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.

Central European Institute of Technology Brno Czech Republic; Faculty of Science Masaryk University Brno Czech Republic

Fresenius Kabi Austria GmbH Graz Austria

Institute of Molecular Biosciences Biophysics Division University of Graz NAWI Graz Graz Austria; BioTechMed Graz Graz Austria

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Mayer M.L., Easton D.M., Hancock R.E.W. Fine tuning host responses in the face of infection: emerging roles and clinical applications of host defence peptides. In: Wang G., editor. Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies. CABI; 2010. pp. 195–220.

Steinstraesser L., Kraneburg U., Al-Benna S. Host defense peptides and their antimicrobial-immunomodulatory duality. Immunobiology. 2011;216:322–333. PubMed

Hancock R.E., Haney E.F., Gill E.E. The immunology of host defence peptides: beyond antimicrobial activity. Nat. Rev. Immunol. 2016;16:321–334. PubMed

Wimley W.C., Hristova K. Antimicrobial peptides: successes, challenges and unanswered questions. J. Membr. Biol. 2011;239:27–34. PubMed PMC

Lohner K. Membrane-active antimicrobial peptides as template structures for novel antibiotic agents. Curr. Top. Med. Chem. 2017;17:508–519. PubMed

Lohner K., Blondelle S.E. Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics. Comb. Chem. High Throughput Screen. 2005;8:241–256. PubMed

Henderson J.M., Lee K.Y.C. Promising antimicrobial agents designed from natural peptide templates. Curr. Opin. Solid State Mater. Sci. 2013;17:175–192.

Hoffmann W., Richter K., Kreil G. A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin. EMBO J. 1983;2:711–714. PubMed PMC

Soravia E., Martini G., Zasloff M. Antimicrobial properties of peptides from Xenopus granular gland secretions. FEBS Lett. 1988;228:337–340. PubMed

Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA. 1987;84:5449–5453. PubMed PMC

Williams R.W., Starman R., Covell D. Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa. Biochemistry. 1990;29:4490–4496. PubMed

Westerhoff H.V., Zasloff M., Juretić D. Functional synergism of the magainins PGLa and magainin-2 in Escherichia coli, tumor cells and liposomes. Eur. J. Biochem. 1995;228:257–264. PubMed

Matsuzaki K., Mitani Y., Miyajima K. Mechanism of synergism between antimicrobial peptides magainin 2 and PGLa. Biochemistry. 1998;37:15144–15153. PubMed

Wieprecht T., Apostolov O., Seelig J. Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects. Biochemistry. 2000;39:442–452. PubMed

Hara T., Mitani Y., Matsuzaki K. Heterodimer formation between the antimicrobial peptides magainin 2 and PGLa in lipid bilayers: a cross-linking study. Biochemistry. 2001;40:12395–12399. PubMed

Nishida M., Imura Y., Matsuzaki K. Interaction of a magainin-PGLa hybrid peptide with membranes: insight into the mechanism of synergism. Biochemistry. 2007;46:14284–14290. PubMed

Tremouilhac P., Strandberg E., Ulrich A.S. Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin. J. Biol. Chem. 2006;281:32089–32094. PubMed

Salnikov E.S., Bechinger B. Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic enhancement of the antimicrobial activities of PGLa and magainin 2. Biophys. J. 2011;100:1473–1480. PubMed PMC

Strandberg E., Zerweck J., Ulrich A.S. Synergistic insertion of antimicrobial magainin-family peptides in membranes depends on the lipid spontaneous curvature. Biophys. J. 2013;104:L9–L11. PubMed PMC

Wilkinson S.G. Gram-negative bacteria. In: Wilkinson S.G., Ratledge C., editors. Microbial Lipids. Volume 1. Academic Press; 1988. pp. 299–488.

