The Metallopeptides Piscidin 1 And Piscidin 3 Employ Membrane And Nuclease Activity To Eradicate Planktonic, Biofilm, And Persister Cells

Piscidin 1 (P1) and piscidin 3 (P3) are highly potent host-defense peptides (HDPs) active against drug resistant bacteria, and therefore important for the design of novel anti-infective therapeutics. Both peptides are cationic and interact with the anionic lipid membranes of bacteria. They also translocate across these membranes and colocalize with intracellular DNA. While both peptides have similar three-dimensional structures, P1 is generally more biologically active than P3 for reasons that are not understood. Here, we compare the disruptive effects of P1 and P3 on lipid bilayers and DNA, and investigate the role of their amino terminal Cu and Ni (ATCUN) binding motif in their mechanism of action. We rely on a combination of complementary methods, including CD- and NMR-monitored titrations, structural solid-state NMR, neutron diffraction with deuterium labeling, surface plasmon resonance, microscopy, biological activity assays, and voltammetry. We find by solid-state NMR that both peptides use an α-helical structure to interact with bacterial cell membrane mimics and isolated DNA. Interestingly, while P1 is more membrane active than P3, the latter is more disruptive to DNA. Through neutron diffraction studies, we demonstrate that P1 inserts more deeply in lipid membranes and induces a major conformational rearrangement of the lipid headgroups, which leads to bilayer defects and significant water penetration into the interstices of the membrane. Remarkably, both peptides bound to Cu2+ act as nucleases that damage isolated DNA within minutes and P3 is significantly more effective than P1. In the context of bacterial cells, we show that copper plays an essential role in planktonic, biofilm, and persister cell eradication. Furthermore, we find that the more membrane-active P1 kills bacteria rapidly while the more DNA-damaging P3 results in slower cell death. Taken together, these results identify host defense metallopeptides with complementary and multi-pronged mechanisms of action as valuable templates to consider for the design of novel strategies against drug resistant bacteria.