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Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

ABSTRACT: Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/Escherichia coli (E. coli) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties.

SUBMITTER: Soblosky L

PROVIDER: S-EPMC4385500 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

Soblosky Lauren L Ramamoorthy Ayyalusamy A Chen Zhan Z

Chemistry and physics of lipids 20150220

Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, ma ...[more]

PMID: 25707312

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Project description:The bacterial membrane represents an attractive target for the design of new antibiotics to combat widespread bacterial resistance. Understanding how antimicrobial peptides (AMPs) and other membrane-active agents attack membranes could facilitate the design of new, effective antimicrobials. Despite intense study of AMPs on model membranes, we do not know how well the mechanism of attack translates to real biological membranes. To that end, we have characterized the attack of AMPs on Escherichia coli cytoplasmic membranes and directly compared this action to model membranes. AMPs induce membrane permeability in E. coli spheroplasts or giant unilamellar vesicles (GUVs) under well-defined concentrations of AMPs and fluorescent molecules. The action of AMPs on spheroplasts is unique in producing an intracellular fluorescence intensity time curve that increases in a sigmoidal fashion to a steady state. This regular pattern is reproducible by melittin, LL37, and alamethicin but not by CCCP or daptomycin, agents known to cause ion leakage. Remarkably, a similar pattern was also reproduced in GUVs. Indeed the steady-state membrane permeability induced by AMPs is quantitatively the same in spheroplasts and GUVs. There are, however, interesting dissimilarities in details that reveal differences between bacterial and lipid membranes. Spheroplast membranes are permeabilized by a wide range of AMP concentrations to the same steady-state membrane permeability. In contrast, only a narrow range of AMP concentrations permeabilized GUVs to a steady state. Tension in GUVs also influences the action of AMPs, whereas the spheroplast membranes are tensionless. Despite these differences, our results provide a strong support for using model membranes to study the molecular interactions of AMPs with bacterial membranes. As far as we know, this is the first time the actions of AMPs, on bacterial membranes and on model membranes, have been directly and quantitatively compared.

Project description:The horseshoe crab cationic antimicrobial peptide polyphemusin I is highly active in vitro but not protective in mouse models of bacterial and LPS challenge, while a synthetic polyphemusin variant, PV5, was previously shown to be protective in vivo. In this study, we investigated the interaction of these peptides with lipid membranes in an effort to propose a mechanism of interaction. The solution structure of PV5 was determined by proton NMR in the absence and presence of dodecylphosphocholine (DPC) micelles. Like polyphemusin I, PV5 is a beta-hairpin but appeared less amphipathic in solution. Upon association with DPC micelles, PV5 underwent side chain rearrangements which resulted in an increased amphipathic conformation. Using fluorescence spectroscopy, both peptides were found to have limited affinity for neutral vesicles composed of phosphatidylcholine (PC). Incorporation of 25 mol % cholesterol or phosphatidylethanolamine into PC vesicles produced little change in the partitioning of either peptide. Incorporation of 25 mol % phosphatidylglycerol (PG) into PC vesicles, a simple prokaryotic model, resulted in a large increase in the affinity for both peptides, but the partition coefficient for PV5 was almost twice that of polyphemusin I. Differential scanning calorimetry studies supported the partitioning data and demonstrated that neither peptide interacted readily with neutral PC vesicles. Both peptides showed affinity for negatively charged membranes incorporating PG. The affinity of PV5 was much greater as the pretransition peak was absent at low peptide to lipid ratios (1:400) and the reduction in enthalpy of the main transition was greater than that produced by polyphemusin I. Both peptides decreased the lamellar to inverted hexagonal phase transition temperature of PE indicating the induction of negative curvature strain. These results, combined with previous findings that polyphemusin I promotes lipid flip-flop but does not induce significant vesicle leakage, ruled out the torroidal pore and carpet mechanisms of antimicrobial action for these polyphemusins.

Project description:Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Dataset Information

Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

Publications

Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

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