Antimicrobial peptides (AMPs) are naturally occurring antibiotics which induce cell death in bacteria by destabilizing their membranes and/or interacting with intracellular targets. Unlike traditional antibiotics, AMPs display low level of bacterial drug resistance and have immunomodulatory properties, making them good candidates as novel agents in anti-infective therapies. In this study, we focused on piscidin 1 (p1) and piscidin 3 (p3), which are twenty-two-residue-long amphipathic, cationic, membrane-active AMPs discovered from hybrid striped bass. Extensive work has been done to characterize the atomic-level structures of both p1 and p3 in magnetically and mechanically aligned bilayers using solid-state NMR. The 3D structures indicate that p1 is more hydrophobic than p3. Here we investigated 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 and analyzed with Sauerbrey and Voigt models to obtain real-time changes in lipid bilayer viscoelastic properties. Binding affinities 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.

Structural flexibility, hydrophobicity and charge regulation in histidine-rich antimicrobial piscidins underpin membrane disruption examined in real time

251st National Meeting (2016)

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