Antibacterial properties of an oligo-acyl-lysyl hexamer targeting Gram-negative species

Abstract

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 μM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

Fig 1

Molecular structure of two OAK analogs bearing 7 positive charges. The upper structure is composed of α₁₂-5α₁₀. The lower structure is of a previously investigated sequence (8) composed of C₁₂K-3β₁₀.

Fig 2

Selectivity of action. (Left) Cytotoxicity of α₁₂-5α₁₀ (black circles) and its deaminated analog, C₁₂K-5α₁₀ (white circles), assessed using a commercial kit (Cell Proliferation XTT kit) after incubation with 5 × 10⁴ human foreskin fibroblasts (3 h at 37°C); (right) hemolytic activity assessed with human blood (10% hematocrit) after incubation in PBS (1 h at 37°C).

Fig 3

Dose dependence of three kinetic assays performed with E. coli 35218: (a) cell permeabilization assessed by EtBr accumulation; (b) cell membrane depolarization assessed by the potential sensitive dye DiSC3 (5); (c) bacterial growth kinetics assessed by CFU count. Symbols: circles, untreated controls; triangles, squares, right triangles, left triangles, and stars, increasing concentrations of α₁₂-5α₁₀ (1.4, 2.9, 5.8, 11.5, and 23 μg/ml, respectively). Error bars represent means ± standard deviations.

Fig 4

Synergy and its molecular basis assessed against a representative E. coli strain. (a) Evolution of erythromycin's MIC values when determined in the presence of a sub-MIC of OAK (white bars) or OAK MIC values in the presence of a sub-MIC of erythromycin (gray bars). (b to d) Assessment of a representative set of synergistic conditions, i.e., 2.9 μg/ml α₁₂-5α₁₀ (triangle), 16 μg/ml erythromycin (inverted triangle), and their combination (diamond) performed to determine growth kinetics (b), depolarization (c), and permeabilization (d) of strain 35218.

Fig 5

Mechanistic studies using an isogenic pair of E. coli strains: acrAB-deletion mutant AG100A and wild-type strain AG100. (Top) Cell permeability to ethidium bromide; (bottom) membrane depolarization in these strains at concentrations of 2.9 μg/ml OAK (triangle) and 16 μg/ml erythromycin (inverted triangle) and with their combination (diamond).