Abstract Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

Author summary Trypanosomes are the etiological agents of human sleeping sickness and animal African trypanosomiases, a range of diseases in cattle, other livestock and horses caused by several Trypanosoma subspecies. The mammalian stage of the parasite circulates in the bloodstream, a nutrient-rich environment with constant temperature and pH and high glucose concentration. Hence, it was unsurprising that bloodstream trypanosomes use glucose in a low-efficiency manner and produce ATP mostly from glycolysis, with simplified mitochondria and metabolism. Recently though, T. brucei were found in abundance in adipose tissue, and also in skin, suggesting the need for flexible and more elaborate metabolic capacity. We show that trypanosomes synthesise sugars de novo from glycerol via gluconeogenesis. Depletion of glucose transporters is rescued by supplementation with glycerol. Moreover, even wild-type parasites, grown in the presence of glucose and glycerol, use both substrates and have active gluconeogenesis. Metabolome analysis also showed utilization of glycerol to feed the pentose phosphate pathway, nucleotide biosynthesis and glycerophospholipid biosynthesis. Trypanosomes do not accumulate storage polysaccharides, but mammalian-infective parasites do assemble a dense surface glycoprotein coat, the glycan components of which incorporate carbons from glycerol. Thus, gluconeogenesis can be used to drive intermediate metabolism and terminal metabolite biosynthesis. Our results reveal metabolic flexibility and adaptability in trypanosomes, which is likely required for survival in multiple host tissue environments. This should be considered when devising metabolically targeted therapies.

Citation: Kovářová J, Nagar R, Faria J, Ferguson MAJ, Barrett MP, Horn D (2018) Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei. PLoS Pathog 14(12): e1007475. https://doi.org/10.1371/journal.ppat.1007475 Editor: Julius Lukeš, Biology Centre, Academy of Sciences of the Czech Republic, CZECH REPUBLIC Received: August 1, 2018; Accepted: November 19, 2018; Published: December 27, 2018 Copyright: © 2018 Kovářová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Metabolomic data uploaded at MetaboLights database with an identifier MTBLS706. Funding: The work was funded by Wellcome Trust (https://wellcome.ac.uk/) Investigator Awards to DH (100320/Z/12/Z) and MAJF (101842/Z13/Z), with additional support from Wellcome Trust Centre Awards to Dundee (203134/Z/16/Z) and Glasgow (104111/Z/14/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction T. brucei is the causative agent of human and animal African trypanosomiases, devastating but neglected tropical diseases. The mammalian-infective form of the parasite, typically referred to as the bloodstream form (BSF), lives in blood of mammalian hosts and enters the central nervous system (CNS), leading to a fatal disease if not treated. In addition, trypanosomes were recently detected in adipose tissue in a mouse model [1] and the skin of both humans [2] and mice [3]. Tsetse flies transmit the parasites; these procyclic forms (PCF) grow in the insect mid-gut, differentiating through other adaptive life-cycle stages, and later migrating to the salivary glands, for transmission in saliva as metacyclic forms. Each of the parasite’s stages is morphologically and metabolically adapted to the respective environmental conditions. Nutrient availability is variable in the tsetse mid-gut and PCF trypanosomes can utilize proline, and generate the majority of their ATP in a reticulated mitochondrion containing canonical functions; although the tricarboxylic acid (TCA) cycle appears to operate in a non-canonical manner [4]. On the other hand, BSF trypanosomes in the bloodstream grow in a stable, nutrient rich environment, with a constant and abundant glucose supply, producing ATP from glycolysis; the mitochondrion, the electron transport chain and the TCA cycle are substantially reduced in BSF cells [5]. The recent identification of trypanosomes in adipose tissue [1] and in the skin of humans [2] and mice [3], indicated that the ‘bloodstream forms’ should now be considered as bloodstream-resident, CNS-resident, adipose-resident