Investigation of Antimicrobial and Lipid Perturbing Properties of Lactoferrin Peptides

Abstract

An increase in bacterial resistance to conventional antibiotics has led to an intense search for alternative treatments. Lactoferricin B (FKCRRWQWRMKKLGAPSITCVRRAF), a peptide with potent broad-spectrum antimicrobial activity, is released by pepsin from bovine lactoferrin. A smaller amidated peptide, (LfB6; RRWQWR-NH2), has been identified as having the core antimicrobial activity (Tomita et al. (1994) Acta Paediatr Jpn. 36:585-91). The exact mechanism by which antimicrobial peptides interact with bacterial cell membranes is not well understood, but it is proposed to depend on lipid composition. In contrast to mammalian membranes which are comprised primarily of neutral lipids, bacterial membranes contain a significant (∼20-25%) fraction of negatively charged lipids. In the case of LfB6, the presence of two tryptophans (W; Trp) and three arginines (R) are thought to promote selective interaction with bacterial cell membranes. Recently, we have shown that the antimicrobial activity of LfB6 peptides is increased by N-acylation and Trp-methylation (Greathouse et al. (2008) J. Pept. Sci. 14:1103-1110).To ascertain whether LfB peptides perturb lipids with negatively charged head groups, macroscopically aligned bilayers composed of lipids to mimic bacterial cell membranes have been prepared in the absence and presence of peptide. The samples are composed of neutral (POPE) and anionic (POPG) lipids (3:1), containing either sn-1 chain perdeuterated POPE-d31 or POPG-d31. The effects of LfB6 and amino acylated LfB peptides on lipid dynamics are being investigated by solid-state deuterium NMR spectroscopy and differential scanning calorimetry. The 2H NMR spectra reveal that the addition of LfB6 results in slight but specific changes in the outer quadrupolar splittings, which result from the methylene groups closest to the lipid head groups. Antimicrobial assays against S. aureus and E. coli demonstrate that the activity of N-acylated LfB peptides increases with acyl chain length.