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.

Fluorescence High-Throughput Screening for Inhibitors of TonB Action

“…However, as the cells transported FeEnt it became depleted from solution, FepA was vacated, and fluorescence rebounded. Control experiments in energy-and TonB-deficient bacteria (27,28,42) confirmed the interpretation of these observations. Thus, transport appeared in real time as sequential quenching and unquenching of fluorescence intensity.…”

“…Considering TonB's essential role in iron acquisition, we sought to identify small molecules that inhibit TonB-dependent ferric siderophore transport in microtiter plates by the FLHTS approach, which spectroscopically measured FeEnt uptake by FepA in live E. coli (42). When bacteria harboring fluorescently labeled FepA were exposed to FeEnt, the ferric siderophore bound and quenched fluorescence intensity.…”

“…For fluoresceination of FepA in live cells, after overnight growth in LB, we subcultured E. coli in MOPS minimal medium and A. baumannii in nutrient broth (NB) with shaking at 37°C until an optical density at 600 nm (OD 600 ) of 1.0 to 1.5 was reached. We labeled the live bacteria with 5 M fluorescein maleimide ( 493 nm ϭ 81,500 M Ϫ1 at pH 8.0) in 50 mM NaHPO 4 , pH 6.7, for 15 min at 37°C, quenched the reaction with 1.3 mM ␤-mercaptoethanol, and pelleted, washed, and suspended the bacteria in phosphate-buffered saline (PBS) plus 0.4% glucose for E. coli (42), or 0.4% 50 mM sodium acetate for A. baumannii.…”

Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens

“…We previously designed fluorescence methods that monitored high-affinity ligand binding and uptake (23,24). By genetically engineering FepA and then labeling it with fluorescein maleimide (FM; Fig.…”

“…When FeEnt bound to FepA-FM, it quenched its fluorescence. Quenching originated from binding-induced conformational motion in the external loops of FepA, which subjected attached fluorophores to collisions with other residue side chains or elements of protein structure or increased interactions with the aqueous environment (23,24). When cellular uptake depleted FeEnt from solution, fluorescence rebounded.…”