or skin-resident forms, potentially with differing metabolic capacities. Glycolysis is the metabolic pathway with the highest flux in BSF T. brucei grown in culture medium; this pathway has been thoroughly studied and has long been considered a promising potential drug target [6]. The majority of the glycolytic enzymes are localized inside glycosomes, specialized peroxisomes harboring glycolysis and additional metabolic pathways [7]. The glycosome membrane is semi-permeable; hence only smaller metabolites can pass freely [8], while ADP/ATP or NAD+/NADH regeneration must be balanced inside the organelle. It has been proposed that compartmentalized glycolysis emerged to facilitate adaptation to different environmental conditions [9]. Other metabolic pathways that are compartmentalized inside glycosomes include the pentose phosphate pathway (PPP), nucleotide sugar biosynthesis, nucleotide biosynthesis and salvage, lipid synthesis, and fatty acid β-oxidation, probably due to their connection to glycolysis [10]. Gluconeogenesis (GNG) is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as glycerol, lactate or glucogenic amino acids. In principle, it is the reverse of glycolysis, as many glycolytic enzymes are reversible, the direction depending on substrate and product concentrations. Only two steps are thought to be unique to GNG in protozoa; requiring fructose-1,6-bisphosphatase (FbPase) and phosphoenolpyruvate carboxykinase activity [11]. PCF T. brucei display GNG capacity fed by proline [12] but GNG was thought to be absent from BSF T. brucei, since FbPase activity was not detected [13]. Metabolomic analysis with labelled glucose also failed to reveal evidence for GNG [11]. Consequently, it is often stated that BSF trypanosomes depend ‘exclusively’ [14,15,16,17] or ‘entirely’ [18,19] on glycolysis, using glucose as a substrate, for ATP production (reviewed in [20,21]). For this reason, the glycolytic pathway has been considered to be a promising target for antitrypanosomal drug discovery [6]. On the other hand, Ryley [22] reported utilization of glycerol for respiration by both BSF and PCF T. b. rhodesiense in the early 1960s. Furthermore, glycerol has been used routinely to sustain trypanosomes in glucose-free media for several hours in radiolabelling experiments [23]. We also recently reported the use of glycerol for ATP production by BSF T. brucei [24]. There are thought to be five copies of each trypanosome hexose transporters gene, THT1 (Tb927.10.8440–8480) and THT2 (Tb927.10.8490–8530), arranged in an array in the T. b. brucei strain 927 reference genome, however the number of copies is variable across different strains [25]. The two gene types are similar, containing some identical domains. THT1 transcripts are substantially more abundant in BSF cells relative to PCF cells, while in PCF cells, THT2 transcripts are substantially more abundant than THT1 transcripts [26,27]. Specific regions in the 3’-untranslated regions contribute to this stage-specific expression pattern [28], while only THT2 transcripts are upregulated after glucose depletion [27]. The THTs comprise twelve trans-membrane domains and a cysteine-rich loop [26]. They are closely related to mammalian hexose transporters, although some substrate selectivity was observed [29]. Overall, the substrate selectivity of THT1 and THT2 is similar, but THT1 is a high capacity, low affinity transporter, whereas THT2 is a lower capacity, higher affinity transporter; this may reflect the conditions under which each of the proteins is expressed (reviewed in [26]). Here, we explore glycerol utilization for GNG in T. brucei depleted of THTs, and find GNG operating even in wild-type cells that have access to glycerol. Carbons from stable isotope labelled glycerol are detected in sugar phosphates, PPP intermediates, VSG glycans and other metabolites. We also detect robust FbPase activity, even after deletion of the annotated FbPase gene. Thus, contrary to prior thinking, GNG is available to mammalian stage T. brucei and may operate in tissue environments where glycerol is available. This metabolic flexibility may be essential for adaptation to environmental conditions and survival in mammalian host tissues.

