Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Richard-Greenblatt, Melissa; Bach, Horacio; Adamson, John; Peña-Díaz, Sandra; Wu, Li; Steyn, Adrie J. C.; Av‐Gay, Yossef

doi:10.1074/jbc.m115.648642

Cited by 47 publications

(53 citation statements)

References 59 publications

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“…Previously, EgtD has been shown to be required for the biosynthesis of ergothioneine as well as being essential for the growth of Mtb in murine macrophages and animal models. 37–39 Analysis of the 446 compounds screened from the NIH clinical collection yielded a hit cutoff of 30.1% activity of EgtD, which produced an initial hit rate of 4.3% (19 compounds, Figure 7A). The hit cutoff was determined by calculating three standard deviations from the average of the entire screening data.…”

Section: Resultsmentioning

confidence: 99%

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

et al. 2018

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S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5′-methylthioadenosine, AMP, and ADP.

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Section: Resultsmentioning

confidence: 99%

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

et al. 2018

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“…To the extent that ERG is a 'secondary' metabolite, defined (88) as a molecule whose synthesis has a relatively restricted distribution in different organisms, the biosynthetic pathways diverge from primary metabolism via the amino acids histidine, cysteine and methionine (89)(90)(91)(92)(93)(94) . Thus (Fig.…”

Section: Biosynthesis and Phylogenetic Distributionmentioning

confidence: 99%

The biology of ergothioneine, an antioxidant nutraceutical

et al. 2020

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Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

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“…However, despite considerable effort, roles for EGT in Mtb and its potential involvement in redox homeostasis and pathogenesis remain unknown. Recently we have shown that EGT levels in Mtb are modulated by protein phosphorylation during transition into late states of growth (Richard-Greenblatt et al, 2015), yet it is still unclear why mycobacteria produce both EGT and MSH to maintain redox homeostasis.…”

Section: Introductionmentioning

confidence: 99%

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis

Cumming²,

et al. 2016

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SUMMARY The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping, but distinct functions of EGT and MSH. Lastly, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

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Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Cited by 47 publications

References 59 publications

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

The biology of ergothioneine, an antioxidant nutraceutical

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis

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