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High-Throughput Screening for Antimicrobial Peptides Using the SPOT Technique

  • Kai Hilpert
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 618)

Abstract

The SPOT technique provides a fast, cost-efficient, and highly parallel method to synthesize peptide arrays on cellulose. Peptides synthesized on cellulose can be easily cleaved from the support and used directly in a screening assay for antimicrobial activity. Depending on the equipment, the synthesis and the screening can be performed in a medium- or high-throughput manner. High-sensitivity screening is achieved using a bacterial strain (e.g., Pseudomonas aeruginosa H1001) in which a luminescence-encoding gene cassette has been introduced. The intensity of light produced is directly dependent on the energy level of the bacteria. This screening supports the development of new drugs against multidrug-resistant bacteria.

Key words

Screening antimicrobial peptides bacteria yeast human pathogens Pseudomonas aeruginosa luminescence SPOT synthesis peptide array 

References

  1. GERMAP (2008), Antibiotika-Resistenz und-Verbrauch, Verlag: Antiinfectives Intelligence, Gesellschaft für klinisch-mikrobiologische Forschung und Kommunikation mbH, Rheinbach, Germany.Google Scholar
  2. 2.
    Rossolini, G. M. and Mantengoli, E. (2008) Antimicrobial resistance in Europe and its potential impact on empirical therapy. Clin. Microbiol. Infect. 14, 2–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Mullen, L. M., Nair, S. P., Ward, J. M., Rycroft, A. N., and Henderson, B. (2006) Phage display in the study of infectious diseases. Trends Microbiol. 14, 141–147.PubMedCrossRefGoogle Scholar
  4. 4.
    Brogden, K. A. (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250.PubMedCrossRefGoogle Scholar
  5. 5.
    Cherkasov, A., Hilpert, K., Jenssen, H., Fjell, C. D., Waldbrook, M., Mullaly, S. C., Volkmer, R., and Hancock, R. E. W. (2008) Use of artificial intelligence in the design of small peptide antibiotics effective against a broad spectrum of highly antibiotic-resistant superbugs. ACS Chem. Biol. 4, 65–74.CrossRefGoogle Scholar
  6. 6.
    Fjell, C. D., Jenssen, H., Hilpert, K., Cheung, W., Panté, N., Hancock, R. E. W., and Cherkasov, A. (2009) Identification of novel antibacterial peptides by chemoinformatics and machine learning. J. Med. Chem., 52(7), 2006–2015.PubMedCrossRefGoogle Scholar
  7. 7.
    Hilpert, K., Winkler, D. F. H., and Hancock, R. E. W. (2007) Cellulose-bound peptide arrays: preparation and applications. Biotechnol. Genet. Eng. Rev. 24, 31–106.Google Scholar
  8. 8.
    Winkler, D. F. H. and Hilpert, K. (2009) Synthesis of antimicrobial peptides using the SPOT technique. Methods Mol. Med., in press.Google Scholar
  9. 9.
    Lewenza, S., Falsafi, R. K., Winsor, G., Gooderham, W. J., McPhee, J. B., Brinkman, F. S. L., and Hancock, R. E. W. (2005) Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res. 15, 583–589.Google Scholar
  10. 10.
    Hilpert, K., Volkmer-Engert, R., Walter, T., and Hancock, R. E. W. (2005) High-throughput generation of small antibacterial peptides with improved activity. Nat. Biotechnol. 23, 1008–1012.PubMedCrossRefGoogle Scholar
  11. 11.
    Hilpert, K., Elliott, M. R., Volkmer-Engert, R., Henklein, P., Donini, O., Zhou, Q., Winkler, D. F. H., and Hancock, R. E. W. (2006) Sequence requirements and an optimization strategy for short antimicrobial peptides. Chem. Biol. 13, 1101–1117.PubMedCrossRefGoogle Scholar
  12. 12.
    Mikut, R. (2009) Computer-based analysis, visualization, and interpretation of antimicrobial peptide activities. Methods Mol. Biol. 618, 287–299.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Kai Hilpert
    • 1
  1. 1.Institute of Biological Interfaces – IBG 2, KIT (Karlsruhe Institute of Technology), Forschungszentrum KarlsruheEggenstein-LeopoldshafenGermany

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