Discussion It has been thought for decades that mammalian forms of T. brucei, the causative agents of African sleeping sickness in humans and nagana in cattle, are exclusively dependent on glycolysis, using glucose as a substrate, for ATP production. We now provide evidence that these cells can utilise glycerol for gluconeogenesis (GNG) and for ATP production. Thus, metabolism in these parasites is not as simplified and reduced as had been thought. Indeed, we demonstrate some GNG even in wild-type cells grown on glycerol, even when glucose is also available. Metabolism may indeed be simple and dependent on glycolysis in blood, but it now seems likely that GNG can be activated in different environments. Creek and colleagues [11] utilised metabolomic analyses to demonstrate extensive utilization of glucose in a wide range of metabolic pathways. We now report the utilization of glycerol for many of those same pathways. FbPase is considered to be one of the rate-limiting activities of GNG in other systems [48], but this activity has not previously been detected in T. brucei and therefore kinetic parameters have not been established. An annotated FbPase (Tb927.9.8720) is present in trypanosome glycosomes [7] and is expressed at similar protein levels in both bloodstream and insect life-cycle stages [44], the transcript levels are similarly not significantly different between culture, blood, or adipose tissue derived BSF [1]. We now show that FbPase activity is present in BSF T. brucei cells and increases when GNG is activated. We also show, however, that this activity is Tb927.9.8720-independent. Thus, the dephosphorylation of F1,6bP to F6P in T. brucei may require reversal of the phosphofructokinase reaction or sedoheptulose-1,7-bisphosphatase (Tb927.2.5800) activity [49]. In addition to FbPase, phosphoenolpyruvate carboxykinase (PEPCK) is the other enzyme specific for GNG, and responsible for its regulation in mammals [48]; this enzyme is also known to be present and active in T. brucei [11]. As expected, a third GNG-specific enzyme in animals, glucose-6-phosphatase, is not present in T. brucei, nor in other protozoa, since loss of the phosphate group would allow free glucose to diffuse out of the cells [48]. Previous modelling and metabolomics of glycolysis suggested high reverse flux of aldolase [11], however, similar information is lacking for the other enzymes. T. brucei glycerol kinase is unique in its bidirectional activity, and is known to lack the classical allosteric regulation [50]. According to canonical biochemistry, glycolysis and GNG cannot operate simultaneously, and the exclusive regulatory mechanisms are well known in mammalian systems. However, this classical regulation is missing in the T. brucei enzymes [34,51]. Compartmentalization in glycosomes would not present a solution if both glycolysis and FbPase activity are localised inside glycosomes [7] and we cannot exclude futile cycling in T. brucei, which has been suggested in Toxoplasma gondii [52]. However, since we now demonstrate Tb927.9.8720-independent FbPase activity, further work will be required to determine whether this FbPase activity is indeed compartmentalized within glycosomes in T. brucei. GNG is essential in other unicellular pathogens. It is vital for the mammalian stage of Leishmania due to the need for mannogen biosynthesis [53]. GNG is also vital for Toxoplasma gondii, regardless of whether infected host cells are rich or poor in glucose [52]. Mycobacterium tuberculosis is also dependent on GNG and harbours two independent FbPase genes [54]. GNG may be particularly important in mammalian form T. brucei to feed the essential pentose-phosphate and glycoprotein glycosylation pathways, since even if the cells produce ATP from the second half of glycolysis when glucose is limiting, G6P is required as a substrate for these other essential pathways. Aquaglyceroporins (AQPs) transport glycerol, but have generally been considered important for glycerol efflux rather than acquisition [33]. AQPs can be used for glycerol uptake to fuel T. brucei metabolism, however [24]. Indeed, our current data are consistent with a report from 1962 on trypanosomes using glycerol as a substrate for respiration [22], and a report from 1977 on pyruvate production from glycerol [55]. Is glycerol the only GNG substrate in T. brucei? Leishmania can also use aspartate, alanine or lactate to feed