Nejvíce citovaný článek - PubMed ID 23528099
We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities-membrane curvature sensing and inducing-is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.
We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.
Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.
- MeSH
- antibakteriální látky chemie izolace a purifikace farmakologie MeSH
- buněčná membrána chemie účinky léků MeSH
- ethanolaminy chemie MeSH
- fixní kombinace léků MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie MeSH
- fosfatidylcholiny chemie MeSH
- fosfatidylethanolaminy chemie MeSH
- fosfatidylglyceroly chemie MeSH
- kationické antimikrobiální peptidy chemie izolace a purifikace farmakologie MeSH
- kůže chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- magaininy chemie izolace a purifikace farmakologie MeSH
- proteiny Xenopus chemie izolace a purifikace farmakologie MeSH
- sloučeniny boru chemie MeSH
- synergismus léků MeSH
- vazba proteinů MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol MeSH Prohlížeč
- 1-palmitoyl-2-oleoylphosphatidylethanolamine MeSH Prohlížeč
- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Prohlížeč
- antibakteriální látky MeSH
- ethanolaminy MeSH
- fixní kombinace léků MeSH
- fluorescenční barviva MeSH
- fosfatidylcholiny MeSH
- fosfatidylethanolaminy MeSH
- fosfatidylglyceroly MeSH
- kationické antimikrobiální peptidy MeSH
- lipidové dvojvrstvy MeSH
- magainin 2 peptide, Xenopus MeSH Prohlížeč
- magaininy MeSH
- PGLa-H peptide, Xenopus MeSH Prohlížeč
- phosphorylethanolamine MeSH Prohlížeč
- proteiny Xenopus MeSH
- sloučeniny boru MeSH
We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding article of this series, we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in the membranes. Here, we focus on the structures of the peptide-lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic, and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles with a collapsed interbilayer spacing resulting from peptide-induced adhesion. Interestingly, the adhesion does not lead to a peptide-induced lipid separation of charged and charge-neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle x-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.
We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.
- MeSH
- buněčná membrána metabolismus MeSH
- dimerizace MeSH
- fosfatidylethanolaminy MeSH
- fosfatidylglyceroly MeSH
- kationické antimikrobiální peptidy metabolismus MeSH
- lipidové dvojvrstvy chemie MeSH
- lipidy chemie MeSH
- magaininy metabolismus MeSH
- simulace molekulární dynamiky MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-palmitoyl-2-oleoylphosphatidylethanolamine MeSH Prohlížeč
- 1,2-dioleoyl-sn-glycero-3-phosphoglycerol MeSH Prohlížeč
- fosfatidylethanolaminy MeSH
- fosfatidylglyceroly MeSH
- kationické antimikrobiální peptidy MeSH
- lipidové dvojvrstvy MeSH
- lipidy MeSH
- magaininy MeSH
- peptide-Gly-Leu-amide MeSH Prohlížeč
Mixtures of the frog peptides magainin 2 and PGLa are well-known for their pronounced synergistic killing of Gram-negative bacteria. We aimed to gain insight into the underlying biophysical mechanism by interrogating the permeabilizing efficacies of the peptides as a function of stored membrane curvature strain. For Gram-negative bacterial-inner-membrane mimics, synergism was only observed when the anionic bilayers exhibited significant negative intrinsic curvatures imposed by monounsaturated phosphatidylethanolamine. In contrast, the peptides and their mixtures did not exhibit significant activities in charge-neutral mammalian mimics, including those with negative curvature, which is consistent with the requirement of charge-mediated peptide binding to the membrane. Our experimental findings are supported by computer simulations showing a significant decrease of the peptide-insertion free energy in membranes upon shifting intrinsic curvatures toward more positive values. The physiological relevance of our model studies is corroborated by a remarkable agreement with the peptide's synergistic activity in Escherichia coli. We propose that synergism is related to a lowering of a membrane-curvature-strain-mediated free-energy barrier by PGLa that assists membrane insertion of magainin 2, and not by strict pairwise interactions of the two peptides as suggested previously.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- biomechanika účinky léků MeSH
- buněčná membrána účinky léků metabolismus MeSH
- Escherichia coli cytologie účinky léků MeSH
- magaininy chemie farmakologie MeSH
- mechanický stres * MeSH
- multimerizace proteinu MeSH
- sekvence aminokyselin MeSH
- synergismus léků MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- magaininy MeSH