Morein S., Andersson A., Lindblom G. Wild-type Escherichia coli cells regulate the membrane lipid composition in a “window” between gel and non-lamellar structures. J. Biol. Chem. 1996;271:6801–6809. PubMed

Chen Y.F., Tsang K.Y., Fan Z.A. Differential dependencies on [Ca2+] and temperature of the monolayer spontaneous curvatures of DOPE, DOPA and cardiolipin: effects of modulating the strength of the inter-headgroup repulsion. Soft Matter. 2015;11:4041–4053. PubMed

Zweytick D., Deutsch G., Lohner K. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides. J. Biol. Chem. 2011;286:21266–21276. PubMed PMC

Kollmitzer B., Heftberger P., Pabst G. Monolayer spontaneous curvature of raft-forming membrane lipids. Soft Matter. 2013;9:10877–10884. PubMed PMC

Cooke I.R., Kremer K., Deserno M. Tunable generic model for fluid bilayer membranes. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2005;72:011506. PubMed

Vácha R., Frenkel D. Relation between molecular shape and the morphology of self-assembling aggregates: a simulation study. Biophys. J. 2011;101:1432–1439. PubMed PMC

Wang F., Landau D.P. Efficient, multiple-range random walk algorithm to calculate the density of states. Phys. Rev. Lett. 2001;86:2050–2053. PubMed

Marsh D. Intrinsic curvature in normal and inverted lipid structures and in membranes. Biophys. J. 1996;70:2248–2255. PubMed PMC

Lohner K., Sevcsik E., Pabst G. Liposome-based biomembrane mimetic systems: implications for lipid-peptide interactions. In: Leitmannova-Liu A., editor. Advances in Planar Lipid Bilayers and Liposomes (Vol. 6) Elsevier; 2008. pp. 103–137.

Meletiadis J., Pournaras S., Walsh T.J. Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus. Antimicrob. Agents Chemother. 2010;54:602–609. PubMed PMC

Zerweck J., Strandberg E., Ulrich A.S. Molecular mechanism of synergy between the antimicrobial peptides PGLa and magainin 2. Sci. Rep. 2017;7:13153. PubMed PMC

Liang H.P., Brophy T.M., Hogg P.J. Redox properties of the tissue factor Cys186-Cys209 disulfide bond. Biochem. J. 2011;437:455–460. PubMed

Zerweck J., Strandberg E., Ulrich A.S. Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes. Eur. Biophys. J. 2016;45:535–547. PubMed

Boggs J.M. Intermolecular hydrogen bonding between lipids: influence on organization and function of lipids in membranes. Can. J. Biochem. 1980;58:755–770. PubMed

Pink D.A., McNeil S., Zuckermann M.J. A model of hydrogen bond formation in phosphatidylethanolamine bilayers. Biochim. Biophys. Acta. 1998;1368:289–305. PubMed

Koller D., Lohner K. The role of spontaneous lipid curvature in the interaction of interfacially active peptides with membranes. Biochim. Biophys. Acta. 2014;1838:2250–2259. PubMed

Dan N., Safran S.A. Effect of lipid characteristics on the structure of transmembrane proteins. Biophys. J. 1998;75:1410–1414. PubMed PMC

Botelho A.V., Gibson N.J., Brown M.F. Conformational energetics of rhodopsin modulated by nonlamellar-forming lipids. Biochemistry. 2002;41:6354–6368. PubMed

Shearman G.C., Ces O., Seddon J.M. Inverse lyotropic phases of lipids and membrane curvature. J. Phys. Condens. Matter. 2006;18:S1105–S1124. PubMed

Matsuzaki K., Sugishita K., Epand R.M. Relationship of membrane curvature to the formation of pores by magainin 2. Biochemistry. 1998;37:11856–11863. PubMed

Glattard E., Salnikov E.S., Bechinger B. Investigations of the synergistic enhancement of antimicrobial activity in mixtures of magainin 2 and PGLa. Biophys. Chem. 2016;210:35–44. PubMed

Marquette A., Salnikov E.S., Bechinger B. Magainin 2-PGLa interactions in membranes - two peptides that exhibit synergistic enhancement of antimicrobial activity. Curr. Top. Med. Chem. 2016;16:65–75. PubMed

White S.H., Wimley W.C. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 1999;28:319–365. PubMed

Almeida P.F., Pokorny A. Mechanisms of antimicrobial, cytolytic, and cell-penetrating peptides: from kinetics to thermodynamics. Biochemistry. 2009;48:8083–8093. PubMed PMC

Beschiaschvili G., Seelig J. Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes. Biochemistry. 1990;29:52–58. PubMed

