Mechanism of a prototypical synthetic membrane-active antimicrobial: Efficient hole-punching via interaction with negative intrinsic curvature lipids

Abstract

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal H(II) phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from approximately 1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.

Fig. 1.

The limitation of dye leakage experiments. (A) Alexa480 fluorescence intensity from tagged dextran encapsulated in antimicrobial-treated DOPG/DOPC = 20/80 GUVs. (B) Alexa480 fluorescence intensity from tagged dextran encapsulated in antimicrobial-treated DOPG/DOPE = 20/80 GUVs is extremely weak because of leakage. Dotted white lines indicate the outline of vesicle membranes. (Scale bars, 10 μm, in A and B.) These dye leakage results and others suggest that a trend of increasing leakage with increasing PE lipid content in membranes after treated with the phenylene ethynylene antimicrobials. (C) Confocal microscopy results show circularly integrated intensities of fluorescently tagged dextran inside antimicrobial treated GUVs for PE-rich membranes composed of DOPG/DOPE = 20/80 and for PC-rich membranes composed of DOPG/DOPC = 20/80. Data from 10 GUVs are averaged into the PE-rich trace and 17 into the PC-rich trace. Fluorescence intensity is normalized so that untreated vesicles have fluorescence intensity of 1.0, so clearly there is significant nonspecific leakage, which complicates interpretation.

Fig. 2.

PE-deficient E. coli mutant out-survives the WT against the membrane-active antimicrobial. (A) Microbicidal assays show that, in the presence of the membrane-active phenylene ethynylene-based antimicrobial, the survival of the fragile PE-deficient mutant strain AD93 is much greater than that of its WT parent strain W3899 (≈3 orders of magnitude at 4 μg/ml) in sterile buffer solution (10 mM Hepes, 60 mM NaCl, 100 μM MgCl₂, and 400 mM sucrose, pH 7.5). (B) When being exposed to tobramycin, a conventional antibiotic that targets the ribosome rather than the bacterial membrane, the WT parent strain (W3899) survived at a drastically higher rate than the mutant strain (AD93) (≈4 orders of magnitude at 1 μg/ml) in sterile buffer solution (10 mM Hepes, 60 mM NaCl, and 200 μM MgCl₂, pH 7.5). Data points are reported as mean ± standard deviation.

Fig. 3.

The structure of induced H_II phase changes with antimicrobial concentration. (A) Structure of phenylene ethynylene-based antimicrobial. (B Top) Formation of a transmembrane pore requires both negative and positive curvatures (black and red arrows, respectively). (Bottom) Schematic representation of how this PE-dependent structural tendency may be realized on a membrane, in which the antimicrobials recruit PE lipids to achieve the negative circumferential curvature necessary for pore formation. The white and green spheres represent headgroups of zero intrinsic curvature (e.g., DOPG, DOPC) and negative intrinsic curvature lipids (e.g., DOPE) respectively. The antimicrobials are represented by blue spherocylinders. (C) Synchrotron SAXS data show that the inverted hexagonal phase induced by antimicrobial in DOPG/DOPE = 20/80 membranes evolves structurally as antimicrobial to lipid molar ratio (A/L) varies from 1/122 to 1/9.5. (D) The integrated diffraction intensities increase in a sigmoidal manner and saturate at A/L ∼ 1/14.

Fig. 4.

Cooperativity of antimicrobial occupation is described by a Potts model. (A) A typical electron density profile of the 2D hexagonal unit cell confirms the formation of an inverted hexagonal structure. Here, we show the 2D plot of the reconstructed electron density profile at A/L = 1/14. The light blue regions have low electron density (≈0.29 e/ų) and correspond to hydrocarbon chains of the lipids, whereas the “rims” in dark red have the highest electron density (≈0.55 e/ų) and correspond to the regions near phospholipid head groups. The rim centers in lighter blue have intermediate electron density (≈0.33 e/ų) and correspond to water within hydrophilic channels. (B and C) The diameter of water channels (B) and the electron density at the hydrophilic surface of lipid membranes (C) change abruptly as A/L increases (red arrows). (D and E) By analogy with exactly solvable 1D magnetic systems, we deduce thermodynamic parameters governing antimicrobial cooperativity in generating the necessary negative membrane curvature, using the antimicrobial occupancy data in the induced H_II phase. We estimated the antimicrobial occupancy based on the relative enhancement in electron density around the hydrophilic membrane surface. Using Bayesian sampling of model fits, we show the range of predictions obtained from 1D 3-Potts and the simpler Ising model fits (D). Both 3-Potts model and Ising model give excellent fits with identical values for E_1–3. We find that the antimicrobial occupation as a function of system-wide antimicrobial concentration is highly nonlinear, but is well described by a simple two-level system of singly and triply occupied states separated by a well defined free energy difference E_1–3 of ≈ 4.68 kT, which quantifies the cooperativity in these antimicrobials. (E) The posterior distribution with normal likelihood from the experimental data are sampled by using Monte Carlo for 10⁵ steps, and an ensemble of 10³ 3-Potts model parameter choices are taken. The median predicted site occupancy with upper and lower quartiles show the range of model predictions. The E_1–3 binding free energy has the narrowest distribution with a full width of 0.04 kT (a measure of the statistical uncertainty of the fit, independent of the experimental error), whereas the other model parameters have variability of at least 1 kT. The histogram of E_1–3 binding parameters from the Bayesian sampling shows this narrow distribution.