Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell–directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.
The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flipflop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.
Although the alpha-toxin from S. aureus was the first pore-forming toxin identified, its mode of interaction with membranes is still not fully understood. The toxin forms heptameric pores on cellular and artificial membranes. The observation that artificial membranes are permeabelized by this toxin indicates that no protein receptor is mandatory. Efficient permeabilisation is only possible in presence of cholesterol and sphingomyelin, which could be interpreted as a preference of the toxin for raft-like structures. However, variation of lipid composition and determination of oligomer formation by pyrene-fluorescence reveals, that the toxin favors specifically sphingomyelin in combination with cholesterol and that the interaction is not raft-specific in a general sense. The mode of action seems also to differ from the primary binding of the toxin lysenin, which binds specifically to sphingomyelin. Under certain conditions also non-lytic oligomers can be formed, for example at low temperatures or for certain mutants. The spectra obtained for pyrenelabelled mutants indicate that also lipid-composition might modulate the probability of lytic versus non-lytic pore formation, which might give a hint why for some cell non-lytic cells are found. Employing both AFM and fluorescence microscopy we aim to determine the molecular basis for efficient pore formation by this toxin. We thank the DFG (SFB 490) for financial support, S.Bhakdi and A.Valeva for production of the toxin and helpful discussions, G. Gimpl for help with fluorescence microscopy and the MPI for Polymer Research for the possibility to use the AFM.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.