Pathak N., Salas-Auvert R., Harrison R.G. Comparison of the effects of hydrophobicity, amphiphilicity, and alpha-helicity on the activities of antimicrobial peptides. Proteins. 1995;22:182–186. PubMed

Huang H.W., Wu Y. Lipid-alamethicin interactions influence alamethicin orientation. Biophys. J. 1991;60:1079–1087. PubMed PMC

Ulmschneider J.P., Smith J.C., Strandberg E. Reorientation and dimerization of the membrane-bound antimicrobial peptide PGLa from microsecond all-atom MD simulations. Biophys. J. 2012;103:472–482. PubMed PMC

Takei J., Remenyi A., Dempsey C.E. Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues. FEBS Lett. 1999;442:11–14. PubMed

Dempsey C.E., Ueno S., Avison M.B. Enhanced membrane permeabilization and antibacterial activity of a disulfide-dimerized magainin analogue. Biochemistry. 2003;42:402–409. PubMed

Lorenzón E.N., Santos-Filho N.A., Cilli E.M. C-terminal lysine-linked magainin 2 with increased activity against multidrug-resistant bacteria. Protein Pept. Lett. 2016;23:738–747. PubMed

Verly R.M., Resende J.M., Bechinger B. Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin. Sci. Rep. 2017;7:40854. PubMed PMC

Frey S., Tamm L.K. Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers. Biochem. J. 1990;272:713–719. PubMed PMC

Gambin Y., Lopez-Esparza R., Urbach W. Lateral mobility of proteins in liquid membranes revisited. Proc. Natl. Acad. Sci. USA. 2006;103:2098–2102. PubMed PMC

Broekhuyse R.M. Phospholipids in tissues of the eye. I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids. Biochim. Biophys. Acta. 1968;152:307–315. PubMed

Buboltz J.T., Feigenson G.W. A novel strategy for the preparation of liposomes: rapid solvent exchange. Biochim. Biophys. Acta. 1999;1417:232–245. PubMed

Rieder A.A., Koller D., Pabst G. Optimizing rapid solvent exchange preparation of multilamellar vesicles. Chem. Phys. Lipids. 2015;186:39–44. PubMed

Alley S.H., Ces O., Templer R.H. X-ray diffraction measurement of the monolayer spontaneous curvature of dioleoylphosphatidylglycerol. Chem. Phys. Lipids. 2008;154:64–67. PubMed

Leikin S., Kozlov M.M., Rand R.P. Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes. Biophys. J. 1996;71:2623–2632. PubMed PMC

Israelachvili J.N. Third Edition. Academic Press; Burlington, MA: 2011. Intermolecular and Surface Forces.

Ellens H., Bentz J., Szoka F.C. H+- and Ca2+-induced fusion and destabilization of liposomes. Biochemistry. 1985;24:3099–3106. PubMed

Ladokhin A.S., Wimley W.C., White S.H. Leakage of membrane vesicle contents: determination of mechanism using fluorescence requenching. Biophys. J. 1995;69:1964–1971. PubMed PMC

Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. PubMed

Frenkel D., Smit B. Academic Press; San Diego, CA: 2002. Understanding Molecular Simulation.

Mueller J.H., Hinton J. A protein-free medium for primary isolation of the gonococcus and meningococcus. Proc. Soc. Exp. Biol. Med. 1941;48:330–333.

Gesell J., Zasloff M., Opella S.J. Two-dimensional 1H NMR experiments show that the 23-residue magainin antibiotic peptide is an α-helix in dodecylphosphocholine micelles, sodium dodecylsulfate micelles, and trifluoroethanol/water solution. J. Biomol. NMR. 1997;9:127–135. PubMed

Bechinger B., Zasloff M., Opella S.J. Structure and dynamics of the antibiotic peptide PGLa in membranes by solution and solid-state nuclear magnetic resonance spectroscopy. Biophys. J. 1998;74:981–987. PubMed PMC

Membrane Adsorption Enhances Translocation of Antimicrobial Peptide Buforin 2

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption

Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids

Effect of helical kink in antimicrobial peptides on membrane pore formation

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation

Magainin 2 and PGLa in Bacterial Membrane Mimics I: Peptide-Peptide and Lipid-Peptide Interactions