An intracellular release peptide display technology unveils an antimicrobial peptide with low probability for resistance development.

Discovery of bioactive peptides, including those acting to permeabilize and/or kill bacterial cells (antimicrobial peptides) has drawn extensive interest in recent years. However, current technologies for their identification are limited. To address these limitations, the Intracellular Release Peptide Display (IRPD) technology allowing the recombinant "display" of intracellular linear peptides was developed. IRPD uses the protease domain of the capsid protein from the Semliki Forest virus as a scaffold to express and liberate linear peptides intracellularly in Escherichia coli. IRPD is a universal platform that allows screening of millions of peptides and the discovery of bioactive peptides from direct target interactions and independent of the cell envelope barrier. Here, we identified peptides that cause increased bacterial cell envelope permeability and lysis. The most promising candidate, P38, effectively kills Gram-negative pathogens by disrupting the inner membrane without detectable resistance development. Thus, P38 constitutes an interesting hit peptide for further development.