Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

Abstract

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

Optical Microscopy of Giant Vesicles as a Tool to Reveal the Mechanism of Action of Antimicrobial Peptides and the Specific Case of Gomesin

“…The formation of dense regions on the membrane surface of GUVs was also induced by the cecropin-melittin hybrid peptide BP100 [68]. Quite often, these domains appeared even before permeabilization/destruction of the membrane structure, as can be seen in the sequences shown in Fig.…”

“…However, burst events of POPC GUVs were also observed occasionally, especially when the addition of the peptides was done with a micropipette, which might create an inhomogeneous concentration of peptide around the vesicle. Light scattering experiments performed with LUVs show that BP100 also induces significant lipid aggregation modulated by membrane charge [68].…”

Shedding light on the puzzle of drug-membrane interactions: Experimental techniques and molecular dynamics simulations

“…Using fluorescence spectroscopy, the release can be monitored over time [40,273]. Other strategies rely on microscopy using inverted optical microscopy for giant unilamellar vesicles (GUVs) or atomic force microscopy (AFM) for supported lipid bilayers [265,274]. Computer simulations have been extensively used to study pore formation by antimicrobial peptides [130,275]; however, as previously mentioned, they will not be discussed in this review since the majority of antimicrobial peptides are not FDA approved.…”

Potential antitumor activity of novel DODAC/PHO-S liposomes

“…The concentration of PHO-S was quantified by determining the phosphate molecules, as described in the literature. 17 Encapsulation efficiency (EE%) was calculated using the l-glutamine (Cultilab, Campinas, SP, Brazil), 10 mM HEPES (Cultilab), 24 mM sodium bicarbonate, 0.01% antibiotics, and 10% fetal bovine serum (Cultilab). Cells were cultivated in 5% CO 2 atmosphere at 37°C as monolayer cultures.…”