Antimicrobial Peptide Piscidin Permeabilizes Bacterial Membranes and Binds Intracellular Targets at Sub-Lethal Concentrations

Abstract

Piscidins 1 (P1) and 3 (P3) are cationic α-helical membrane active antimicrobial peptides originally isolated from the hybrid striped seabass. The peptides, which possess broad-spectrum activity against bacteria, fungi, and viruses, bind to anionic membranes and lyse them once a sufficient concentration has been reached. In this study, we used confocal microscopy and FITC-labeled piscidins to show that at sub-inhibitory concentrations (0.75 μM), P1 and P3 are able to translocate across the membrane of Gram-positive and -negative bacteria. The FITC signal of the peptides was co-localized with the DAPI signal of the nucleoid region of the bacteria. CD using a 15 base pair piece of DNA and a gel retardation assay using a 1782 base pair piece of DNA confirmed that P1 and P3 could bind to DNA in vitro. It appears that P3 binding was more disruptive to the structure of DNA, and this may be due to the extra arginine residue in P3's sequence. However, these results did not indicate whether the peptides translocated through the membrane or entered the cells via pores formed in the membrane. To probe this question, we used the bacterial strain E. coli ML35 in a permeabilization assay to determine at which peptide concentration the membrane became permeabilized. Our data indicate that at the lowest concentration tested (0.1 μM) the membrane of ML35 cells became permeabilized by P1 and P3. Our results agree with prior findings that permeabilization of bacterial cell membranes can occur prior to lysis and cell death. The implications of piscidin's ability to translocate into bacterial cells and bind to intracellular targets at sub-lethal concentrations will be discussed in terms of its mechanism of action, which was previously believed to be based on its membrane activity.