Proline-rich antimicrobial peptides (PrAMPs) are promising agents to combat multi-drug resistant pathogens due to a high antimicrobial activity, yet low cytotoxicity. A library of derivatives of the PrAMP Bac5(1–17) was synthesized and screened to identify which residues are relevant for its activity. In this way, we discovered that two central motifs -PIRXP- cannot be modified, while residues at N- and C- termini tolerated some variations. We found five Bac5(1–17) derivatives bearing 1–5 substitutions, with an increased number of arginine and/or tryptophan residues, exhibiting improved antimicrobial activity and broader spectrum of activity while retaining low cytotoxicity toward eukaryotic cells. Transcription/translation and bacterial membrane permeabilization assays showed that these new derivatives still retained the ability to strongly inhibit bacterial protein synthesis, but also acquired permeabilizing activity to different degrees. These new Bac5(1–17) derivatives therefore show a dual mode of action which could hinder the selection of bacterial resistance against these molecules.
