High-throughput and facile assay of antimicrobial peptides using pH-controlled fluorescence resonance energy transfer

Amphipathic antimicrobial peptides can destroy bacteria cells by inducing membrane permeabilization, forming one strategy for innate defense by various organisms. However, although the antimicrobial peptides are considered a promising alternative for use against multidrug-resistant bacteria, large-scale screening of potential candidate antimicrobial peptides will require a simple, rapid assay for antimicrobial activity. Here, we describe a novel fluorescence resonance energy transfer (FRET)-based assay system for antimicrobial peptides which takes advantage of pH-related changes in FRET efficiency due to the instability of enhanced yellow fluorescent protein versus the stability of enhanced cyan fluorescent protein in a reduced-pH environment. We successfully showed that quantification of antimicrobial activity is possible through a difference of FRET efficiency between ECFP-EYFP fusion molecules released from disrupted Escherichia coli in an extracellular environment (pH 6) and those retained in an intracellular environment (pH approximately 7). Thus, we herein suggest a new simple, effective, and efficient pH-controlled FRET-based antimicrobial peptide screening method applicable to high-throughput screening of candidate peptide libraries.