Mechanism and Kinetics of δ-Lysin Interaction with Phospholipid Vesicles

Abstract

Delta-lysin is a 26 amino acid, hemolytic peptide toxin secreted by Staphylococcus aureus. It has been reported to form an amphipathic helix upon binding to lipid bilayers and is often cited as a typical example of the barrel-stave model for pore formation in lipid bilayer membranes. However, the exact mechanism by which it lyses cells and the physical basis of its target specificity are still unknown. Moreover, the evidence for delta-lysin insertion and pore formation in the membrane stems largely from theoretical modeling of the toxin and lacks experimental confirmation. We investigated binding and insertion of delta-lysin into phospholipid bilayer vesicles. The kinetics of these processes were studied by stopped-flow fluorescence with two types of experiments: (a) carboxyfluorescein release from the vesicles upon peptide-vesicle interaction, with concomitant relief of dye self-quenching; (b) fluorescence energy transfer from the intrinsic tryptophan of the peptide to a membrane-bound lipid probe. We formulated a detailed kinetic mechanism with explicit molecular rate constants for peptide binding, association, and insertion, obtaining a quantitative description of the experimental results. delta-Lysin insertion is strongly dependent on the peptide-to-lipid ratio, suggesting that association of a critical number of monomers on the membrane is required for activity. However, we found no evidence for a stable membrane-inserted pore. Rather, the peptide appears to cross the membrane rapidly and reversibly and cause release of the lipid vesicle contents in this process.

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes,

“…While the data presented in this paper support only steps 1 and 2 of this mechanism, it serves to further define the aggregate model of membrane translocation previously proposed by our group (43). It should also be noted that the aggregate model is similar in nature to the sinking-raft model proposed by Pokorny et al (44,45).…”

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches

“…Pore models account for the formation of membrane-spanning pores, namely the 'barrel stave pore model' (Rapaport and Shai 1991), in which AMPs interact to form a hydrophilic channel, and the 'toroidal pore model' (Ludtke et al 1996), in which AMPs affect the curvature of the membrane. In turn, non-pore models comprise: the 'carpet model' (Gazit et al 1996), which is the most cited model and accounts for the parallel deposition of AMPs on the membrane, causing global bilayer destabilization due to a detergent-like effect; the 'detergent model' (Ostolaza et al 1993) that explains catastrophic collapse of the membrane using high concentrations of AMPs; the 'molecular shape models' (Bechinger and Lohner 2006), in which AMP-lipid interactions can be portrayed with phase diagrams; the 'lipid clustering model' (Epand and Epand 2009), in which AMPs induce lipid phase separation; the 'sinking raft model' (Pokorny et al 2002), in which AMP activity is described in terms of binding, insertion and perturbation; and the 'interfacial activity model' (Rathinakumar and Wimley 2008), which is used to explain, predict and engineer the activity of AMPs. All the aforementioned models imply the need to reach a certain threshold concentration of AMPs in the membrane prior to disruption (Melo et al 2009).…”

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

“…In these pores the outer and the inner leaflets of the bilayer are fused and the lipids are then curved inward; these pores are toroidal holes lined by lipids and peptides (Matsuzaki et al 1996b;Huang et al 2004). In an alternative model, the adsorbed peptides induce curvature strain in the bilayer, which is relieved by their aggregation into the bilayer, enabling the aggregate to be translocated to the vesicle lumen by a sinking-raft process (Pokorny et al 2002). Depending on the lipid composition of the bilayer, the peptide can induce a non-lamellar phase and be translocated across the membrane by the formation of non-bilayer intermediates in which the peptide is trapped inside inverted micelle (Haney et al 2010).…”

“…This method allows the amount of dye released and the amount that remained entrapped after the peptide action to be monitored. With this alternative experiment, it is possible to estimate whether the leakage process is graded or all-or-none (Parente et al 1990;Pokorny et al 2002). In addition, Heerklotz and coauthors (Patel et al 2009) developed a method using time-resolved fluorescence approach to monitor dye release (calcein) from LUVs.…”

“…A wealth of information regarding the mode of action of these peptides has been gathered over recent decades (Parente et al 1990;Matsuzaki et al 1996a;Pokorny et al 2002;Huang et al 2004;Sengupta et al 2008;Epand et al 2009;Haney et al 2010;Nguyen et al 2011). Some of this information indicates that the bactericide activities of all L-residue peptides are the same as for all D-residue peptides.…”