Insights into the mechanism of action of two analogues of aurein 2.2

The naturally occurring host defense peptide (HDP), aurein 2.2, secreted by the amphibian Litoria aurea, acts as a moderate antibacterial, affecting Gram positive bacteria such as Staphylococcus aureus by forming selective ion pores. In a quest to find more active analogues of aurein 2.2, peptides 73 and 77 were discovered. These peptides were rich in arginine and tryptophan and found to have MICs of 4 μg/mL. Here we examined what impact the increased charge from +2 to +3 and a slight increase in hydrophobic moment relative to aurein 2.2 had on the mechanism of action of these two analogues. Using a time-kill assay, both peptides 73 and 77 were found to kill bacteria more effectively than the parent peptide. Using solution CD and NMR, the peptides were found to not adopt a continuous α-helical structure, i.e. the analogues were not helical from residue 1–13 like the parent peptide. Results obtained from oriented CD (OCD), DiSC35 and pyranine assays and a gel retardation experiment showed that the peptides did not function by membrane perturbation and further showed that peptide 73 and 77 did not interact with DNA. Overall, the data were consistent with these peptides acting as cell penetrating peptides with intracellular targets, which did not appear to be DNA. •A library of Arg and Trp rich peptides was derived from the parent peptide, aurein 2.2-Δ3.•Two analogues, peptides 73 and 77, were found to be more active than the parent peptide.•Time-kill assay data shows that peptides 73 and 77 are bactericidal.•Peptides 73 and 77 adopt an α-helical structure for part of the sequence.•These analogues do not function by perturbing membranes, nor by interacting with DNA.