Research Graduate William & Mary Williamsburg, Virginia, United States
Mary Rooney (William & Mary)| Yawei Xiong (William & Mary)| Steven Paredes (William & Mary)| Jessica Hill (William & Mary)| Marlaina Horewitz (William & Mary)| David Giles (University of Tennessee, Chattanooga)| Ella Mihailescu (University of Maryland)| Myriam Cotten (William & Mary)
Piscidin 1 (P1) and piscidin 3 (P3), isolated from the mast cells of hybrid striped bass, are host defense peptides (HDPs) that are active against several types of pathogens, including viruses and bacteria, as well as some tumor cells. They contain an amino-terminal copper and nickel binding (ATCUN) motif, and have enhanced antimicrobial functionality when metal-bound. Studies suggest that P1’s preferred method of attack is disruption of cell membranes and P3 is more damaging to DNA, but their mechanisms of action, particularly in the metallated state, are not yet well characterized. Resilience of cell membranes under adverse conditions is known to be greater when a portion of their constituent lipids are polyunsaturated fatty acids (PUFAs). It has also been shown that both peptide activity and structure respond to changes in lipid environment. Here, we posit that P1 and P3 retain their permeabilizing function in PUFA-containing membranes by chelating a redox metal ion that enhances their ability to disrupt membranes physically as well as chemically by oxidizing double bonds. We take a multi-faceted approach utilizing both functional assays (dye leakage, antimicrobial assays) and experimental and computational structural studies including circular dichroism, solid-state NMR, neutron diffraction, and molecular dynamics simulations, to examine the complex interplay between P1 and P3 and cell membrane mimics with different lipid compositions. One model mimics bacterial cells with incorporated PUFAs and the other mimics mammalian cells under oxidative stress conditions. We show that in the presence of PUFAs, membrane lytic activity of both P1 and P3 decreases, but increases as much as five fold upon metallation and twofold when membranes contain oxidized lipids. We investigate how changing peptide-lipid interactions in these different membranes could explain these results. The combined findings give insight into protein lipid-interactions and valuable information for the design of novel therapeutics for combating drug-resistant infections.