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Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

['Stefan Allmann', 'Microbiologie Fondamentale Et Pathogénicité', 'Umr', 'Bordeaux University', 'Cnrs', 'Bordeaux', 'Centre De Résonance Magnétique Des Systèmes Biologiques', 'Marion Wargnies', 'Nicolas Plazolles', 'Edern Cahoreau']

Date: 2021-08

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.

Funding: 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).

Here we report a novel molecular mechanism for management of available resources, named “metabolic contest,” that does not require complex sensory and signal transduction systems, as opposed to “catabolite repression.” This is illustrated by the PCF trypanosomes, which prefer glycerol, a gluconeogenic carbon source, to glucose. The glycerol preference is due to the approximately 80-fold excess of glycosomal glycerol kinase (GK) activity (EC 2.7.1.30; the first step of glycerol assimilation) compared to glycosomal hexokinase (HK) activity (EC 2.7.1.1; the first glycolytic step), which compete for the same glycosomal pool of ATP ( Fig 1A ).

(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- 13 C]-glycerol ( 13 C-Glyc) and/or glucose (Glc), as measured by proton nuclear magnetic resonance ( 1 H-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- 13 C]-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 13 C 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) RNAi GK 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 × 10 6 (1), 5 × 10 5 (/10), and 10 5 (/50) parental cells and 5 × 10 6 RNAi GK.ni and RNAi GK.i cells, as well as the corresponding GK activity. ND, not detectable. (F and G) Glucose and glycerol consumption by the (F) tetracycline-induced RNAi GK ( RNAi GK.i) and (G) uninduced RNAi GK ( RNAi GK.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), RNAi GK.ni, and RNAi GK.i cell lines from [U- 13 C]-glycerol ( 13 C-Glyc) and/or glucose (Glc), as measured by 1 H-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 .

Trypanosoma brucei is a unicellular eukaryote that causes human African trypanosomiasis, also known as sleeping sickness [ 7 ]. Parasite transmission between mammals (bloodstream form [BSF] of T. brucei) is ensured by a hematophagous insect vector of the genus Glossina (tsetse fly). When grown in vitro in standard rich medium, the procyclic form (PCF) of T. brucei, present in the digestive tract of the insect vector, metabolizes glucose, which is converted by aerobic fermentation into partially oxidized end products, succinate, acetate, and alanine [ 8 – 10 ]. One unique particularity of trypanosome glycolysis is the occurrence of the first 6 glycolytic steps in specialized peroxisomes called glycosomes (see Fig 1A ), while this pathway is cytosolic in all other eukaryotes [ 11 ]. No exchange of nucleotides has been described so far between the glycosomal and cytosolic compartments. Consequently, consumption and production of ATP are tightly balanced within the organelle [ 12 ].

The diauxic growth observed in microorganisms consists of the sequential use of carbon sources when several are available, with the first one consumed, often glucose, being the one that ensures the highest growth rate. This concept emerged in the 1940s with the description in prokaryotes of preference for certain sugars, such as glucose over lactose or maltose, followed by the first description in 1964 of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) [ 1 ]. PTS is a carbohydrate transport and phosphorylation system composed of 3 protein complexes that regulates numerous cellular processes by either phosphorylating target proteins or interacting with them in a phosphorylation-dependent manner [ 2 ]. The diauxic growth pattern also occurs in yeasts, which first consume glucose; then, the fermentative product ethanol is oxidized in a noticeably slower second growth phase, if oxygen is available [ 3 ]. In addition, the presence of glucose suppresses molecular activity of yeasts involved in the use of alternate carbon sources [ 4 ]. Whether a carbon source behaves as a preferred or non-preferred one is not defined by its chemical structure but by the rate at which it enters metabolism. The mechanisms by which repression is imposed are quite variable; however, they follow a general pattern, with complex sensory systems relying mostly on protein kinases and phosphatases [ 4 – 6 ]. These carbon “catabolite repression” processes prevent expression of enzymes for catabolism of less preferred carbon sources when the preferred substrate is present.

