Insight into the Lytic Mechanism of Antimicrobial Piscidin 1 and 3 using QCM-D

Abstract

In this work, we focused on piscidin 1 (P1) and 3 (P3), which are twenty-two-residue-long amphipathic, cationic antimicrobial peptides (AMPs) isolated from hybrid striped bass. Extensive work has been reported on characterizing the atomic-level structure of both P1 and P3 in magnetically and mechanically aligned bilayers using solid-state NMR. Here we investigated the mechanism of action of both P1 and P3 with supported lipid bilayers (SLB), using a quartz crystal microbalance with dissipation monitoring (QCM-D). In order to mimic bacterial cell membranes, we selected a 3:1 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Liposomes were deposited onto a QCM-D silicon oxide crystal until SLB formation, then P1 and P3 were introduced into the system and the interaction between the peptide and the lipid system was monitored by changes in frequency (Δf) and energy dissipation (ΔD). Different concentrations (2-20 μM) of the peptide and buffer conditions (pH 6.0 and 7.4) were tested. In particular we observed a Δf increase (∼ mass removal) as we increased the concentration of P1 at pH 7.4, suggesting lipid removal, membrane thinning, or pore formation; while at pH 6.0 the Δf decrease (∼ mass addition) was probably due to the electrostatic interaction between the more cationic peptide and the anionic POPG. For a quantitative assessment of the effects on the SLB, we have obtained the shear viscosity and shear modulus as a function of piscidin type and concentration via a fit of the well-known Voigt model to the data. In all cases, ΔD values were a clear indication of the destabilization of the SLB. Overall, this study, which demonstrates quantitative P1 and P3 disruption of a bacterial membrane mimic, assists in better understanding the mode of action of piscidins and related AMPs.