Bacterial membrane activity of α-peptide/β-peptoid chimeras: Influence of amino acid composition and chain length on the activity against different bacterial strains

<p>Abstract</p> <p>Background</p> <p>Characterization and use of antimicrobial peptides (AMPs) requires that their mode of action is determined. The interaction of membrane-active peptides with their target is often established using model membranes, however, the actual permeabilization of live bacterial cells and subsequent killing is usually not tested. In this report, six α-peptide/β-peptoid chimeras were examined for the effect of amino acid/peptoid substitutions and chain length on the membrane perturbation and subsequent killing of food-borne and clinical bacterial isolates.</p> <p>Results</p> <p>All six AMP analogues inhibited growth of twelve food-borne and clinical bacterial strains including Extended Spectrum Beta-Lactamase-producing <it>Escherichia coli</it>. In general, the Minimum Inhibitory Concentrations (MIC) against Gram-positive and -negative bacteria were similar, ranging from 1 to 5 μM. The type of cationic amino acid only had a minor effect on MIC values, whereas chain length had a profound influence on activity. All chimeras were less active against <it>Serratia marcescens </it>(MICs above 46 μM). The chimeras were bactericidal and induced leakage of ATP from <it>Staphylococcus aureus </it>and <it>S. marcescens </it>with similar time of onset and reduction in the number of viable cells. EDTA pre-treatment of <it>S. marcescens </it>and <it>E. coli </it>followed by treatment with chimeras resulted in pronounced killing indicating that disintegration of the Gram-negative outer membrane eliminated innate differences in susceptibility. Chimera chain length did not influence the degree of ATP leakage, but the amount of intracellular ATP remaining in the cell after treatment was influenced by chimera length with the longest analogue causing complete depletion of intracellular ATP. Hence some chimeras caused a complete disruption of the membrane, and this was parallel by the largest reduction in number of viable bacteria.</p> <p>Conclusion</p> <p>We found that chain length but not type of cationic amino acid influenced the antibacterial activity of a series of synthetic α-peptide/β-peptoid chimeras. The synthetic chimeras exert their killing effect by permeabilization of the bacterial cell envelope, and the outer membrane may act as a barrier in Gram-negative bacteria. The tolerance of <it>S. marcescens </it>to chimeras may be due to differences in the composition of the lipopolysaccharide layer also responsible for its resistance to polymyxin B.</p

Delivery of siRNA Complexed with Palmitoylated α‑Peptide/β-Peptoid Cell-Penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-Based Nanocarrier System

Proteolytically stable α-peptide/β-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing <i>in vitro</i>. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/β-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs

High in vitro antimicrobial activity of β-peptoid–peptide hybrid oligomers against planktonic and biofilm cultures of Staphylococcus epidermidis

An array of β-peptoid-peptide hybrid oligomers displaying different amino acid/peptoid compositions and chain lengths was studied with respect to antimicrobial activity against Staphylococcus epidermidis both in planktonic and biofilm cultures, comparing the effects with those of the common antibiotic vancomycin. Susceptibility and time-kill assays were performed to investigate activity against planktonic cells, whilst confocal laser scanning microscopy was used to investigate the dynamics of the activity against cells within biofilms. All tested peptidomimetics were bactericidal against both exponentially growing and stationary-phase S. epidermidis cells with similar killing kinetics. At the minimum inhibitory concentration (MIC), all peptidomimetics inhibited biofilm formation, whilst peptidomimetics at concentrations above the MIC (80-160μg/mL) eradicated young (6-h-old) biofilms, whilst even higher concentrations were needed to eradicate mature (24-h-old) biofilms completely. Chiral and guanidinylated hybrids exhibited the fastest killing effects against slow-growing cells and had more favourable antibiofilm properties than analogues only containing lysine or lacking chirality in the β-peptoid residues. However, the results of the mature biofilm killing assay indicated more complex structure-activity relationships. Cytotoxicity assays showed a clear correlation between oligomer length and cell toxicity within each subclass of peptides, but all possessed a high differential toxicity favouring killing of bacterial cells. This class of peptidomimetics may constitute promising antimicrobial alternatives for the prevention and treatment of multidrug-resistant S. epidermidis infections