Results and discussion

Glycerol down-regulates glucose catabolism The PCF of T. brucei catabolizes glucose and glycerol within glycosomes [13] (see Fig 1A). To determine their preferred carbon source, we first measured the consumption of glucose or glycerol by PCF trypanosomes maintained in culture in SDM79 medium supplemented with either glycerol or glucose or both. As expected, glycerol (compound with 3 carbons) was consumed by PCF faster than glucose (compound with 6 carbons). The rate of glycerol consumption was not affected by the presence of glucose. In contrast, the latter was not consumed as long as glycerol was present in the medium (Fig 1B). After glycerol exhaustion, glucose was consumed at a rate similar to under glucose-alone conditions. This absence of glucose consumption in the presence of glycerol clearly showed a strong catabolic-repression-like effect exerted by glycerol on glucose. It is noteworthy that the consumption of glucose started as soon as glycerol was exhausted, with no delay. To confirm this glycerol preference in PCF trypanosomes, we monitored the metabolic fate of [13C]-labeled glycerol ([U-13C]-glycerol) alone or in combination with equimolar amounts of unlabeled glucose. The analysis of metabolic end products by proton nuclear magnetic resonance (1H-NMR) spectroscopy (Fig 1C; S1 Table) allowed determining the respective contribution of [U-13C]-glycerol (labeled compounds) and glucose (unlabeled compounds) [14–16]. Trypanosomes mostly excreted acetate and succinate from glycerol or glucose (see Fig 1A). The rate at which glycerol was converted into these compounds was only slightly modified by the presence of glucose. In contrast, the conversion of glucose into acetate and succinate was reduced by approximately 30-fold in the presence of [U-13C]-glycerol. Moreover, the small production of lactate and alanine from glucose observed in the absence of glycerol was abolished in its presence. This significant reduction in the conversion of glucose into end products in the presence of glycerol was correlated with an approximately 20-fold decrease in glucose consumption in this experimental setup (see S1 Table), confirming that glucose metabolism was strongly down-regulated in the presence of glycerol. Since production of glucose 6-phosphate (G6P) through gluconeogenesis is essential in the absence of glucose (see Fig 1A), we measured by ion chromatography–high-resolution mass spectrometry (IC-HRMS) the incorporation of [13C] label into glycolytic intermediates in PCF trypanosomes incubated with [U-13C]-glycerol. In this experiment, most hexose phosphate glycolytic intermediates were fully [13C]-labeled (88.2% ± 1.8% of total molecules on average) after 2 h of incubation with [U-13C]-glycerol as the sole carbon source (Fig 1D). Addition of an equal amount of unlabeled glucose only slightly reduced 13C incorporation into hexose phosphates, with an average of 69.9% ± 9.7% fully [13C]-labeled molecules (Fig 1D). To confirm this preference for glycerol over glucose, the equivalent experiment was performed with [U-13C]-glucose (Fig 1D). Addition of an equal amount of unlabeled glycerol abolished incorporation of 13C from [U-13C]-glucose into triose phosphates and fructose 1,6-bisphosphate (F1,6BP). The 13C incorporation into G6P was strongly reduced (40% ± 0.9% versus 98% ± 0.1% fully [13C]-labeled molecules in the presence and absence of glycerol, respectively). Altogether, these data demonstrate that PCF trypanosomes significantly prefer glycerol to glucose for the production of hexose phosphates, including the first glycolytic intermediate, i.e., G6P. These data also suggest that HK, which produces G6P from glucose and/or some glucose transporters, may be the target of the glycerol-induced down-regulation of glucose metabolism. As far as we are aware, PCF trypanosomes are the only extracellular microorganisms described to date showing a preference for glycerol over glucose. T. brucei PCF is also the only known glycolytic-competent lower eukaryote performing gluconeogenesis in the presence of glucose.

