Structure and association of human lactoferrin peptides with Escherichia coli lipopolysaccharide.
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 beta-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.
Chapple
Daniel S
DS
Metalloprotein Research Group, Randall Centre for Molecular Cell Biology, King's College, London, United Kingdom.
Hussain
Rohanah
R
Joannou
Christopher L
CL
Hancock
Robert E W
RE
Odell
Edward
E
Evans
Robert W
RW
Siligardi
Giuliano
G
D023181:Antimicrobial Cationic Peptides; D002942:Circular Dichroism; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins; D007781:Lactoferrin / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry*; D008566:Membranes / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D010539:Permeability; D013050:Spectrometry, Fluorescence
D023181:Antimicrobial Cationic Peptides; C406269:HLP-2 polypeptide; C487654:HLP-6 peptide; D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D010455:Peptides; D007781:Lactoferrin