Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide

Abstract

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides.

Figure 1. Antibacterial activity of lactoferricin-derived peptides LF11 (○) and lauryl-LF11 (●), compared to synthetic bee venom melittin (△) against S. enterica serovar Minnesota strains with variation in the LPS carbohydrate structures

(A) Strain R595 (LPS Re); (B) strain R60 (LPS Ra); (C) wild-type (S-form LPS).

Figure 2. Concentration-dependent inhibition of the biological activity of various LPS by synthetic peptides

LF11 (○) and lauryl-LF11 (●) were incubated with 0.5 ng/ml LPS Re (A), 0.5 ng/ml LPS Ra (B) or 1 ng/ml S-form LPS (C) at the indicated peptide concentrations. The mixtures were used to activate human MNCs. As a marker of LPS-induced cell activation, the production of the cytokine TNFα was determined by ELISA.

Figure 3. Displacement of ⁴⁵Ca from LPS Re monolayers by cationic peptides

LF11 (○) and lauryl-LF11 (●) were injected in the subphase under an LPS Re monolayer saturated with ⁴⁵Ca. Peptide binding resulted in the displacement of calcium accompanied by a decrease in radioactivity.

Figure 4. Dependence of zeta potential of LPS preparations (A, LPS Re; B, LPS Ra) on different peptide/LPS molar ratios

LF11 (○) and lauryl-LF11 (●) were titrated against LPS suspensions and the zeta potential was determined from the electrophoretic mobility analysed by laser-Doppler anemometry. Note the charge overcompensation for lauryl-LF11.

Figure 5. Enthalpy change of the peptide-LPS (○, LPS Re; ●, LPS Ra; □, S-form LPS) binding reaction as a function of various peptide/LPS molar ratios from calorimetric titration curves

(A) LF11; (B) lauryl-LF11. Positive and negative ΔH values indicate endothermic and exothermic reactions respectively.

Figure 6. Phase-transition behaviour of LPS Re and LPS Ra in the presence of LF11 and lauryl-LF11, monitored over the peak position of the symmetric stretching vibration of the methylene groups of LPS acyl chains at different LPS/peptide molar ratios

In the gel (β) phase of the acyl chains, the peak lies at approx. 2850 cm⁻¹; in the liquid-crystalline (α) phase, the peak lies at approx. 2852.5 cm⁻¹.

Figure 7. Heat-capacity curves, Cp_diff, against temperature of various LPS Ra/peptide mixtures from DSC scans

(A) LF11; (B) lauryl-LF11.

Figure 8. Small-angle X-ray diffraction patterns of lipid A from S. enterica (serovar Minnesota) R595 LPS Re at 90% water content and 40 °C without peptide (A) and in the presence of LF11 (B) and lauryl-LF11 (C), at a peptide/LPS molar ratio of 10:1