Enterobactin-mediated delivery of β-lactam antibiotics enhances antibacterial activity against pathogenic Escherichia coli

Abstract

The design, synthesis, and characterization of enterobactin-antibiotic conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactin scaffold via a stable poly(ethylene glycol) linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide enhanced antibacterial activity against Escherichia coli strains, including uropathogenic E. coli CFT073 and UTI89, enterohemorrhagic E. coli O157:H7, and enterotoxigenic E. coli O78:H11, compared to the parent β-lactams. Studies with E. coli K-12 derivatives defective in ferric enterobactin transport reveal that the enhanced antibacterial activity observed for this strain requires the outer membrane ferric enterobactin transporter FepA. A remarkable 1000-fold decrease in minimum inhibitory concentration (MIC) value is observed for uropathogenic E. coli CFT073 relative to Amp/Amx, and time-kill kinetic studies demonstrate that Ent-Amp/Amx kill this strain more rapidly at 10-fold lower concentrations than the parent antibiotics. Moreover, Ent-Amp and Ent-Amx selectively kill E. coli CFT073 co-cultured with other bacterial species such as Staphylococcus aureus, and Ent-Amp exhibits low cytotoxicity against human T84 intestinal cells in both the apo and iron-bound forms. These studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens utilizing enterobactin for iron acquisition.

Figure 1

Structures of enterobactin (1, Ent) and a generalized enterobactin–cargo conjugate.

Scheme 1. Syntheses of the l and d Forms of Ent-Amp/Amx

Figure 2

Antibacterial activity of Ent-Amp/Amx against various E. coli strains that include human pathogens. (A) Laboratory test strain E. coli ATCC 25922. (B) Uropathogenic E. coli UTI89. (C) Uropathogenic E. coli CFT073. (D) Non-pathogenic clinical isolate E. coli H9049. (E) Pathogenic ETEC E. coli ATCC 35401. (F) Pathogenic EHEC E. coli ATCC 43895. All assays were performed in 50% MHB medium supplemented with 200 μM DP to provide iron-limiting conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DP are presented in Figures S2–S7.

Figure 3

Antibacterial activity of Ent-Amp/Amx against wild-type and mutant E. coli K-12. (A,B) Growth inhibition of E. coli K-12 by Amp/Amx and Ent-Amp/Amx in the absence (A) and presence (B) of DP. (C) Growth inhibition of E. coli K-12 treated with a 1:1 molar ratio of Ent/Amp and Ent/Amx. (D) Growth inhibition of E. coli K-12 treated with ferric Ent-Amp/Amx. (E–G) Growth inhibition of fepA- (E), fepC- (F), and fes- (G) by Amp/Amx and Ent-Amp/Amx. (H,I) Growth of E. coli K-12 in the presence of 1 μM Ent-Amp/Amx (“conjugate”) and mixtures of Ent-Amp/Amx (1 μM) and 1, 5, or 20 equiv of exogenous Ent in the absence (H) and presence (I) of DP. The ** indicates OD₆₀₀ < 0.01. All assays were performed in 50% MHB medium with or without 200 μM DP (see panels) (mean ± SEM, n ≥ 3). The data for additional assays performed in the absence of DP are presented in Figure S8.

Scheme 2. Syntheses of Ent-Hydro-Amp/Amx

Figure 4

β-Lactam is required for Ent-Amp/Amx antimicrobial activity. (A,B) Antibacterial activity assays against E. coli K-12 (A) and CFT073 (B) using Ent-Amp/Amx and Ent-Hydro-Amp/Amx. (C) Antibacterial activity assays against E. coli ATCC 35218, which expresses a class A serine β-lactamase, using Ent-Amp/Amx in the absence and presence of the β-lactamase inhibitors potassium clavulanate (PC) and sulbactam (SB). All assays were performed in 50% MHB supplemented with 200 μM DP (mean ± SEM, n ≥ 3). Additional data are presented in Figure S14.

Figure 5

Time-kill kinetic assays for treatment of E. coli K-12 (top panel) and CFT073 (bottom panel) with Amp/Amx and Ent-Amp/Amx. E. coli K-12 (∼10⁸ CFU/mL) was treated with 50 μM of Amp/Amx or 50 μM Ent-Amp/Amx. E. coli CFT073 (∼10⁸ CFU/mL) was treated with 50 μM of Amp/Amx or 5 μM Ent-Amp/Amx. The assays were conducted in 50% MHB medium containing 200 μM DP at 37 °C (mean ± SEM, n = 3).

Figure 6

Ent-Amp/Amx selectively kill E. coli CFT073 in the presence of S. aureus ATCC 25923. (A,B) Antimicrobial activity assays against S. aureus ATCC 25923 in the absence (A) and presence (B) of 200 μM DP. (C,D) Bacterial growth monitored by OD₆₀₀ for cultures of E. coli only, S. aureus only, and 1:1 E. coli/S. aureus mixtures treated with Amp/Amx or Ent-Amp/Amx in the absence (C) and presence (D) of 200 μM DP. The * indicates OD₆₀₀ < 0.01. (E) Representative photographs of colonies from mixed cultures of E. coli CFT073 and S. aureus ATCC 29523 treated with Ent-Amp/Amx (1 μM) or Amp/Amx (1 μM) in the presence of 200 μM DP. All assays were conducted in 50% MHB medium (t = 19 h, 30 °C) (mean ± SEM, n ≥ 3 for A–D).

Figure 7

Ent-Amp exhibit negligible cytotoxicity toward human T84 intestinal epithelial cells. Percent cell survival quantified by MTT assay after a 24 h treatment with apo or iron-bound Ent, Ent-Amp, and the parent antibiotic Amp in the absence and presence of 1 equiv of Fe(III) (mean ± SEM, n = 3).

Figure 8

Antibacterial activity of Ent-Amp against E. coli CFT073 in the presence of lcn2 or BSA. (A) E. coli CFT073 treated with 100 nM Ent-Amp and varying concentrations of lcn2 or BSA control. (B) E. coli CFT073 treated with Ent-Amp, varying concentration of Ent, and varying concentrations of lcn2 or BSA control. The assays were performed in M9 minimal medium (24 h, 37 °C) (mean ± SEM, n ≥ 3). The ** indicates OD₆₀₀ < 0.01.