Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes

Abstract

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide-β-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.

Keywords: Antimicrobial peptidomimetics; Bacterial membrane; Guanidinium cation; Peptide–peptoid chimeras; Phosphatidylglycerol; X-ray scattering.

Figure 1

Molecular structures of the tested chimeras (A) and lipids used for modeling bacterial cell membranes (B).

Figure 2. Epifluorescence images of DPPG monolayers after injection of N_speK (A) and N_speR (B) at concentrations corresponding to 20% of their MIC values observed against S. aureus respectively

Lipid-linked Texas Red-DHPE fluorescence probe (1 mol %) was added to the phospholipid solutions for EFM experiments. Because of steric hindrance, the dye is located in the liquid-disordered phase, rendering it bright whereas the liquid-ordered phase remains dark.

Figure 3. Electron density profiles and corresponding Fresnel-divided reflectivity curves against the scattering vector (q) in the z direction (q_z) for DPPG (A) and Kdo2-Lipid A (B) monolayers at 30 mN × m^–1

Electron density profiles were normalized to the electron density of the subphase buffer. On the XR graphs, the scatter plots are experimental values and solid lines are the best fits of the models to the experimental data. Fresnel reflectivity is the reflectivity from ideal smooth surface.

Figure 4

Bragg peaks plot of scattering vector Q_xy as a function of intensity.

Figure 5. Cartoon schematic of possible interactions of KβN_speK and RβN_speR with (A) DPPG and (B) Kdo-2 Lipid A monolayers at 30 mN × m^–1

Chimeras carrying amino groups are solely located in the polar head-moieties of DPPG accompanied with considerable thinning of the entire monolayer, whereas their guanidino-substituted analogues form an extra layer on the surface of lipid film resulting in more compact distribution of inserted molecules within the model membrane. Unlike DPPG, the insertion mechanisms of KβN_spe and RβN_spe into Kdo-2 Lipid A model look nearly identical.