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Antimicrobial Agents and Chemotherapy, June 2004, p. 2190-2198, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2190-2198.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Structure and Association of Human Lactoferrin Peptides with Escherichia coli Lipopolysaccharide

Daniel S. Chapple,1 Rohanah Hussain,2 Christopher L. Joannou,1 Robert E. W. Hancock,3 Edward Odell,4 Robert W. Evans,1 and Giuliano Siligardi2*

Metalloprotein Research Group, Randall Centre for Molecular Cell Biology, Guy's Campus, King's College London, London SE1 1UL,1 Drug Interaction Activity Screening Unit, Department of Pharmacy, King's College London, London SE1 8WA,2 Department of Oral Medicine and Pathology, King's College London, Guys Hospital, London SE1 9RT, United Kingdom,4 Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada3

Received 30 June 2003/ Returned for modification 25 November 2003/ Accepted 10 February 2004

An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity. The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays. These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6. Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement. Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E. coli LPS. In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a ß-strand conformation rather than an {alpha}-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy. Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity.

* Corresponding author. Mailing address: Department of Pharmacy, King's College London, Franklin Wilkins Building, 150 Stanford St., London SE1 8WA, United Kingdom. Phone: 44 20 7848 4786. Fax: 44 20 7848 4940. E-mail: giuliano.siligardi{at}kcl.ac.uk.

Antimicrobial Agents and Chemotherapy, June 2004, p. 2190-2198, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2190-2198.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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