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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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Antibacterial activity of Ent-Amp/Amx against various E.coli strains that include human pathogens. (A) Laboratorytest 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 assayswere performed in 50% MHB medium supplemented with 200 μM DPto provide iron-limiting conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DPare presented in Figures S2–S7.
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fig2: Antibacterial activity of Ent-Amp/Amx against various E.coli strains that include human pathogens. (A) Laboratorytest 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 assayswere performed in 50% MHB medium supplemented with 200 μM DPto provide iron-limiting conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DPare presented in Figures S2–S7.

Mentions: To ascertain whether Ent-Amp/Amx provide antibacterial activityagainst E. coli, including pathogenic strains,76 we preformed antimicrobial activity assays usingsix strains (Table S1, Figures 2, S2–S7). E. coli ATCC 25922 is a laboratory susceptibility test strainoriginally obtained as a clinical isolate. E. coli H9049 is a non-pathogenic clinical isolate.77E. coli UTI8978 andCFT07379 are both pathogens of the humanurinary tract (UPEC).80E. coli ATCC 35401 (serotype O78:H11) is an enterotoxigenic (ETEC) strainthat was isolated from human feces. E. coli ATCC43895 (serotype O157:H7) is an enterohemorrhagic (EHEC) strain thatwas isolated from raw hamburger meat implicated in a hemorrhagic colitisoutbreak. Both ETEC and EHEC strains produce virulence factors andtoxins and cause diarrhea in humans.76 All E. coli strains biosynthesize Ent for iron acquisition andexpress the Ent receptor FepA. Some E. coli strainsemployed in this work also have the capacity to produce and utilizesalmochelins, C-glucoyslated Ent derivatives.81 These molecules are produced by Salmonella spp.and pathogenic E. coli strains for iron acquisition.The iroA gene cluster (iroBCDEN)encodes proteins required for the biosynthesis and transport of salmochelins,and IroN is the outer membrane receptor for salmochelins encoded bythe iroA cluster.82,83 Studies with Salmonella indicate that IroN has the ability to transportEnt as well as its glucosylated forms.84 Of the strains considered in this work, E. coli CFT073 and UTI89 harbor the iroA gene cluster. E. coli H9049 does not produce salmochelins,85 and a BLAST search using available E.coli genomes reveals that the E. coli 43985genome does not contain the iroA cluster. The genomefor E. coli ATCC 25922 is unpublished; however, thisstrain is reported to be sensitive to lipocalin-2 (vide infra),86 which suggests that its genome doesnot encode the iroA cluster. Whether E. coli 35401 produces salmochelins is unclear from the available literature.Lastly, it should be noted that E. coli CFT073 iscelebrated for having redundant iron import machineries, and thisstrain also harbors the iha gene, which encodes theouter membrane Ent receptor Iha that is distinct from FepA.87


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

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

Antibacterial activity of Ent-Amp/Amx against various E.coli strains that include human pathogens. (A) Laboratorytest 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 assayswere performed in 50% MHB medium supplemented with 200 μM DPto provide iron-limiting conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DPare presented in Figures S2–S7.
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fig2: Antibacterial activity of Ent-Amp/Amx against various E.coli strains that include human pathogens. (A) Laboratorytest 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 assayswere performed in 50% MHB medium supplemented with 200 μM DPto provide iron-limiting conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DPare presented in Figures S2–S7.
Mentions: To ascertain whether Ent-Amp/Amx provide antibacterial activityagainst E. coli, including pathogenic strains,76 we preformed antimicrobial activity assays usingsix strains (Table S1, Figures 2, S2–S7). E. coli ATCC 25922 is a laboratory susceptibility test strainoriginally obtained as a clinical isolate. E. coli H9049 is a non-pathogenic clinical isolate.77E. coli UTI8978 andCFT07379 are both pathogens of the humanurinary tract (UPEC).80E. coli ATCC 35401 (serotype O78:H11) is an enterotoxigenic (ETEC) strainthat was isolated from human feces. E. coli ATCC43895 (serotype O157:H7) is an enterohemorrhagic (EHEC) strain thatwas isolated from raw hamburger meat implicated in a hemorrhagic colitisoutbreak. Both ETEC and EHEC strains produce virulence factors andtoxins and cause diarrhea in humans.76 All E. coli strains biosynthesize Ent for iron acquisition andexpress the Ent receptor FepA. Some E. coli strainsemployed in this work also have the capacity to produce and utilizesalmochelins, C-glucoyslated Ent derivatives.81 These molecules are produced by Salmonella spp.and pathogenic E. coli strains for iron acquisition.The iroA gene cluster (iroBCDEN)encodes proteins required for the biosynthesis and transport of salmochelins,and IroN is the outer membrane receptor for salmochelins encoded bythe iroA cluster.82,83 Studies with Salmonella indicate that IroN has the ability to transportEnt as well as its glucosylated forms.84 Of the strains considered in this work, E. coli CFT073 and UTI89 harbor the iroA gene cluster. E. coli H9049 does not produce salmochelins,85 and a BLAST search using available E.coli genomes reveals that the E. coli 43985genome does not contain the iroA cluster. The genomefor E. coli ATCC 25922 is unpublished; however, thisstrain is reported to be sensitive to lipocalin-2 (vide infra),86 which suggests that its genome doesnot encode the iroA cluster. Whether E. coli 35401 produces salmochelins is unclear from the available literature.Lastly, it should be noted that E. coli CFT073 iscelebrated for having redundant iron import machineries, and thisstrain also harbors the iha gene, which encodes theouter membrane Ent receptor Iha that is distinct from FepA.87

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