title: High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport.
abstract: The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8-1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
authors.forename: Mathew
authors.initials: M
authors.lastname: Hanson
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
authors.forename: Lorne D
authors.initials: LD
authors.lastname: Jordan
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shipelskiy
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
authors.forename: Salete M
authors.initials: SM
authors.lastname: Newton
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA peklebba@ksu.edu.
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Klebba
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects*; D055808:Drug Discovery / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008565:Membrane Proteins; C057616:tonB protein, Bacteria; D007501:Iron
keywords: Gram-negative bacteria; antibacterial drugs; cell-based assays; fluorescence methods; iron