Glycerol metabolism is critical for glucose catabolism repression To further study glycerol metabolism in PCF trypanosomes, the expression of the first enzyme of the glycerol pathway was down-regulated by an RNA interference (RNAi) silencing approach simultaneously targeting the 5 tandemly arranged GK genes (Tb927.9.12550–Tb927.9.12630) under control of a tetracycline-inducible system. In the absence of tetracycline, the uninduced RNAiGK (RNAiGK.ni) cell line presented strong constitutive leakage of the RNAi silencing system, with a 50-fold reduction of GK protein content, reducing overall GK enzyme activity to an undetectable level (Fig 1E). The residual GK protein level could be further reduced after tetracycline induction (RNAiGK.i). Thus, the direct involvement of GK in glycerol metabolism in these cells was determined by measuring glycerol consumption and release of metabolic end products under glycerol conditions (Fig 1F–1H; S1 Table). Both glycerol consumption (Fig 1F) and acetate/succinate production from glycerol metabolism (Fig 1H; S1 Table) were almost abolished in the RNAiGK.i mutant, demonstrating that there is no alternative to GK for glycerol breakdown in PCF trypanosomes. Interestingly, the presence of glycerol did not affect glucose consumption by the RNAiGK.i mutant (Fig 1F), indicating that the presence of glycerol in the medium is not per se responsible for glucose metabolism repression. In other words, glycerol does not directly affect glucose uptake and metabolism, which implies that intracellular glycerol metabolism is required to repress glucose metabolism. It is also important to mention that replacing glucose by glycerol did not affect growth of the RNAiGK.i mutant (S1 Fig), given that proline was the main carbon source used in these conditions, as in the insect vector midgut [8,10,17]. It is worth mentioning that knocking down the 10 identical GK genes is far easier than doing the alternative experiment consisting on knocking down/out the 3 different aquaglyceroporin genes (AQP1–AQP3) responsible for glycerol uptake in T. brucei [18]. The analysis of the RNAiGK.ni cell line also provided relevant information regarding the unexpected role of GK activity in the preference for glycerol over glucose. First, the consumption of glycerol (Fig 1G) and its conversion into end products (Fig 1H; S1 Table) were reduced only by 3.5-fold and 3.1-fold, respectively, in the RNAiGK.ni mutant as compared to the parental cells, while GK expression was approximately 50-fold down-regulated (Fig 1E). One can extrapolate from these data that a reduction of GK activity by at least 90% would not affect the glycerol metabolism flux, which would highlight a large excess of GK activity in PCF trypanosomes (in the range of 10-fold). Second, glucose metabolism was no longer repressed by glycerol in the RNAiGK.ni cells, which consumed glucose at the same rate as the parental cells, without any glycerol-induced delay, although glycerol consumption remained constant over the 10 h of incubation (Fig 1G). This strongly suggests that the abolition of the glycerol preference in the RNAiGK.ni cells could be the consequence of the 50-fold reduction of GK activity.

The glucose catabolism repression is due to a large excess of GK activity Interestingly, GK activity was approximately 80-fold higher than HK activity in total PCF extracts (Fig 2B), using the enzymatic assays described in Fig 2A. Since these 2 glycosomal enzymes can compete for the same ATP pool (glycosomal), we hypothesized that this significant difference in activity would favor glycerol metabolism and disfavor glucose metabolism, and would hence explain the unique repression of glucose by glycerol. To test this hypothesis, we measured HK activity in the presence or absence of glycerol under incubation conditions compatible with HK and GK activity. Importantly, the enzymatic assay included 0.6 mM ATP, which corresponds to the measured glycosomal concentration [19]. The presence of glycerol in the assay induced a 15-fold reduction of HK activity in the parental cell extracts, but not in the RNAiGK.ni and RNAiGK.i cell extracts (Fig 2B), which demonstrates that the conversion of glycerol into Gly3P, but not the presence of glycerol per se, inhibits HK activity and thus glucose metabolism. In contrast, GK activity was not impaired by the presence of glucose in the parental cell line extract (Fig 2B). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. The glycerol preference is the consequence of the high excess of GK activity. 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. https://doi.org/10.1371/journal.pbio.3001359.g002 We took advantage of the fact that both the parental and RNAiGK.i cell extracts displayed similar HK activity (see Fig 2B) to further characterize the GK-derived inhibition effect on HK activity as a function of the HK/GK activity ratio, by diluting the parental cell extract with different volumes of the RNAiGK.i cell extract (Fig 2C). As expected, HK activity was equivalent in all the samples in the presence of 10 mM glucose (S2 Fig). However, the addition of 10 mM glycerol decreased HK activity, and this effect was dependent on the HK/GK ratio (Fig 2C). Indeed, a reverse correlation between HK and GK activity was observed (Fig 2D), which was consistent with our hypothesis that both enzymes are competing for the same ATP pool. To confirm that this inhibitory effect was due to GK activity rather than to any other activities or biochemical properties of the enzyme, GK and glycerol were replaced by acetate kinase and acetate in the same HK activity assay. As anticipated, HK activity was inhibited concomitantly with increasing amounts of acetate kinase (Fig 2E). Altogether, these data demonstrate that the preference for glycerol over glucose is the consequence of a competition between HK and GK for their common substrate (ATP), which we named “metabolic contest.”

