title: Inhibitors of TonB function identified by a high-throughput screen for inhibitors of iron acquisition in uropathogenic Escherichia coli CFT073.
abstract: The urinary tract is one of the most common sites of infection in humans, and uropathogenic Escherichia coli (UPEC) is the main causative agent of urinary tract infections. Bacteria colonizing the urinary tract face extremely low iron availability. To counteract this, UPEC expresses a wide variety of iron acquisition systems. To exploit iron acquisition in UPEC as a global target for small-molecule inhibition, we developed and carried out a whole-cell growth-based high throughput screen of 149,243 compounds. Our primary assay was carried out under iron-limiting conditions. Hits in the primary screen were assayed using two counterscreens that ruled out iron chelators and compounds that inhibit growth by means other than inhibition of iron acquisition. We determined dose-response curves under two different iron conditions and purchased fresh compounds for selected hits. After retesting dose-response relationships, we identified 16 compounds that arrest growth of UPEC only under iron-limiting conditions. All compounds are bacteriostatic and do not inhibit proton motive force. A loss-of-target strategy was employed to identify the cellular target of these inhibitors. Two compounds lost inhibitory activity against a strain lacking TonB and were shown to inhibit irreversible adsorption of a TonB-dependent bacteriophage. Our results validate iron acquisition as a target for antibacterial strategies against UPEC and identify TonB as one of the cellular targets. IMPORTANCE Half of women will suffer at least one episode of urinary tract infection (UTI) during their lifetime. The current treatment for UTI involves antibiotic therapy. Resistance to currently used antibiotics has steadily increased over the last decade, generating a pressing need for the development of new therapeutic agents. Since iron is essential for colonization and scarce in the urinary tract, targeting iron acquisition would seem to be an attractive strategy. However, the multiplicity and redundancy of iron acquisition systems in uropathogenic Escherichia coli (UPEC) make it difficult to pinpoint a specific cellular target. Here, we identified 16 iron acquisition inhibitors through a whole-cell high-throughput screen, validating iron acquisition as a target for antibacterial strategies against UPEC. We also identified the cellular target of two of the inhibitors as the TonB system.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Yep
authors.forename: Thomas
authors.initials: T
authors.lastname: McQuade
authors.forename: Paul
authors.initials: P
authors.lastname: Kirchhoff
authors.forename: Martha
authors.initials: M
authors.lastname: Larsen
authors.forename: Harry L T
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects*; D003090:Coliphages / Q000502:physiology; D004353:Drug Evaluation, Preclinical; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D056689:Uropathogenic Escherichia coli / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D053585:Virus Attachment
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C489181:tonB protein, E coli; D007501:Iron