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. 2021 Aug 13;19(8):e3001359.
doi: 10.1371/journal.pbio.3001359. eCollection 2021 Aug.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

Stefan Allmann  1   2 , Marion Wargnies  1   2 , Nicolas Plazolles  1 , Edern Cahoreau  3   4 , Marc Biran  2 , Pauline Morand  2 , Erika Pineda  1 , Hanna Kulyk  3   4 , Corinne Asencio  1 , Oriana Villafraz  1 , Loïc Rivière  1 , Emmanuel Tetaud  1 , Brice Rotureau  5 , Arnaud Mourier  6 , Jean-Charles Portais  3   4   7 , Frédéric Bringaud  1   2
Affiliations

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

Stefan Allmann et al. PLoS Biol. .

Abstract

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Procyclic trypanosomes prefer glycerol to glucose.
(A) Schematic representation of glycerol (black) and glucose (blue) metabolism in procyclic form (PCF) trypanosomes. The metabolic end products are shown in rectangles, and metabolites analyzed by ion chromatography high-resolution mass spectrometry (IC-HRMS) are underlined and in italic (a–g). The ATP molecules consumed and produced by substrate-level phosphorylation are shown, as well as the enzymes hexokinase (HK) and glycerol kinase (GK). (B) Glucose and glycerol consumption by PCF trypanosomes incubated in glucose (2 mM), glycerol (2 mM) and glucose + glycerol (2 mM each) conditions. (C) Metabolic end products produced by PCF trypanosomes from [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc), as measured by proton nuclear magnetic resonance (1H-NMR) spectroscopy (the values are calculated from the data presented in S1 Table). (D) IC-HRMS analyses of intracellular metabolites collected from PCF trypanosomes after incubation with 2 mM [U-13C]-labeled carbon sources in the presence or not of unlabeled carbon sources, as indicated on the right margin. The figure shows the proportion (%) of molecules having incorporated 0 to 6 13C atoms (m0 to m6, color code indicated on the left margin). G6P (a), glucose 6-phosphate; F6P (b), fructose 6-phosphate; M6P (c), mannose 6-phosphate; F1,6BP (d), fructose 1,6-bisphosphate; Gly3P (e), glycerol 3-phosphate; 2/3PG, 2- or 3-phosphoglycerate (which are not undistinguished by IC-HRMS); PEP (g), phosphoenolpyruvate. (E) Western blot analysis of total protein extracts from the parental (wild-type [WT]) and tetracycline-induced (.i) or uninduced (.ni) RNAiGK cell line probed with anti-GK (αGK) and anti-paraflagellar-rod (αPFR) immune sera. The table below the blots shows the relative levels of GK expression in 5 × 106 (1), 5 × 105 (/10) and 105 (/50) parental cells, and 5 × 106 RNAiGK.ni and RNAiGK.i cells, as well as the corresponding GK activity. ND, not detectable. (F and G) Glucose and glycerol consumption by the (F) tetracycline-induced RNAiGK (RNAiGK.i) and (G) uninduced RNAiGK (RNAiGK.ni) mutant cell lines incubated in glucose (2 mM), glycerol (2 mM) and glucose + glycerol (2 mM each) conditions. (H) Production of metabolic end products by the parental (WT), RNAiGK.ni, and RNAiGK.i cell lines from [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc), as measured by 1H-NMR spectroscopy (the values are calculated from the data presented in S1 Table). Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.
Fig 2
Fig 2. The glycerol preference is the consequence of the high excess of GK activity.
(A) Enzymatic assays used for the quantification of hexokinase (HK) and glycerol kinase (GK) activity. The bold and underlined substrates and enzymes are included in the assay for production of NADPH (HK assay) and consumption of NADH (GK assay) that are detected by spectrometry at 350 nm. 6PG, 6-phosphogluconate; G6PDH, glucose-6-phosphate dehydrogenase; Gly3P, glycerol 3-phosphate; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PYK, pyruvate kinase. (B) HK (left panel) and GK (right panel) activity in total cell extracts (wild-type [WT], RNAiGK.i and RNAiGK.ni) determined in the presence of glucose (Glc), glycerol (Glyc) or equal amounts of glucose and glycerol (Glc/Glyc). (C) GK and HK activity in different combinations (indicated in the table below the graph) of total cell extracts from the parental (WT) and the RNAiGK.i cell lines. The amount of HK remained the same in all samples (see S2 Fig), while the amount of GK present in the parental samples was diluted with the GK-depleted RNAiGK.i samples. The HK and GK activity were determined in the presence of both glucose and glycerol, as in the Glc/Glyc condition (see [B]). (D) Expression of HK activity as a function of GK activity. The values in parentheses indicate the rate of glycerol consumption in the RNAiGK.ni and RNAiGK.i cells compared to the parental cells (100%) (see Fig 1F and 1G). (E) HK activity in the presence of 10 mM acetate and increasing amounts of acetate kinase. Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.
