Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

Abstract

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.

Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

“…In atomic force microscopy, transformation of LPS assembly from ribbon-like structures into large and densely packed multilamellar aggregates was seen upon the addition of AMPs (67). In our experiments, the full absorption of B2088 into the LPS was detected.…”

(Lipo)polysaccharide interactions of antimicrobial peptides

“…Despite this, small cationic peptides are able to penetrate LPS layers to the lipid A moiety. Through this, lipid A disordering may occur, as reported, e.g., for Pep19 variants (22) and polymyxin B (23).…”

Preclinical Investigations Reveal the Broad-Spectrum Neutralizing Activity of Peptide Pep19-2.5 on Bacterial Pathogenicity Factors

“…Sepsis was induced in mice by cecal ligation and puncture and, subsequently, the animals were treated by continuous infusion with Pep19-2.5 intravenously for 24 h. To compare efficacy, further groups were treated with polymyxin B or with Pep19-4 or Pep19-8. The latter two peptides were selected since they have been shown to represent compounds with medium and weak efficacy in the inhibition of the LPS-induced cytokine secretion of human mononuclear cells (20,21). Minnesota R60 or heat-killed methicillin-resistant Staphylococcus aureus (MRSA) by isothermal titration calorimetry.…”

“…We have taken the LALF protein as the template, but we rationally designed a library with linear peptides, in which sequences were improved on the basis of their ability to bind and neutralize the hydrophobic moiety of LPS, the lipid A, which is responsible for the pathophysiological effects of the molecule (19)(20)(21).…”