New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Abstract

Antimicrobial peptides of the mastoparans family exert their bactericidal activity by binding to lipid membranes, inducing pores or defects and leaking the internal contents of vesicles and cells. However, this does not seem to be the only mechanism at play, and they might be important in the search for improved peptides with lower undesirable side effects. This work deals with three mastoparans peptides, Polybia-MP-1(MP-1), N2-Polybia-MP-1 (N-MP-1), and Mastoparan X (MPX), which exhibit high sequence homology. They all have three lysine residues and amidated C termini, but because of the presence of two, one, and no aspartic acid residues, respectively, they have +2, +3, and +4 net charges at physiological pH. Here we focus on the effects of these mastoparans peptides on anionic model membranes made of palmitoleyoilphosphatidylcholine (POPC) and palmitoleyoilphosphatidylglycerol (POPG) at 1:1 and 3:1 molar ratios in the presence and in the absence of saline buffer. Zeta potential experiments were carried out to measure the extent of the peptides' binding and accumulation at the vesicle surface, and CD spectra were acquired to quantify the helical structuring of the peptides upon binding. Giant unilamellar vesicles were observed under phase contrast and fluorescence microscopy. We found that the three peptides induced the leakage of GUVs at a gradual rate with many characteristics of the graded mode. This process was faster in the absence of saline buffer. Additionally, we observed that the peptides induced the formation of dense regions of phospholipids and peptides on the GUV surface. This phenomenon was easily observable for the more charged peptides (MPX> N-MP-1 > MP-1) and in the absence of added salt. Our data suggest that these mastoparans accumulate on the bilayer surface and induce a transient interruption to its barrier properties, leaking the vesicle contents. Next, the bilayer recovers its continuity, but this happens in an inhomogeneous way, forming a kind of ply with peptides sandwiched between two juxtaposed membranes. Eventually, a peptide-lipid aggregate forming a lump is formed at high peptide-to-lipid ratios.

Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

“…Similar observations have been made before for daptomycin (14) and mastoparans (37). It could be argued that these areas represent folds of the bilayer (37, 38), lipid–peptide aggregates on the membrane (14), or nonbilayer structures.…”

Single-Vesicle Detection and Analysis of Peptide-Induced Membrane Permeabilization

“…Thus, as mentioned in the Introduction, mastoparan X has been suggested to induce graded leakage, [35][36][37] and magainin 2 has been suggested to induce all-or-none leakage. [38][39][40][41] For melittin, the reports are more unclear.…”

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

“…Several studies on antimicrobial peptides have revealed dynamic equilibriums between monomeric and oligomeric states and the transient character of membrane-destabilizing forms [65,66]. It is thus possible that the GXXXG motifs of PTCs may have a more dynamical role in favoring only transient close contacts between helices.…”