Fig 3
Fig 3. Glycerol down-regulates GK expression but does not affect preference for glycerol over glucose.
(A) Glycerol kinase (GK), hexokinase (HK), and malic enzyme (ME) activity determined in total cell extracts of EATRO1125.T7T procyclic trypanosomes grown in glucose-rich (Glc/−), glycerol-rich (−/Glyc) or glucose/glycerol-depleted (−/−) conditions. (B) Western blot analysis of procyclic cells grown in glucose-rich medium (lane 0), then in glycerol-rich medium (in the absence of glucose and in the presence of N-acetyl-D-glucosamine) for 48 h, before reintroducing glucose (without glycerol and N-acetyl-D-glucosamine) for 48 h. The immune sera used against GK (αGK), pyruvate phosphate dikinase (αPPDK), and glyceraldehyde-3-phosphate dehydrogenase (αGAPDH) are indicated on the left margin. The bottom panel is a quantitative analysis of the GK signal indicated by an arrow (n = 4). (C) Metabolic end products of PCF trypanosomes from metabolism of [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc) measured by proton nuclear magnetic resonance spectrometry (the values are calculated from the data presented in S2 Table). (D) Expression of the recoded (GKrec) and native GK in the wild-type (WT), RNAiGKcst, and tetracycline-induced (.i) and uninduced (.ni) RNAiGKcst/OEGKrec cell lines monitored by Western blotting on total cell extracts using immune sera against GK (αGK) and paraflagellar rod (αPFR) as control (top panel), and GK activity assay normalized with malic enzyme activity and expressed as a percentage of activity in the WT cells (bottom panel, n = 2). Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.
Fig 4
Fig 4. Analysis of intracellular ATP and metabolites.
(A) The top panel shows hexokinase (HK) activity determined at 350 nm (NADPH production) over the incubation time of trypanosome extracts in the presence of 10 mM glucose and 10 mM glycerol and 0.2 to 3.0 mM ATP. The dashed lane corresponds to background HK activity measured without ATP. The arrows indicate the calculated ATP amounts (mM) remaining in the assay at the time of HK activity inhibition, taking into account glycerol kinase (GK) and HK activity. The asterisk indicates the time when 0.6 mM ATP is consumed by GK (deduced from the bottom panel). The bottom panel shows NADH consumption (GK activity) and NADPH production (HK activity) in the presence of 0.6 mM ATP (GK activity) or 0.2 to 3.0 mM ATP (HK activity). The dashed lane corresponds to HK activity measured without ATP. (B) Schematic drawing of the ATeam probe from [24]. Variants of cyan fluorescent protein (CFP; mseCFP) and yellow fluorescent protein (YFP; cp173-mVenus) were connected by the ε subunit of Bacillus subtilis FoF1-ATP synthase. In the ATP-free form (top), extended and flexible conformations of the ε subunit separate the 2 fluorescent proteins, resulting in a low fluorescence resonance energy transfer (FRET) efficiency. In the ATP-bound form, the ε subunit retracts to draw the 2 fluorescent proteins close to each other, which increases FRET efficiency. (C) The expression of ATeam-Myc-GPDH was controlled by Western blotting on total cell extracts of tetracycline-induced (.i) and uninduced (.ni) OEATeam-Myc-GPDH cells using anti-GPDH (αGPDH) and anti-Myc (αMyc) immune sera, and as control anti-enolase (αENO) immune serum. (D) The subcellular localization of ATeam-Myc-GPDH was confirmed by immunofluorescence assays on the OEATeam-Myc-GPDH.i cell line using an anti-aldolase immune serum as a glycosomal marker (top panel; the yellow YFP signal was converted to green to merge it with the red fluorescence corresponding to aldolase) or by observing the fluorescence activity of CFP and YFP (FRET) (bottom panel). (E) The ratio of YFP emission (FRET) and CFP emission after excitation at 435 nm in the OEATeam-Myc-GPDH.i cell