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Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB)
Open accessAbstract
Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
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Examples include screening strategies to identify small-molecule inhibitors of FeoB, or antibodies that target IsdA and IsdB and prevent S. aureus from binding to haem, which has led to protection in mouse models of infection.
“Examples include screening strategies to identify small- molecule inhibitors of FeoB 183-185 , or antibodies that target IsdA and IsdB and prevent S. aureus from binding to haem, which has led to protection in mouse models of infection 186,187 .”
From Nutritional immunity: the battle for nutrient metals at the host–pathogen interface (2022) · cites this paper as [185]
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Research works printed but not linked: 18
- Payne, 2016, Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments, Microbiol. Mol. Biol. Rev., 80, 69, 10.1128/MMBR.00046-15 · DOI
- Guilfoyle, 2009, Structural Basis of GDP Release and Gating in G Protein Coupled Fe2+ Transport, EMBO J., 28, 2677, 10.1038/emboj.2009.208 · DOI
- Hantke, 2003, Is the Bacterial Ferrous Iron Transporter FeoB a Living Fossil?, Trends Microbiol., 11, 192, 10.1016/S0966-842X(03)00100-8 · DOI
- Marlovits, 2002, The Membrane Protein FeoB Contains an Intramolecular G Protein Essential for Fe(II) Uptake in Bacteria, Proc. Natl. Acad. Sci. U.S.A., 99, 16243, 10.1073/pnas.242338299 · DOI
- Hung, 2010, Structural Fold, Conservation and Fe(II) Binding of the Intracellular Domain of Prokaryote FeoB, J. Struct. Biol., 170, 501, 10.1016/j.jsb.2010.01.017 · DOI
- Hohenwallner, 1973, The Malachite Green Micromethod for the Determination of Inorganic Phosphate, Clin. Chim. Acta., 45, 169, 10.1016/0009-8981(73)90406-3 · DOI
- Ash, 2010, Potassium-Activated GTPase Reaction in the G Protein-Coupled Ferrous Iron Transporter B, J. Biol. Chem., 285, 14594, 10.1074/jbc.M110.111914 · DOI
- Zhang, 1999, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J. Biomol. Screen., 4, 67, 10.1177/108705719900400206 · DOI · Paper page
- Cogan, 1999, A Robotics-Based Automated Assay for Inorganic and Organic Phosphates, Anal. Biochem., 271, 29, 10.1006/abio.1999.4100 · DOI
- Baykov, 1988, A Malachite Green Procedure for Orthophosphate Determination and Its Use in Alkaline Phosphatase-Based Enzyme Immunoassay, Anal. Biochem., 171, 266, 10.1016/0003-2697(88)90484-8 · DOI
- Bharat, 2013, A High-Throughput Screen of the GTPase Activity of Escherichia coli EngA to Find an Inhibitor of Bacterial Ribosome Biogenesis, J. Biomol. Screen., 18, 830, 10.1177/1087057113486001 · DOI
- Mishra, 2015, High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins, Methods Mol. Biol., 1298, 47, 10.1007/978-1-4939-2569-8_4 · DOI
- Surviladze, 2010, Identification of a Small GTPase Inhibitor Using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay, J. Biomol. Screen., 15, 10, 10.1177/1087057109352240 · DOI
- Mohanakrishnan, 2017, A Highly-Sensitive High Throughput Assay for Dynamin’s Basal GTPase Activity, PLoS One, 12, e0185639, 10.1371/journal.pone.0185639 · DOI
- Reichman, 2015, A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay, J. Biomol. Screen., 20, 1294, 10.1177/1087057115596326 · DOI
- Hong, 2015, A Pan-GTPase Inhibitor as a Molecular Probe, PLoS One, 10, e0134317, 10.1371/journal.pone.0134317 · DOI
- Ohashi, 1999, The Lethal Effect of a Benzamide Derivative, 3-Methoxybenzamide, Can Be Suppressed by Mutations within a Cell Division Gene, FtsZ, in Bacillus subtilis, J. Bacteriol., 181, 1348, 10.1128/JB.181.4.1348-1351.1999 · DOI
- Harrison, 2003, The [35S]GTPgammaS Binding Assay: Approaches and Applications in Pharmacology, Life Sci., 74, 489, 10.1016/j.lfs.2003.07.005 · DOI
Cited by
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Nutritional immunity: the battle for nutrient metals at the host–pathogen interface
Murdoch, C. C., & Skaar, E. P. (2022) · Nature Reviews Microbiology · doi:10.1038/s41579-022-00745-6 · Cited by 701
Metadata
- Authors
- Veloria, J., Shin, M., Devkota, A. K., Payne, S. M., Cho, E. J., & Dalby, K. N.
- Affiliations
- The University of Texas at Austin (US)
- Corresponding author
- Cho, Eun Jeong
- Dalby, Kevin N.
- Venue
- SLAS Discovery
- Publisher
- Elsevier BV
- Published
- 2019-06
- Volume
- 24
- Issue
- 5
- Pages
- 597-605
- ISSN
- 2472-5552 (ISSN-L)
- PMC
- PMC6714053
- OpenAlex
- W2943028438
- Cited by
- 9
- References
- 0
- Citations by year
- 2026: 1 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 1
- FWCI
- 0.90
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- SLAS DISCOVERY (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Allergy and Infectious Diseases (R01AI091957)
- Cancer Prevention and Research Institute of Texas (RP160657)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Keywords
- FeoB, GTPase activity, ferrous iron transport, GTP hydrolysis, malachite green, colorimetric assay, luminescence assay, high-throughput screening, small molecule screening
- MeSH terms
- Protein Domains, Amino Acid Sequence, Colorimetry, Escherichia coli, Guanosine Triphosphate, Hydrolysis, Models, Molecular, GTP Phosphohydrolases, Cation Transport Proteins, Escherichia coli Proteins, Luminescence, Small Molecule Libraries, High-Throughput Screening Assays
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