Evaluation of the antimicrobial efficiency of three novel chimeric peptides through biochemical and biophysical analyses

Abstract

Three chimeric membrane-active antimicrobial peptides (AMPs) were designed from previously characterized parental molecules, namely pandinin-2, ascaphin-8, and maximin-3. The aim of constructing these chimeras was to obtain sequences with improved therapeutic indices or increased activity, while simultaneously investigating the functional roles of key segments of the parental peptides. Chimera-1 was the most active peptide against clinically relevant bacterial species, followed by chimera-2, and chimera-3, respectively, with no clear preference towards Gram-negative or Gram-positive strains. Escherichia coli and Pseudomonas aeruginosa were the most sensitive bacteria, while Klebsiella pneumoniae and Staphylococcus aureus were resistant to AMP activity. All peptides presented significantly lower activities towards human erythrocytes, with chimera-1 being the most selective. Additionally, only chimera-2 showed cytotoxicity towards Vero cells. Calcein leakage and dynamic light scattering assays using liposomal formulations indicated that the chimeras conserved the pore forming membrane perturbation mechanisms of the parental molecules. Peptide interaction also reduced membrane fluidity. Circular dichroism (CD) data showed disordered peptides in aqueous solution that transitioned into alpha helical structures lipid bilayer environments. In silico assessments correlated well with microbiological and in vitro experimental data. All peptides established greater contact with the bacterial biomimetic membrane compared to the erythrocyte system, as analyzed by distance from membrane surface, number of contacts, solvent accessible surface area, and number of hydrogen bonds. Additionally, the presence of the bilayer lipid patches favored peptide folding, consistent with CD experiments. Molecular dynamics simulations of peptide aggregation revealed that chimera-2 formed the largest oligomers, consistent with the predicted aggregation propensities and the predicted physico-chemical properties. Interaction with membrane surfaces resulted in smaller clusters while low or lack of interaction favored larger aggregates. Overall, the chimeric peptides displayed higher activity and selectivity compared to the parental ones. The contribution of the flanking regions of pandidin-2 and maximin-3 with respect to the core region of ascaphin-8 was not clear.

Keywords: Biophysical characterization; Chimeric antimicrobial peptides; Lipid-peptide interaction microbial assays; Molecular dynamics simulations.