There are countless studies showing that AMPs interact with and perturb lipid bilayer membranes in vitro
. Within these many experiments, lipid compositions, buffer conditions, peptide and lipid concentrations vary widely. Even the phase state of the lipids and the degree of bilayer hydration vary between experiments. Combined with the inherently variable nature of AMP activity, these factors confuse the interpretation and comparison of results. While lipid compositions vary widely, mixtures of anionic and zwitterionic lipids are often used to mimic microbial membranes. For example, some laboratories use mixtures of phosphatidylglycerol and phosphatidylethanolamine to specifically mimic the E. coli inner membrane(33
), while others use pure phosphatidylglycerol to specifically mimic Gram positive bacteria(34
). Other commonly used systems include phosphatidylcholine mixed with phosphatidylglycerol, phosphatidylserine or cardiolipin to broadly mimic the anionic surface of a microbe(12
). Experiments have also been performed with lipids extracted from E. coli or other organisms(35
). To mimic exposed mammalian or host membranes, zwitterionic phosphatidylcholine (PC) or PC-cholesterol membranes are often used(8
). Because of the variety of protocols and experimental conditions used, a single most appropriate lipid composition has never emerged from the literature.
In model membranes and living cells, lipid composition will affect peptide binding as well as the inherent susceptibility of the bilayer to permeabilization. Other than electrostatic effects, there are few, if any, examples of specific binding of an AMP to a particular lipid species. Cationic/hydrophobic peptides with good interfacial activity are expected to perturb any fluid phase lipid bilayer membrane to which they bind well enough, although bilayers with some lipid compositions may be inherently more or less stable than others. In practical terms AMPs that are less hydrophobic and more cationic will require anionic lipids for binding. For example, human defensins, which are highly cationic, must be studied in bilayers with anionic lipids, or else they are inactive due to poor binding(36
). The more hydrophobic AMPs, such as the magainins, are active in zwitterionic lipid bilayers as well as anionic bilayers. It is likely that any model system that comprises a fully hydrated, fluid phase bilayer to which one can achieve reasonable bound peptide concentration is appropriate as a model system. Recently we showed that large unilamellar vesicles(11
) composed of 90% phosphatidylcholine and 10% phosphatidylglycerol in 50 mM sodium phosphate buffer could be used to specifically select non-hemolytic, broad-spectrum antimicrobial peptides from combinatorial libraries, suggesting that this may be a good consensus membrane composition to study AMPs.
Upon binding of AMPs to anionic lipid vesicles through electrostatic and hydrophobic interactions, many types of perturbations can occur, including membrane permeabilization. But AMPs can also drive vesicle aggregation, vesicle fusion, formation of non-bilayer phases, lipid phase separation, transbilayer movement (flip-flop) of lipids and complete solubilization of membranes. Some of these effects may not relate to antimicrobial activity and should be considered experimental artifacts. Although not often reported in the literature, vesicle aggregation and fusion are very common occurrences when cationic peptides are added to anionic vesicles(13
), as evidenced by rapid increases in turbidity upon peptide addition. It is likely that some (or many) reports of peptide-induced “leakage” from anionic vesicles observed at very high P:L ratios (such as P:L = 1:10) include leakage that is due to leaky fusion/aggregation events in addition to (or instead of) direct membrane permeabilization. Few attempts have been made to distinguish leakage that is purely a consequence of fusion from legitimate leakage that occurs without vesicle fusion. In a recent paper(13
) we examined AMP-induced fusion and leakage independently and found that leakage and vesicle fusion both occurred at P:L ≥ 1:50 while only leakage occurred at lower P:L (up to 1:500). We concluded that leakage measured at P:L = 1:50 may have included a contribution from fusion as well as membrane permeabilization.
As shown by the example data in , most antimicrobial peptides are active against vesicles when bound peptide to lipid ratios are from about 1:500 to 1:50, or about 200–2000 peptides per vesicle. In contrast, ideal pore-forming peptides can permeabilize lipid vesicle with as few as 10 peptides bound per vesicle, or 1 peptide per 10,000 lipids(37
). We speculate that AMPs are not more potent because the very potent peptides lack selectivity. On the other end of the spectrum, any membrane-interacting molecule can disrupt membranes at very high concentrations. For this reason, it is likely that some published results for putative AMPs permeabilizing vesicles are essentially artifacts that arise from extremely high peptide:lipid ratios. The literature contains many experiments which show significant leakage of vesicle entrapped contents at P:L of 1:10, 1:1 or even at P:L = 10:1. Any in vitro
membrane permeabilization measurements that show activity only
at more than 1 peptide bound
per 50 lipids (P:L = 1:50) should be viewed with caution.
Figure 3 Peptide activity against lipid vesicles. True transmembrane pore-forming peptides, such as alamethicin, permeabilize vesicles at very low peptide:lipid ratios. The green line is based on experimental measurements. Antimicrobial peptides, on the other (more ...)