By combining vesicles of a particular lipid composition that contain the fluorescent dye calcein with calcein-free vesicles, the activity of an antimicrobial peptide having a known pore-forming activity has been investigated within a competitive lipid environment. This novel approach allowed demonstrating the AMP’s preferential affinity for a negatively charged membrane, such as bacterial membranes, than for a neutral membrane, such as prokaryotic cell membranes, in a single experiment despite the AMP was dramatically more active in eukaryotic-like membrane environment only than prokaryotic-like only. This approach can also be used to probe the binding strength in a competitive lipid environment. By pre-incubating the peptides with a calcein-free lipid environment and then introducing another lipid environment containing the dye, the pore-forming AMP was shown to traffic from the eukaryotic-like membrane interface to the prokaryotic-like interface, although it had formed a pore in the previous environment.
This result questions the value of the therapeutic index, which are often measured in infected and healthy cells separately. It also opens further possibilities in testing for instance the impact of membrane curvature in modulating membrane-active peptides or proteins activity.