GNG [56]. Although the same enzymatic repertoire is theoretically available in T. brucei [4], our findings suggest that amino acid supply in growth medium fails to fuel GNG in cultured bloodstream form T. brucei. On the other hand, glycerol may activate GNG, thereby allowing other substrates to be used. Futile cycling between glycolysis and GNG would be energetically disadvantageous, but it is consistent with our observation that GNG is associated with a fitness cost. Depletion of triosephosphate isomerase, leading to production of one ATP per molecule of glucose, caused a severe growth defect in BSF T. brucei [57]. Similarly, one molecule of ATP is produced per molecule of glycerol when GNG is operative, while two molecules of ATP are produced per molecule of glucose in glycolysis. However, this may be compensated for by upregulating glycerol uptake. Decrease in glycolytic intermediates indicates decrease of flux in glycolysis, which may be a consequence of futile cycling. In addition, glycerol must be phosphorylated in the initial step of GNG, potentially inhibiting glycolysis by depleting the ATP required to drive the hexokinase and the phosphofructokinase reactions. In parallel with GNG, we do see evidence for metabolic compensation to balance ATP production. Additional ATP may be produced in the mitochondrion in the acetate:succinyl-CoA shunt, the TCA cycle directly by succinyl-CoA synthetase, or indirectly by feeding the electron transport chain with reduced cofactors from the TCA cycle. Specifically, labelling patterns observed in fumarate, malate, and aspartate support activity of the TCA cycle, as proposed previously in adipose tissue trypanosomes [1]; pyruvate is converted into acetyl-CoA, which then introduces two labelled carbons into the TCA cycle. An alternative explanation for this labelling pattern is that the enzymes, malate dehydrogenase and fumarase, are working in both directions and shuffling carbons, as a consequence of GNG disrupting the usual metabolic steady state. The glycerol-derived 13C 3 component of fumarate, malate, and aspartate; comprising about 8% in wild-type, increased three-fold following hexose transporter knockdown. This may reflect glycosomal succinate shunt activity or the reverse action of TCA cycle enzymes. Notably, increased activity of the glycosomal succinate shunt would also serve to regenerate NAD+ and maintain NAD+/NADH balance inside the glycosome. Various mammalian host tissues may provide glycerol in quantities sufficient for GNG in trypanosomes. Infection in the cerebrospinal fluid is well known. However, little is known about parasite metabolism in the central nervous system and glycerol concentration is lower than in blood [36]. Adipose tissue contains glucose at concentrations about seven fold lower than in plasma [58] and glycerol at concentrations about four fold higher than in plasma [35,36], although these levels may be variable. Adipose tissue form trypanosomes may possess metabolically active or upregulated pathways, which are silent in the BSF, i.e. the TCA cycle and fatty acid β-oxidation [1], and we show here that GNG is active in the presence of glycerol. T. brucei are also present in skin, but their metabolism has yet to be scrutinised in this tissue [2,3]. Trypanosomes may never encounter environments completely lacking glucose under physiological conditions in mammalian hosts. Notably, in this regard, GNG is operative when both glucose and glycerol are present. Incomplete labelling in our glycerol-fed metabolomics analyses suggests that, although glucose uptake was below the detection limit of our assay in the absence of hexose transporters, some glucose was likely still imported by endocytosis. An ability to use both substrates may be crucial for adaptation to particular tissue environments or during transitions between tissues. Our metabolomics analysis indicates that, in the presence of a suitable substrate, GNG does operate in mammalian form T. brucei. The pathway even operates, albeit at a relatively low level, in wild-type cells in the presence of glycerol. Cells that have limited access to glucose, in this case following hexose transporter knockdown, display a major increase in flux through GNG. We conclude that GNG in T. brucei, using glycerol taken up via aquaglyceroporins [24], could be important for colonization of, and survival in, different host tissue environments.

