The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles

Abstract

Cationic antimicrobial peptides (CAMPs) offer a promising strategy to counteract bacterial resistance, mostly due to their membrane-targeting activity. W-BP100 is a potent broad-spectrum cecropin-melittin CAMP bearing a single N-terminal Trp, which was previously found to improve its antibacterial activity. W-BP100 has high affinity toward anionic membranes, inducing membrane saturation at low peptide-to-lipid (P/L) ratios and membrane permeabilization, with the unique property of promoting the aggregation of anionic vesicles only at specific P/L ratios. Herein, we aimed to investigate this unusual behavior of W-BP100 by studying its aggregation and fusion properties with negatively-charged large (LUVs) or giant (GUVs) unilamellar vesicles using biophysical tools. Circular dichroism (CD) showed that W-BP100 adopted an α-helical conformation in anionic LUVs, neutralizing its surface charge at the aggregation P/L ratio. Its fusion activity, assessed by Förster resonance energy transfer (FRET) using steady-state fluorescence spectroscopy, occurred mainly at the membrane saturation/aggregation P/L ratio. Confocal microscopy studies confirmed that W-BP100 displays aggregation and detergent-like effects at a critical P/L ratio, above which it induces the formation of new lipid aggregates. Our data suggest that W-BP100 promotes the aggregation and fusion of anionic vesicles at specific P/L ratios, being able to reshape the morphology of GUVs into new lipid structures.

Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes

“…113 This process is facilitated by fusogenic peptide sequences, typically found within larger proteins. 113,118 Additionally, certain membrane-active peptides, including cell-penetrating peptides (CPPs), possess the capability to induce fusion of membranes and vesicles. 113,119,120 In interactions between CPPs and complex lipid mixtures such as phosphatidylcholine/PE/bis(monoacylglycerol), not only fusion and leakage, but also leaky fusion (simultaneous fusion and leakage) have been observed.…”

“…113,118 Additionally, certain membrane-active peptides, including cell-penetrating peptides (CPPs), possess the capability to induce fusion of membranes and vesicles. 113,119,120 In interactions between CPPs and complex lipid mixtures such as phosphatidylcholine/PE/bis(monoacylglycerol), not only fusion and leakage, but also leaky fusion (simultaneous fusion and leakage) have been observed. 111,121 Recently, Allolio et al have shown that arginine-rich CPPs enter vesicles by inducing membrane multilamellarity and fusion.…”

“…Membrane fusion plays a crucial role in various biological processes such as the entry of enveloped viruses into host cells, fertilization, cell division, and synaptic transmission. 113 This process is facilitated by fusogenic peptide sequences, typically found within larger proteins. 113,118 Additionally, certain membrane-active peptides, including cell-penetrating peptides (CPPs), possess the capability to induce fusion of membranes and vesicles.…”

Deciphering the Mechanism of Action of the Antimicrobial Peptide BP100

“…The groups of M. Castanho, P. Gameiro, and I. M. Cuccovia synthesized BP100 analogues with the aim of analysing the effect of incorporating structural modifications on the mode of action ( Table 2 ) [ 38 , 39 , 40 , 41 ]. In particular, M. Castanho and coworkers studied the activity against Gram-negative and Gram-positive bacteria as well as the mechanistic features of two BP100 analogues: R-BP100 (RRLFRRILRYL-NH 2 ) and RW-BP100 (RRLFRRILRWL-NH 2 ) [ 38 ].…”

“…Later, the group of P. Gameiro [ 39 , 40 ] studied the antibacterial activity and the biophysical properties of W-BP100 (WKKLFKKILKYL-NH 2 ), incorporating an additional tryptophan residue at the N-terminus. The Edmunson wheel projection of W-BP100 showed that this tryptophan was located at the hydrophobic face and therefore, similarly to BP100, this analogue had an amphipathic character.…”