The GK/HK activity ratio is optimal for glycerol preference in procyclic trypanosomes cultured in glycerol-rich medium As mentioned above, procyclic trypanosomes multiply in medium containing glycerol instead of glucose; however, all the biochemical experiments presented so far were performed on cells grown in standard glucose-rich conditions. Transferring the procyclic cells from glucose-rich to glycerol-rich conditions (without glucose) induced a 2.3-fold reduction of GK activity and a 1.4-fold increase of HK activity, with the GK/HK ratio reduced by 3.3-fold in glycerol-rich medium (Fig 3A). These changes in GK and HK activity were not observed under glucose/glycerol-depleted conditions, indicating that the presence of glycerol, but not the absence of glucose, is responsible for this adaptation. The glycerol-induced down-regulation of GK expression was confirmed by Western blotting, with a 3.5-fold reduction of the GK protein level 2 d after cell transfer to glycerol-rich conditions (Fig 3B). Interestingly, this phenomenon was reverted when replacing glycerol with glucose (Fig 3B), which suggests that a high level of GK expression is required for the cells to grow under glucose-rich conditions or that reduced GK expression is optimal for glycerol metabolism. To determine the effect of GK down-regulation on glycerol preference, we monitored by 1H-NMR spectrometry the metabolic fate of [U-13C]-glycerol alone or in combination with equimolar amounts of unlabeled glucose. As expected, the rate of end product excretion from glycerol catabolism was not affected by the reduced expression of GK (Fig 3C). More importantly, the repression exerted by glycerol on glucose degradation was similar regardless of the growing conditions of the parasite (Fig 3C). PPT PowerPoint slide

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larger image TIFF original image Download: 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 [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. https://doi.org/10.1371/journal.pbio.3001359.g003 In order to modulate GK expression, a GK gene recoded for resisting RNAi silencing (GKrec), was introduced in the RNAiGKcst cell line, in which the expression of the endogenous GK genes is constitutively down-regulated (Fig 3D). Because of the strong constitutive leakage of the RNAi silencing system, as observed above for the RNAiGK.ni cell line (Fig 1E), the resulting uninduced RNAiGKcst/OEGKrec.ni cell line expressed GKrec with a GK activity lowered by 35% compared to that of the parental EATRO1125.T7T cells (Fig 3D). This reduced GK activity did not affect the preference for glycerol over glucose, as deduced from 1H-NMR analysis of excreted end products from glucose and/or glycerol metabolism (Fig 3C). As expected, the RNAiGKcst/OEGKrec.i cell line showed a 2-fold increase in GK activity upon tetracycline induction (Fig 3D) and maintained a preference for glycerol over glucose (Fig 3C). Altogether, these data show that a 3.3-fold reduction of the GK/HK activity ratio in the presence of glycerol does not affect the preference for glycerol over glucose. It is noteworthy that we also previously reported glycerol-induced down-regulation of GK expression (7-fold) in the BSF of T. brucei [20].

GK and HK compete for glycosomal ATP To further understand the mechanisms underlying the metabolic contest, we determined the ATP concentrations required to prevent HK activity in an excess of GK. The HK activity of trypanosome extracts was monitored over a period of incubation in the presence of glycerol and different amounts of ATP. In the presence of GK activity, HK activity was maintained until ATP concentrations reached the millimolar range (1.1 to 1.5 mM depending on the initial amounts of ATP, i.e., 1.2 to 3 mM, respectively) (Fig 4A, top panel). However, in the presence of lower amounts of ATP such as 0.6 mM, no HK activity was detected, while GK was active for 1.5 min, until all ATP was consumed (Fig 4A, bottom panel). These data demonstrate that HK activity is inhibited by an excess of GK in the presence of physiological amounts of ATP (0.6 mM in glycosomes [19]); however, at higher ATP concentrations, HK is active until ATP levels drop below 1.5 mM. It is noteworthy that T. brucei HK and GK have the same affinity for ATP (Km = 0.28 mM and 0.24 mM, respectively [21,22]), suggesting that the preference for GK over HK at ATP concentrations below 0.6 mM is primarily due to the large excess of GK activity. This competition between HK and GK for glycosomal ATP was previously anticipated based on a kinetic model of trypanosome glycolysis [23]. PPT PowerPoint slide

