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.
Kim
Young Soo
YS
Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea.
Cha
Hyung Joon
HJ
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D054806:Magainins; D008826:Microbial Sensitivity Tests / Q000379:methods*; D029867:Xenopus Proteins / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D054806:Magainins; D029867:Xenopus Proteins; C054562:yellow fluorescent protein, Bacteria; C053301:magainin 2 peptide, Xenopus; D049452:Green Fluorescent Proteins