Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1-21) Confers Activity against , Including Drug-Resistant and Biofilm Phenotype.

, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) () that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide were designed by replacing Gly with α-aminoisobutyric acid (Aib), Pro, and dPro (-, respectively). The single substitution Gly → Aib in peptide makes it active against the planktonic form of Gram-positive bacterial strains, especially , including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide showed a higher antibiofilm activity than peptide against both reference and clinical isolates of . . Peptide was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide evidenced that the improved biological activity of peptide is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide , and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.