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, alpha-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.

FIG. 1.

Schematic structures of S. enterica LPSs used in this study. The molecular masses of lipid A, LPS Re, LPS Ra, and S-form LPS are 2.3, 2.65, 4.3, and 10 kDa, respectively. GlcNac, N-acetyl-d-glucosamine; Glc, d-glucose; Gal, d-galactose; Hep, l-glycero-d-manno-heptose; Kdo, 3-deoxy-d-manno-oct-2-ulopyranosonic acid; GlcN, d-glucosamine; P, phosphate; C, carboxylate.

FIG. 2.

Primary structure of the NK-2 peptide and tertiary structure of its parental protein, NK-lysin (in two different views). The nuclear magnetic resonance imaging structure of NK-lysin (32) was taken from the protein data bank (1NKL.pdb) and was displayed by use of the Rasmol program. NK-2 spans the third and the fourth helical domains (in gray) of NK-lysin, including the (mostly positively charged) adjacent amino acid residues. Lysine and arginine residues within the NK-2 structure are darkened. Note that the sequence of NK-2 differs from that of NK-lysin by the exchange of the underlined amino acid residues (37).

FIG. 3.

Activity of NK-2 against S. enterica strains with various LPS carbohydrate structures: strain R595 (LPS Re; circles), strain R60 (LPS Ra; squares), and wild type (S-form LPS; triangles). The data shown were taken from a representative experiment. Inhibitory concentrations obtained from two independent experiments, each performed in duplicate, were exactly within 1 dilution step. OD620, optical density at 620 nm.

FIG. 4.

Concentration-dependent inhibition of the biological activities of various LPSs by NK-2. LPS Re (circles), LPS Ra (squares), and S-form LPS (triangles) were incubated with NK-2 at the indicated peptide:LPS molar ratios and were used to activate human MNCs. As a marker of LPS-induced cell activation, the level of production of the cytokine TNF-α was determined by ELISA. Similar results were observed when macrophages instead of MNCs were used (data not shown).

FIG. 5.

Influence of NK-2 on the supramolecular structure of lipid A aggregates. Small-angle X-ray diffraction patterns were determined for pure lipid A (A) and a lipid A-NK-2 mixture (molar ratio, 1 to 0.01) (B) at the indicated temperatures.

FIG. 6.

Zeta potentials of LPS preparations (circles, LPS Re; squares, LPS Ra; triangles, S-form LPS) on the basis of different NK-2:LPS molar ratios. Zeta potentials were determined from electrophoretic mobilities by laser Doppler anemometry (A). For clarity, the zeta potential of S-form LPS is shown on an expanded scale (B).

FIG. 7.

Enthalpy change (ΔH) of the peptide-LPS (circles, Re LPS; squares, Ra LPS; triangles, S-form LPS) binding reaction on the basis of various NK-2:LPS molar ratios. The enthalpy changes were determined from calorimetric titration curves. Positive and negative enthalpy change values indicate endothermic and exothermic reactions, respectively.