line incubated in the presence of glucose (Glc) or glycerol (Glyc) (mean ± SD, n = 2 independent experiments, ****p < 0.0001). (F) The CFP fluorescence lifetime of the same cell line incubated in the same conditions (mean ± SD, n = 3 independent experiments, ****p < 0.0001). (G) Intracellular concentrations of metabolites in procyclic form trypanosomes grown in the presence of 10 mM glucose or glycerol (letters in parentheses refer to Fig 1A). The concentrations of the 2 last metabolites (asterisk) cannot be calculated, and the ratio between the 12C (sample metabolite) and 13C (standard) area was considered. G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; 6PG, 6-phosphogluconate; Pentose5P, pentose 5-phosphate (ribose 5-phosphate, xylulose 5-phosphate, and xylose 5-phosphate are not distinguished by ion chromatography high-resolution mass spectrometry); S7P, sedoheptulose 7-phosphate;M6P, mannose 6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; Gly3P, glycerol 3-phosphate; 2/3PG, 2- or 3-phosphoglycerate; PEP, phosphoenolpyruvate. (H) Enzymatic determination of intracellular Gly3P concentration in the parental (wild-type [WT]), RNAiGK.ni and RNAiGK.i cell lines grown in 10 mM glucose (blue), 10 mM glycerol (black) or both (grey). The absence of detectable amounts of Gly3P in cellular extracts from the RNAiGK.i mutants maintained in glycerol (last column) is probably due to cell quiescence caused by the impossibility of this mutant to metabolize glycerol. The intracellular concentrations of metabolites are calculated with the assumption that the total cellular volume of 108 cells is equal to 5.8 μL [27]. (I) Effect of increasing amounts of Gly3P and F1,6BP on HK activity determined in total extracts of procyclic form T. brucei. Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.
Fig 5
Fig 5. Glycerol metabolism in other trypanosomatids.
(A) Glycerol kinase (GK) and hexokinase (HK) activity in total cell extracts of the procyclic form (PCF) of T. congolense in the presence of glucose (Glc), glycerol (Glyc) or equal amounts of glucose and glycerol (Glc/Glyc). (B) Consumption of glucose (left) and glycerol (right) by the T. congolense PCF incubated in glucose-rich (2 mM), glycerol-rich (2 mM) and glucose/glycerol (2 mM each) conditions. (C) Correlation between high GK/HK activity ratio and preference for glycerol over glucose. BSF, bloodstream form. aHK activity in the presence of equimolar amounts of glucose and glycerol. bRatio between GK and HK activity. cRate of glucose (Glc) or glycerol (Glyc) consumption. dCulture in the presence of 2 mM glucose (+Glc), 2 mM glycerol (+Glyc) or both (+Glc, +Glyc). eData from Fig 1. fND, not detectable. gData from (B). hData from [20]. Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.
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FB's team is supported by the Centre National de la Recherche Scientifique (CNRS, https://www.cnrs.fr/) (financial support for consumables and salary of permanent positions), the Université de Bordeaux (https://www.u-bordeaux.fr/) (financial support for consumables and salary of permanent positions), the Agence National de Recherche (ANR, https://anr.fr/) through the grants GLYCONOV (grant number ANR-15-CE15-0025-01) and ADIPOTRYP (grant number ANR19-CE15-0004-01) (financial support for consumables and PM and EP salary) and the Laboratoire d’Excellence (https://www.enseignementsup-recherche.gouv.fr/cid51355/laboratoires-d-excellence.html) through the LabEx ParaFrap (grant number ANR-11-LABX-0024) (financial support for consumables and SA salary), the ParaMet PhD programme of Marie Curie Initial Training Network (https://ec.europa.eu/research/mariecurieactions/) (FP7-PEOPLE-2011-ITN-290080) (financial support for consumables and MW salary) and the "Fondation pour le Recherche Médicale" (FRM, https://www.frm.org/) ("Equipe FRM", grant n°EQU201903007845) (financial support for consumables and EP salary). BR is supported by and the Institut Pasteur (financial support for consumables and salary of permanent positions). JCP's team from Metabolomics & Fluxomics facilities (Toulouse, France, http://www.metatoul.fr) is supported by the Agence National de Recherche (ANR, https://anr.fr/) (grant MetaboHUB-ANR-11-INBS-0010) (financial support for consumables and salary of permanent positions).