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larger image TIFF original image Download: 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 with no 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 F o F 1 -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. https://doi.org/10.1371/journal.pbio.3001359.g004 To estimate the impact of glycerol metabolism on glycosomal ATP levels, we used an ATP-specific fluorescence resonance energy transfer (FRET)–based indicator, named ATeam, that is composed of a bacterial F o F 1 -ATP synthase ε subunit sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) [24]. In the ATP-bound form, the ε subunit retracts to bring the 2 fluorescent proteins close to each other, which increases FRET efficiency and allows detection of changes in ATP level upon fluorescence quantification (Fig 4B). To focus on the glycosomal ATP levels, the ATeam cassette was fused to the N-terminal extremity of a Myc-tagged glycosomal protein containing a C-terminal peroxisomal targeting motif (PTS1), i.e., glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8; Tb927.8.3530), which was recently used to target a cytosolic protein exclusively inside this organelle [25]. Upon tetracycline induction, the anti-GPDH and anti-Myc immune sera recognized a 111-kDa protein corresponding to the expected size of the ATeam-Myc-GPDH recombinant protein (Fig 4C). Immunofluorescence analyses showed that the ATeam-Myc-GPDH recombinant protein was located in glycosomes, as confirmed by the colocalization of the ATeam-Myc-GPDH detected signal (YFP) and the aldolase glycosomal marker (Fig 4D, top panel), as well as the colocalization of CFP and YFP FRET signals, forming glycosomal-like images (Fig 4D, bottom panel). FRET efficiency was significantly decreased in the OEATeam-Myc-GPDH.i cells incubated in the presence of glycerol compared to glucose conditions, which indicates that glycerol metabolism induced a reduction of the intraglycosomal ATP level compared to the standard glucose conditions (Fig 4E). This was confirmed by an increase in CFP fluorescence lifetime corresponding to a FRET decrease (Fig 4F). We concluded that this glycerol-induced reduced glycosomal ATP level favors glycerol preference. To investigate whether parasite metabolic profiles were dependent on carbon source availability, we determined the absolute intracellular concentrations of metabolites by IC-HRMS by adding an internal standard ([U-13C]-labeled Escherichia coli extract) to the T. brucei cell extracts, as described before [26]. Among the glycolytic intermediates analyzed, F1,6BP and Gly3P accumulated approximately 5-fold and 49-fold more, respectively, in parental cells grown on glycerol as compared to those grown on glucose (Fig 4G). This significant Gly3P accumulation was confirmed by using an enzymatic determination (Fig 4H). It is noteworthy that this Gly3P accumulation persisted in the parental cells incubated with equal amounts of glycerol and glucose, while it was abolished for the RNAiGK.ni mutant (Fig 4H). This huge accumulation of Gly3P, due to the large excess of glycosomal GK activity that consumes ATP to produce Gly3P, is probably responsible for the observed reduction of glycosomal ATP level (Fig 4E and 4F). It may also be considered that the accumulation of intracellular amounts of Gly3P could inhibit HK and prevent G6P production from glucose. To test this hypothesis, HK activity was measured in the presence of Gly3P in RNAiGK.i mutant extracts, rather than parental cells, in order to prevent any interference of glycerol metabolism in the assay. Addition of up to 40 mM Gly3P did not significantly affect in vitro HK activity (Fig 4I), which is consistent with previously published data [22]. It is also noteworthy that HK activity was not inhibited by adding up to 5 mM F1,6BP (Fig 4I). This shows that the accumulation of glycerol 3-phosphate per se or F1,6BP is not responsible for the preference for glycerol over glucose.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001359

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