High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport
| Autor: | Yan Shipelskiy, Mathew Hanson, Phillip E. Klebba, Salete M. C. Newton, Lorne D. Jordan |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Siderophore Gram-negative bacteria Iron medicine.disease_cause Biochemistry Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Enterobactin Bacterial Proteins Drug Discovery Gram-Negative Bacteria FepA medicine Escherichia coli Microbial Viability biology Chemistry Membrane Proteins Reproducibility of Results Biological Transport Periplasmic space biology.organism_classification Anti-Bacterial Agents High-Throughput Screening Assays 030104 developmental biology Spectrometry Fluorescence bacteria Molecular Medicine Bacterial outer membrane Bacteria Biotechnology |
| Zdroj: | Journal of biomolecular screening. 21(3) |
| ISSN: | 1552-454X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|