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.

Structural Features Governing the Activity of Lactoferricin-Derived Peptides That Act in Synergy with Antibiotics against Pseudomonas aeruginosa In Vitro and In Vivo

“…In this regard, our results confirm that to maximize antimicrobial activity against Gram-negative bacteria, it is favorable to have larger amounts of hydrophobic residues, which cause distortions in the membrane (12,21,22). On the other hand, activity is decreased by an excessive concentration of basic residues in the molecule and by large peptide size, with the latter factor probably reflecting the steric hindrance that high-molecular weight peptides will encounter when crossing the hydrophilic carbohydrate chains of LPS (1).…”

Structural Origin of Endotoxin Neutralization and Antimicrobial Activity of a Lactoferrin-based Peptide

“…Longer peptides can form transmembrane pores, whereas the proposed mechanism of membrane disruption by shorter peptides is initiated by peptide association with the outer membrane layer, its translocation across the outer membrane by the self-promoted peptide uptake, and formation of defects in the bacterial cytoplasmic membrane (33). The LF11 peptide is too short to cross the bilayer; it can, however, destabilize the bacterial membrane, as observed by permeabilizing bacterial cells to hydrophobic probes (such as N-phenyl-1-naphthylamine (data not shown)) and its action on artificial membranes composed of anionic lipids (39).…”

Ultrashort Antimicrobial Peptides with Antiendotoxin Properties

“…Some AMPs have been shown to bind to LPS and neutralize LPS-stimulated proinflammatory responses (13)(14)(15). Related to this, different studies have shed light on AMPs with antiendotoxin properties (13,14,(16)(17)(18)(19)(20)(21)(22)(23)(24). However, the rules governing the design of AMPs with antiendotoxin properties are still not very clear (13)(14)(15).…”

“…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).…”