(722g) Lytic Mechanism of Histidine-Rich Antimicrobial Piscidins: Structural Flexibility, Hydrophobicity and Charge Regulation Examined in Real Time
Antimicrobial peptides (AMPs) are known to induce cell death in bacteria by destabilizing their membranes and/or interacting with intracellular targets, making them good candidates as novel agents in anti-infective therapies. Here we focused on piscidin 1 (p1) and piscidin 3 (p3), naturally occurring antibiotics from hybrid striped bass. Their atomic-level 3D structures were characterized using solid-state NMR, and showed that p1 is more hydrophobic than p3. In this study, we investigated in real time the mechanism of action of both p1 and p3 on bacterial cell mimics made of 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) lipid bilayers. Membrane disruption by p1 and p3 was monitored using lipid vesicle leakage assays at pH 6.0 and 7.4. More pronounced release of calcein entrapped in the vesicles at pH 7.4 compared with that at pH 6.0, especially for p3, motivated the NMR-monitored titrations of the four and three histidine side chains of p1 and p3, respectively. Since the pKa values were determined to be around 6.0, the titrations provided a correlation between the neutral state of the histidine side chains with stronger ability of the peptides to disrupt membrane bilayers; the lower pKa values for p1 than p3 also showed that stronger charge regulation occurred in p1. Disruption of PC/PG supported lipid bilayers (SLB) by p1 and p3 was measured as a function of time, dosage and pH using a Quartz Crystal Microbalance with Dissipation. Binding affinities and kinetics constants between p1 or p3 and PC/PG SLB, at both pH 6.0 and 7.4, were estimated using Surface Plasmon Resonance. Molecular dynamics showed that deeper penetration occurs when the histidine side chains are neutral, which helps explain the stronger effects at higher pH. Taken together these results show that membrane disruption occurs only when reduced peptide charge allows more peptides to accumulate and insert in the membrane to disrupt it.