Eradication of established biofilm communities of pathogenic bacteria is one of the pending challenges in the development of new antimicrobial agents. In particular, the dreaded nosocomial Pseudomonas aeruginosa forms microbial communities that offer an enhanced resistance to conventional antibiotics. Recently, we have described an engineered antimicrobial peptide derived from the human RNase3, also named the eosinophil cationic protein (ECP), RN3 (5-36), which combines bactericidal activity with high cell agglutination and lipopolysaccharide (LPS) affinity. Through a single replacement scan library using the SPOT methodology we have evaluated both the contribution of sequence positioning and amino acid singularity towards the peptide biological and physicochemical properties. Results indicate that the ECP N-terminus has already been extensively improved through evolution to provide high antimicrobial activity; hence most substitutions improving its antimicrobial performance are in detriment of safety towards host tissues. Only three positions were identified, occupied by polar residues on the first α-helix of the protein and replaceable by a hydrophobic residue, allowing an extended N-terminal patch that mediates bacterial agglutination. Among the best candidates, an Ile replacement proved best in improving the peptide therapeutic window. The novel engineered peptides encompass both the LPS-binding and aggregation-prone regions of parental ECP, providing the appropriate structural features for peptide attachment to the bacterial exopolysaccharide layer and bacterial cell membrane destabilization, thereby promoting biofilm removal at micro molar concentrations. We conclude that the novel engineered peptides are promising lead candidates against Gram-negative biofilms.
Keywords: Antimicrobial peptides; Biofilms; Pseudomonas aeruginosa; Single replacement scan library.
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