An Insect Defensin-Derived β‑Hairpin Peptide with Enhanced Antibacterial Activity

Insect defensins are a class of small, cysteine-rich antimicrobial peptides primarily active on Gram-positive bacteria. Their roles in maggot therapy for treating chronic wound infection have been reported recently. However, a relatively narrow antibacterial spectrum together with the lack of a cost-effective means of commercial-scale production has limited their application. To further exploit the therapeutic potential of these molecules, we engineered the carboxyl-terminal β-sheet of navidefensin2-2, an insect defensin from <i>Nasonia vitripennis</i>, based on its structural similarity to naturally occurring microbicidal β-hairpin peptides. The designed peptide of 14 residues, referred to as <i>Nv</i>BH, spans the β-sheet region of the defensin with two amino acids substituted for assembly of a disulfide-bonded amphipathic β-hairpin structure. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) combined with circular dichroism (CD) analysis shows that the oxidized <i>Nv</i>BH (o<i>Nv</i>BH), produced from the synthetic peptide by air oxidization in an alkaline environment, folds into a typical β-hairpin structure linked by two disulfide bridges (Cys1–Cys4; Cys2–Cys3). However, such a structure appears not to be functionally necessary as synthetic <i>Nv</i>BH with a spontaneously oxidized disulfide bridge (Cys2–Cys3) (termed po<i>Nv</i>BH) displayed similar antibacterial potency to o<i>Nv</i>BH. In comparison with o<i>Nv</i>BH, po<i>Nv</i>BH exhibited higher serum stability and more resistance on tryptic digestion. These two forms of peptides are capable of killing an array of Gram-positive (including antibiotic-resistant strains of <i>Staphylococcus</i>) and Gram-negative bacterial pathogens at low micromolar concentrations through a membrane disruptive mode of action. Our work indicates that the β-sheet region of insect defensins is a promising subdomain of proteins in anti-infective drug discovery.