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

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