Antibacterial mechanism of action of arylamide foldamers

Abstract

Small arylamide foldamers designed to mimic the amphiphilic nature of antimicrobial peptides (AMPs) have shown potent bactericidal activity against both Gram-negative and Gram-positive strains without many of the drawbacks of natural AMPs. These foldamers were shown to cause large changes in the permeability of the outer membrane of Escherichia coli. They cause more limited permeabilization of the inner membrane which reaches critical levels corresponding with the time required to bring about bacterial cell death. Transcriptional profiling of E. coli treated with sublethal concentrations of the arylamides showed induction of genes related to membrane and oxidative stresses, with some overlap with the effects observed for polymyxin B. Protein secretion into the periplasm and the outer membrane is also compromised, possibly contributing to the lethality of the arylamide compounds. The induction of membrane stress response regulons such as rcs coupled with morphological changes at the membrane observed by electron microscopy suggests that the activity of the arylamides at the membrane represents a significant contribution to their mechanism of action.

Fig. 1.

Antimicrobial activity of arylamide foldamers. (A) Structures and MICs of PMX 10070 and PMX 10072 against E. coli D31; (B) bactericidal activity of PMX 10070, PMX 10072, and PmB at MIC against E. coli D31; (C to E) E. coli D31 growth in 1× (red), 2× (blue), and 4× (green) MIC treatment with PMX 10070 (C), PMX 10072 (D), and polymyxin B (E).

Fig. 2.

Outer and inner membrane permeabilization induced by arylamide treatment. (A) Control normalized rate of nitrocefin hydrolysis (s⁻¹) upon treatment of E. coli D31 cultures for 5 min, 30 min, and 60 min with the indicated concentrations of polymyxin B, PMX 10070, and PMX 10072; (B) control-normalized A₄₂₀ upon treatment of E. coli D31 cultures for 5 min, 30 min, and 60 min with the indicated concentrations of polymyxin B and 5 min, 30 min, 60 min, and 120 min with the indicated concentrations of PMX 10070 and PMX 10072.

Fig. 3.

Changes in cell morphology upon treatment with PMX 10072. Cultures of E. coli D31 were incubated with 62.5 μg/ml (10× MIC) of PMX 10072 for the indicated times, stained with uranyl acetate, and visualized by TEM. (A) The membrane is distinct and uniform prior to treatment. (B) After 1 min exposure, the cytoplasm appears dark due to increased stain accessibility. (C) A diffuse halo appears around the cell 2 min after exposure. (D) By 4 min after treatment, the cells reestablish cellular integrity, although they continue to be permeable to stain. (E) The membrane becomes more nonuniform with extensive ruffling with exposure for 8 min. (F) Vesiculation of the outer membrane occurs after 20 min of exposure. (G and H) Vesiculation continues to worsen (G) and results in the total loss of membrane integrity (H).

Fig. 4.

Arylamides cause defects in β-lactamase protein translocation. Cultures of E. coli were treated with inhibitory concentrations of PMX 10070 and PMX 10072 (25 μg/ml) for the indicated times, and β-lactamase (bla) was detected by Western blotting. Twenty-six amino acids are cleaved from the N terminus of the precursor bla (unprocessed) upon translocation, yielding mature bla in the periplasm (processed). (A) Arylamide treatment causes precursor accumulation due to inefficient translocation. CCCP treatment was used as a positive control. (B) Growth attenuation caused by PMX 10070, PMX 10072, and CCCP treatment is comparable.

Fig. 5.

DNA microarray showing upregulation of stress-induced genes by PMX 10070 and polymyxin B. E. coli genes upregulated >1.7-fold by PMX 10070 (8.75 μg/ml, 0.7× MIC) and polymyxin B (0.39 μg/ml, 1× MIC) treatment 20 and 60 min after exposure. (A) Upregulated genes in response to polymyxin B treatment for 20 min (a) and 60 min (b) and PMX 10070 treatment for 20 min (c) and 60 min (d). rpoS-regulated stress response genes are highlighted in red. (B) Upregulated stress response genes are regulated by rcs, marA-soxS, cpxAR-baeSR, kdp, and rpoS. (C) Most upregulated genes by exposure time (20 min and 60 min), treatment (polymyxin B and PMX 10070), and time and treatment belong to the rcs phosphorelay.

Fig. 6.

RT-PCR quantification of rcs upregulation by polymyxin B and PMX 10070 treatment. Cultures of E. coli D31 were treated with 0.7× MIC of PMX 10070 and 1× MIC of polymyxin B. RT-PCR shows the time course of the upregulation of 3 downstream rcs genes, rcsA (A), wza (B), and osmB (C). Fold change is normalized to the cDNA abundance of the housekeeping gene 16S rRNA.

Fig. 7.

rcs induction by arylamides is rcsF dependent. β-Gal activity of cpsB-lacZ reporter in wild-type, ΔrcsB, ΔrcsC, and ΔrcsF E. coli strains in response to treatment with 12.5 μg/ml PMX 10070 (A) and 12.5 μg/ml PMX 10072 (B) for 60 min and 120 min.