Comparison of the Effects of Daptomycin on Bacterial and Model Membranes.

Daptomycin is a phosphatidylglycerol specific, calcium-dependent membrane-active antibiotic that has been approved for the treatment of Gram-positive infections. A recent Bacillus subtilis study found that daptomycin clustered into fluid lipid domains of bacterial membranes and the membrane binding was correlated with dislocation of peripheral membrane proteins and depolarization of membrane potential. In particular, the study disproved the existence of daptomycin ion channels. Our purpose here is to study how daptomycin interacts with lipid bilayers to understand the observed phenomena on bacterial membranes. We performed new types of experiments using aspirated giant vesicles with an ion leakage indicator, making comparisons between daptomycin and ionomycin, performing vesicle-vesicle transfers, and measuring daptomycin binding to fluid phase versus gel phase bilayers and bilayers including cholesterol. Our findings are entirely consistent with the observations for bacterial membranes. In addition, daptomycin is found to cause ion leakage through the membrane only if its concentration in the membrane is over a certain threshold. The ion leakage caused by daptomycin is transient. It occurs only when daptomycin binds the membrane for the first time; afterward, they cease to induce ion leakage. The ion leakage effect of daptomycin cannot be transferred from one membrane to another. The level of membrane binding of daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also weakens the membrane binding of daptomycin. The combination of membrane concentration threshold and differential binding is significant. This could be a reason why daptomycin discriminates between eukaryotic and prokaryotic cell membranes.

Action of Antimicrobial Peptides on Bacterial and Lipid Membranes: A Direct Comparison.

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.