Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling

Forte, Gabriella; Davie, Elizabeth A. Colby; Lie, Shervi; Franz‐Wachtel, Mirita; Ovens, Ashley J.; Wang, Tingting; Oakhill, Jonathan S.; Maček, Boris; Hagan, Iain; Petersen, Janni

doi:10.1242/jcs.223925

Cited by 19 publications

(19 citation statements)

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“…S2A and B in the supplemental material). This agrees with the recent observation that eukaryotic cells can uptake ATP and exploit it as an energy source (48). Interestingly, AMP supplementation also reconstituted H_OFF growth in restrictive conditions to wild-type levels (Fig.…”

Section: Fungal Methionine Synthase In Vivo Target Validationsupporting

confidence: 92%

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Scott

Sueiro-Olivares

Thornton

et al. 2020

mBio

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There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo. Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals. IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

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Section: Fungal Methionine Synthase In Vivo Target Validationsupporting

confidence: 92%

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Scott

Sueiro-Olivares

Thornton

et al. 2020

mBio

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“…This result suggests that TOR is indeed inhibited by AMPK kinase upon blocking respiration. Consistently, regulation of TOR via AMPK in response to environmental perturbations has been reported in fission yeast [50,51], and lowering the Krebs cycle flux leads to AMPK-mediated TOR inhibition in budding yeast [42]. After blocking respiration, the cells may bounce back from the transient TOR inhibition, through a retrograde response and redirection of energy metabolism to fermentation, until the levels of arginine and derived amino acids become limiting for rapid proliferation.…”

Section: Transcriptome Regulation In Response To Respiratory Chain Blsupporting

confidence: 58%

Mitochondrial respiration is required to provide amino acids during fermentative proliferation of fission yeast

Małecki

Kamrad

Ralser

et al. 2020

Preprint

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When glucose is available, many organisms repress mitochondrial respiration in favour of aerobic glycolysis, or fermentation in yeast, that suffices for ATP production. Fission yeast cells, however, rely partially on respiration for rapid proliferation under fermentative conditions. Here we determined the limiting factors that require respiratory function during fermentation. When the electron transport chain was inhibited, supplementation with arginine was necessary and sufficient to restore rapid cell proliferation. Accordingly, a systematic screen for mutants growing poorly without arginine identified not only mutants defective in arginine synthesis but also mutants defective in mitochondrial oxidative metabolism. Genetic or pharmacological inhibition of respiration triggered a drop in intracellular levels of arginine and amino acids derived from the Krebs-cycle metabolite alpha-ketoglutarate: glutamine, lysine and glutamic acid. Conversion of arginine into these amino acids was required for rapid proliferation when the respiratory chain was blocked. The respiratory block triggered an immediate gene-expression response diagnostic of TOR inhibition, which was muted by arginine supplementation or without the AMPK-activating kinase Ssp1. The TOR-controlled proteins featured biased composition of amino acids reflecting their shortage after respiratory inhibition. We conclude that respiration supports rapid proliferation in fermenting cells of fission yeast by boosting the supply of Krebs-cycle derived amino acids.

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“…3C). This agrees with the recent observation that eukaryotic cells can uptake ATP and exploit it as an energy source (47). In conclusion, a decrease in cell energetics developed in the absence of methionine synthase seems to explain MetH essentiality for growth.…”

Section: Methionine Synthase Repression Triggers a Metabolic Imbalancsupporting

confidence: 92%

Targeting methionine synthase in a fungal pathogen causes a metabolic imbalance that impacts cell energetics, growth and virulence

Scott

Sueiro-Olivares

Thornton

et al. 2020

Preprint

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29There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose 30 to human health. In this work, we have performed a comprehensive characterisation and 31 validation of the promising target methionine synthase (MetH). We uncover that in Aspergillus 32 fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a 33 reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. 34Interestingly, growth can be recovered in the presence of certain metabolites, which evidences 35 that conditional essentiality, defined as genes whose deficiency can be overcome in specific 36 conditions, is present in pathogenic fungi. As this concept must be considered for correct target 37 validation, we have optimised a genetic model to mimic treatment of established infections 38 using the tetOFF system. We show that repression of metH in growing hyphae halts growth in 39 vitro, which translates into a beneficial effect when targeting established infections using this 40 model in vivo. Finally, a structural-based virtual screening of methionine synthases reveals key 41 differences between the human and fungal structures and unravels features in the fungal 42 enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is 43 a valuable target for the development of new antifungals. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59Fungal pathogens represent an increasing risk to human health 1 , with over one billion people 60 worldwide affected by mycoses annually. Many of these mycoses are superficial infections of 61 the skin, nails or mucosal membranes and although troublesome are usually not life-62 threatening. However, some fungi cause devastating chronic and invasive fungal infections, 63 which result in an estimated 1.6 million deaths per year 2 . Incidences of invasive infections 64 caused by Aspergillus, Candida, Cryptococcus and Pneumocystis species are increasing 3 , a cause 65 for serious concern as these genera are responsible for 90% of deaths caused by mycoses 4 . 66Despite the availability of antifungal drugs, mortality rates for invasive aspergillosis, invasive 67 candidiasis, cryptococcal meningitis and Pneumocystis jirovecii pneumonia are intolerably high, 68reaching over 80%, 40%, 50% and 30% respectively 2,5 . There are currently only four classes of 69 antifungals in clinical use to treat invasive infections (azoles, echinocandins, polyenes and 70 flucytosine), all suffering from pharmacological drawbacks including toxicity, drug-drug 71 interactions and poor bioavailability 6,7 . With the sole exemption of flucytosine, which is only 72 used in combinatory therapy with amphotericin B for cryptococcal meningitis and Candida 73 endocarditis 7 , the current antifungals target critical components of the fungal cell membrane 74 or cell wall 8 , which represents a very limited druggable space. The rise of antifungal resistance 75 presents an additional challenge as mortality rates in pat...

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Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling

Cited by 19 publications

References 53 publications

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Mitochondrial respiration is required to provide amino acids during fermentative proliferation of fission yeast

Targeting methionine synthase in a fungal pathogen causes a metabolic imbalance that impacts cell energetics, growth and virulence

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