Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin

Abstract

NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, ␣-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

“…below MIC) can be attributed, at least initially, to electrostatics. Several studies investigating the interaction of cationic AMPs with model bacterial membranes have shown that an overcompensation in zeta potential at high peptide concentrations is associated with membrane insertion via hydrophobic interactions (22)(23)(24). A similar trend was also reported for the interaction of the cationic peptide rBPI 21 with LPS aggregates (24).…”

(Lipo)polysaccharide interactions of antimicrobial peptides

“…Similarly, Andrä et al reported on electrostatically driven LPS binding of NK-2, but also that hydrophobic interactions are important for LPS neutralization in this system (13). In line with the latter, Rosenfeldt et al found adsorption to LPS-containing liposomes to increase with increasing length of the hydrophobic conjugation of K/L peptides (14).…”

Ultrashort Antimicrobial Peptides with Antiendotoxin Properties

“…Hydrophobicity has been known as a key factor in the development of effective LPS-neutralizing AMPs (15,16,38). Studies of NK-2 and N-acylated lactoferricin-derived LF11 indicated that neutralization of the LPS surface charges is a requirement but is not sufficient for LPS detoxification (15).…”