ABSTRACT
Human neutrophilpeptide 1 (HNP1) is a human defensin with antimicrobial activity against different bacteria (both Gram-positive and negative), fungi, and viruses. HNP1 is stored in the cytoplasmic azurophilic granules of neutrophils. To elucidate the mode of action of this antimicrobial peptide, studies based on its lipid selectivity were carried out. Large unilamellar vesicles with different lipid compositions were used as biomembranes model systems (mammal, fungal, and bacterial models). Changes on the intrinsic fluorescence of HNP1 upon membrane binding/insertion show that HNP1 has quite distinct preferences for mammalian and fungal membrane model systems. HNP1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a higher partition for ergosterol-containing membranes (as fungal membranes) and lower interaction with cholesterol-containing membranes (as in mammalian cells). These results reveal that lipid selectivity is a determinant step for HNP1 action. Fluorescence quenching data obtained using acrylamide indicate that HNP1 interacts with membranes without a full insertion in the lipid bilayer. Generalized polarization of laurdan indicates a change in membrane fluidity in the presence of HNP1 for POPC membranes but not for ergosterol-enriched membranes.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/metabolism , Lipids/chemistry , alpha-Defensins/chemistry , alpha-Defensins/metabolism , Cholesterol/chemistry , Ergosterol/chemistry , Humans , Lipid Bilayers/chemistry , Membrane Fluidity , Protein Binding , Unilamellar Liposomes/chemistryABSTRACT
Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.
