article  CAS  Google Scholar 

  8. Hilpert, K., Hansen, G., Wessner, H., Schneider-Mergener, J. & Hohne, W. Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis. J. Biochem. 128, 1051–1057 (2000).

    Article  CAS  Google Scholar 

  9. Kramer, A. & Schneider-Mergener, J. Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods Mol. Biol. 87, 25–39 (1998).

    CAS  PubMed  Google Scholar 

  10. Lewenza, S. et al. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res. 15, 583–589 (2005).

    Article  CAS  Google Scholar 

  11. Blondelle, S.E. & Houghten, R.A. Novel antimicrobial compounds identified using synthetic combinatorial library technology. Trends Biotechnol. 14, 60–65 (1996).

    Article  CAS  Google Scholar 

  12. Kramer, A. et al. Combinatorial cellulose-bound peptide libraries: screening tool for the identification of peptides that bind ligands with predefined specificity. Comp. Meth. Enzymol. 6, 388–395 (1994).

    Article  CAS  Google Scholar 

  13. Kamradt, T. & Volkmer-Engert, R. Cross-reactivity of T lymphocytes in infection and autoimmunity. Mol. Divers. 8, 271–280 (2004).

    Article  CAS  Google Scholar 

  14. Wu, M. & Hancock, R.E.W. Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J. Biol. Chem. 274, 29–35 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

R.E.W.H. holds a Canada Research Chair. We thank the Applied Food and Materials Network of Centers of Excellence for funding and Oreola Donini for her technical support in demonstrating how to determine the IC50 values.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Robert E W Hancock.

Ethics declarations

Competing interests

The peptide sequences and research results described in this paper are part of a US provisional patent application by the University of British Columbia.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hilpert, K., Volkmer-Engert, R., Walter, T. et al. High-throughput generation of small antibacterial peptides with improved activity. Nat Biotechnol 23, 1008–1012 (2005). https://doi.org/10.1038/nbt1113

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1113

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing