Structural determinants of the scorpion venom peptide Uy234 govern bactericidal activity and membrane-disruptive properties

Introduction: The growth-inhibiting effect of the peptide Uy234, present in the venom of the scorpion Urodacus yaschenkoi, has been investigated in two bacterial pathogens: Staphylococcus aureus ATCC 25923 and Acinetobacter baumannii AE12, the latter being a multidrug-resistant clinical isolate. With the aim of determining the possible role of specific residues in the bioactivity of this peptide, we studied a proline residue at position 9 and the C-terminal amidation of this peptide. Methods: Two inactivated variants were analyzed: Uy234-C, a non-amidated peptide, and Uy234-A, a P9A mutant. In addition to quantifying in detail the minimum inhibitory and bactericidal concentrations for each microorganism, membrane-damaging effects were assessed through bacterial cell viability assays with SYTO9/PI fluorophores. In addition, AFM, electroforming, and GUV microaspiration were used to determine the effects of each peptide in terms of permeabilization. Molecular dynamics (MD) simulations were also performed for the wild-type peptide and its P9A mutant. Results: Only the native peptide Uy234 showed bacteriostatic and bactericidal activity, whereas the P9A mutant and non-amidated variant lost antimicrobial activity, demonstrating the essential role of the Pro-9 residue and C-terminal amidation in Uy234 bioactivity against both pathogens. SYTO9/PI assays in S. aureus infection showed membrane damage only with native Uy234, while AFM and GUV studies revealed membrane thinning, lateral expansion, and dose-dependent permeabilization of lipid bilayers. Discussion: Our study provides clear evidence of a damaging effect on the membrane associated with the bioactivity of Uy234. This bioactivity is directly associated with the presence of residue P9 and the presence of C-terminal carboxyamidation. The mutant peptide P9A is unable to permeabilize GUVs, which is consistent with the persistence of a greater degree of structural order, according to MD simulations in the aqueous phase. This study provides a framework for the rational design of bactericidal peptides targeting multidrug-resistant bacteria.