Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Smallwood, Chuck R.; Jordan, Lorne D.; Trinh, Vy; Schuerch, Daniel W.; Gala, Amparo; Hanson, Mathew; Hanson, Mark D.; Shipelskiy, Yan; Majumdar, Aritri; Newton, S. A.; Klebba, Phillip E.

doi:10.1085/jgp.201311159

Cited by 20 publications

(44 citation statements)

References 48 publications

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“…1A). Control experiments in energy-and TonB-deficient bacteria 3,11 confirmed the interpretation of these observations. Thus, transport appears as real-time quenching and unquenching of fluorescence intensity, and the extent and duration of quenching depend on the concentrations of bacteria and FeEnt in the cuvette, the temperature, and the incubation time.…”

Section: Fluorescence Spectroscopic Measurement Of Tonb-dependent Trasupporting

confidence: 72%

“…We previously defined conditions that maximize specific labeling of the engineered Cys residues in FepA surface loops: 5 µM FM in 50 mM NaHPO4, pH 6.5, 5 min at 37 °C. 3 To observe the dose-response behavior of FeEnt in the fluorescence assay of iron transport through FepA, we tested a range of concentrations (1,2,4,8,16, and 32 nM) of the ferric siderophore (Fig. 1B) in the SLM/OLIS fluorometer.…”

Section: Fluorescence Spectroscopic Measurement Of Tonb-dependent Tramentioning

confidence: 99%

“…2 Ferric enterobactin (FeEnt; 719 Da) utilizes FepA, an outer membrane (OM) protein that binds and internalizes it by active transport. 3 The detailed mechanism of FeEnt transport through FepA remains uncertain, but the process is controlled by TonB. 4 Active transport across the OM poses an energetic challenge: How does accumulation occur in a membrane containing open porin channels (e.g., OmpF) that preclude an electrochemical gradient?…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

et al. 2016

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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.

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Section: Fluorescence Spectroscopic Measurement Of Tonb-dependent Trasupporting

confidence: 72%

Section: Fluorescence Spectroscopic Measurement Of Tonb-dependent Tramentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

et al. 2016

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“…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.…”

Section: Resultssupporting

confidence: 70%

“…Fluorescence modification of site-directed Cys sulfhydryls in FepA enables spectroscopic observations of FeEnt transport in real time in live bacteria (27,28). FeEnt binding quenches fluorescence, but as the bacteria internalize FeEnt and deplete it from solution, fluorescence recovers.…”

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confidence: 99%

Fluorescence High-Throughput Screening for Inhibitors of TonB Action

et al. 2017

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Gram-negative bacteria acquire ferric siderophores through TonBdependent outer membrane transporters (TBDT). By fluorescence spectroscopic hghthroughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through Escherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonBdependent activity in all tests. Comparison of their effects on [ 59 Fe]Ent and [ 14 C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonBExbBD. The facility of FLHTS against E. coli led us to adapt it to Acinetobacter baumannii. We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii. Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with A. baumannii, FLHTS produced Z= factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenemresistant Enterobacteriaceae)/ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries.IMPORTANCE Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that include Acinetobacter baumannii, a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against Escherichia coli to find inhibitors of TonB-dependent iron transport, tested them against A. baumannii, and then adapted the FLHTS technology to allow direct screening against A. baumannii. This methodology is expandable to other drugresistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.

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Iron Acquisition by Bacterial Pathogens: Beyond Tris‐Catecholate Complexes

2020

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Sequestration of the essential nutrient iron from bacterial invaders that colonize the vertebrate host is a central feature of nutritional immunity and the “fight over transition metals” at the host‐pathogen interface. The iron quota for many bacterial pathogens is large, as iron enzymes often make up a significant share of the metalloproteome. Iron enzymes play critical roles in respiration, energy metabolism, and other cellular processes by catalyzing a wide range of oxidation‐reduction, electron transfer, and oxygen activation reactions. In this Concept article, we discuss recent insights into the diverse ways that bacterial pathogens acquire this essential nutrient, beyond the well‐characterized tris‐catecholate FeIII complexes, in competition and cooperation with significant host efforts to cripple these processes. We also discuss pathogen strategies to adapt their metabolism to less‐than‐optimal iron concentrations, and briefly speculate on what might be an integrated adaptive response to the concurrent limitation of both iron and zinc in the infected host.

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Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Abstract: The loops of the bacterial outer membrane iron transporter FepA move at different rates to adsorb and grasp the substrate ferric enterobactin before transporting it into the periplasm.

Cited by 20 publications

References 48 publications

High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

Fluorescence High-Throughput Screening for Inhibitors of TonB Action

Iron Acquisition by Bacterial Pathogens: Beyond Tris‐Catecholate Complexes

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