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

Zheng T, Nolan EM - J. Am. Chem. Soc. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

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.

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Time-kill kinetic assays for treatment of E. coli K-12 (top panel) and CFT073 (bottom panel) withAmp/Amx and Ent-Amp/Amx. E. coli K-12 (∼108 CFU/mL) was treatedwith 50 μM of Amp/Amx or 50 μM Ent-Amp/Amx. E.coli CFT073 (∼108 CFU/mL) was treated with50 μM of Amp/Amx or 5 μM Ent-Amp/Amx. The assays wereconducted in 50% MHB medium containing 200 μM DP at 37 °C(mean ± SEM, n = 3).
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fig5: Time-kill kinetic assays for treatment of E. coli K-12 (top panel) and CFT073 (bottom panel) withAmp/Amx and Ent-Amp/Amx. E. coli K-12 (∼108 CFU/mL) was treatedwith 50 μM of Amp/Amx or 50 μM Ent-Amp/Amx. E.coli CFT073 (∼108 CFU/mL) was treated with50 μM of Amp/Amx or 5 μM Ent-Amp/Amx. The assays wereconducted in 50% MHB medium containing 200 μM DP at 37 °C(mean ± SEM, n = 3).

Mentions: The remarkable sensitivity of E. coli CFT073 toEnt-Amp/Amx (Figure 2C) motivated us to investigatethe relative cell-killing kineticsof Ent-Amp/Amx and Amp/Amx to determine whether these conjugates kill E. coli CFT073 more rapidly than the unmodified drugs. Forcomparison between E. coli strains, we also evaluatedthe time-kill kinetics for E. coli K-12. Ent-Amp/Amxprovide more rapid cell death than unmodified Amp/Amx (Figure 5), and this behavior is most apparent for E. coli CFT073, where the OD600 value was almostreduced to the baseline value after 1 h incubation with 5 μMEnt-Amp/Amx, corresponding to a 2-fold log reduction in CFU/mL. Incontrast, the change in OD600 and CFU/mL for E.coli CFT073 treated with 50 μM unmodified Amp/Amx isnegligible over this time period. The time-kill kinetics for E. coli K-12, conducted with 50 μM of both unmodifiedand modified β-lactams, indicate a slight increase in kill kineticsfor Ent-Amp/Amx relative to Amp/Amx, and that the kinetics of cell-killingare slower for K-12 than CFT073 (Figure 5).These results support a model whereby Ent modification facilitatesuptake of Amp/Amx relative to the unmodified drugs. This effect ismore dramatic for E. coli CFT073 than K-12, whichis in accordance with the enhanced antibacterial activity observedfor CFT073 relative to the other E. coli strainsconsidered in this work.


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

Zheng T, Nolan EM - J. Am. Chem. Soc. (2014)

Time-kill kinetic assays for treatment of E. coli K-12 (top panel) and CFT073 (bottom panel) withAmp/Amx and Ent-Amp/Amx. E. coli K-12 (∼108 CFU/mL) was treatedwith 50 μM of Amp/Amx or 50 μM Ent-Amp/Amx. E.coli CFT073 (∼108 CFU/mL) was treated with50 μM of Amp/Amx or 5 μM Ent-Amp/Amx. The assays wereconducted in 50% MHB medium containing 200 μM DP at 37 °C(mean ± SEM, n = 3).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4353011&req=5

fig5: Time-kill kinetic assays for treatment of E. coli K-12 (top panel) and CFT073 (bottom panel) withAmp/Amx and Ent-Amp/Amx. E. coli K-12 (∼108 CFU/mL) was treatedwith 50 μM of Amp/Amx or 50 μM Ent-Amp/Amx. E.coli CFT073 (∼108 CFU/mL) was treated with50 μM of Amp/Amx or 5 μM Ent-Amp/Amx. The assays wereconducted in 50% MHB medium containing 200 μM DP at 37 °C(mean ± SEM, n = 3).
Mentions: The remarkable sensitivity of E. coli CFT073 toEnt-Amp/Amx (Figure 2C) motivated us to investigatethe relative cell-killing kineticsof Ent-Amp/Amx and Amp/Amx to determine whether these conjugates kill E. coli CFT073 more rapidly than the unmodified drugs. Forcomparison between E. coli strains, we also evaluatedthe time-kill kinetics for E. coli K-12. Ent-Amp/Amxprovide more rapid cell death than unmodified Amp/Amx (Figure 5), and this behavior is most apparent for E. coli CFT073, where the OD600 value was almostreduced to the baseline value after 1 h incubation with 5 μMEnt-Amp/Amx, corresponding to a 2-fold log reduction in CFU/mL. Incontrast, the change in OD600 and CFU/mL for E.coli CFT073 treated with 50 μM unmodified Amp/Amx isnegligible over this time period. The time-kill kinetics for E. coli K-12, conducted with 50 μM of both unmodifiedand modified β-lactams, indicate a slight increase in kill kineticsfor Ent-Amp/Amx relative to Amp/Amx, and that the kinetics of cell-killingare slower for K-12 than CFT073 (Figure 5).These results support a model whereby Ent modification facilitatesuptake of Amp/Amx relative to the unmodified drugs. This effect ismore dramatic for E. coli CFT073 than K-12, whichis in accordance with the enhanced antibacterial activity observedfor CFT073 relative to the other E. coli strainsconsidered in this work.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

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.

Show MeSH
Related in: MedlinePlus