Materials and methods Cell culture T. b. brucei Lister 427 bloodstream form cells were cultured in the standard HMI-11 medium (Gibco), supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37°C, 5% CO 2 [32]. Phleomycin (Invivogen) was used at 1 μg/ml, hygromycin (Sigma-Aldrich) at 1 μg/ml for bloodstream and 50 μg/ml for insect-stage, puromycin (Sigma-Aldrich) at 1 μg/ml, and blasticidin (Melford) at 5 μg/ml, as appropriate. RNAi was induced using tetracycline (Sigma-Aldrich) at 1 μg/ml. Differentiation into insect-stage cells was performed as described [59]. Briefly, 2 x 107 cells were washed in DTM medium, and resuspended in 5 ml of DTM supplemented with 15% heat-inactivated fetal bovine serum, 3 mM cis-aconitate (Sigma-Aldrich) and 3 mM sodium isocitrate (Sigma-Aldrich). Cells were cultivated for at least 7 days at 27°C prior to analysis. Established insect-stage cells were cultured in SDM-79 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), GlutaMAX (Gibco) and 2 mg/l hemin (Sigma-Aldrich) as described [60]. Genetic manipulation was performed by electroporation in cytomix using an Amaxa nucleofector (Lonza) for BSF cells, and a Gene Pulser (BioRad) for insect-stage cells. Plasmids and Cas9-based editing A codon-optimised mNeonGreen (mNG) sequence was cloned using AvrII and PacI (NEB) restriction sites in the pNAT vector [61]. The THT1/THT2 gene specific targets (nucleotides 4–359 for THT1, and 4–180 for THT2; GeneScript) were cloned using SmaI and XhoI (NEB) restriction sites. The resulting pNATmNGTHT1 was linearized with BaeI and pNATmNGTHT2 with BsrGI (NEB) prior to transfection. The pRPaiSL vector [61] was used to assemble the RNAi constructs. The inserts comprised a THT1-specific sequence for pRPaiSLTHT1 (nucleotides 145–392, 459–600, 704–793; GeneScript) and a common region targeting both THT1 and THT2, for pRPaiSLTHT1+2 (nucleotides 839–1338); these ‘stem-loop’ constructs were assembled using BamHI and XhoI (NEB) restriction sites. Cas9-based editing of Tb927.9.8720 was carried out using a previously described editing system [45]. Briefly, a Tb927.9.8720-specific sgRNA construct was assembled following annealing of the FbPgRNA5 and FbPgRNA3 oliogonucleotides. The resulting construct was linearised with NotI prior to transfection into 2T1T7-Cas9 cells. The NPT58720 / NPT38720 and PAC58720 / PAC38720 primer pairs were used to amplify repair-templates encoding the antibiotic selection markers and with terminal 25-bp Tb927.9.8720 untranslated region-specific targeting sequences. Cas9-based editing was induced for 24 h, at which point both repair templates (~5 μg of each) were transfected. Both antibiotics (G418 [Sigma-Aldrich] and puromycin [Sigma-Aldrich] at 2 μg/ml) were used to select for populations that lacked the Tb927.9.8720 gene, as demonstrated using a series of PCR-assays. FbPgRNA5: AGGGAAGGTGCTCCCGCGCCTCTC FbPgRNA3: AAACGAGAGGCGCGGGAGCACCTT NPT58720: TAACGACACCACTCTTCCCAGATTTCGGGTGCTCAAGCTGTGT NPT38720: CACACGCATCGAAGCAACCATTGGCGGGGAAGGAAACCAACTTG PAC58720: TAACGACACCACTCTTCCCAGATTTATGGGTCCCATTGTTTGCC PAC38720: CACACGCATCGAAGCAACCATTGGCACTATTTTCTTTGATGAAAGGG Western blot analysis For western blot analysis, cells were harvested (1,000 g, 10 min), washed with 1 x PBS and lysed in Laemmli buffer (62 mM Tris pH 6.8, 10% glycerol, 2.3% SDS, 5% β-mercaptoethanol, bromphenol blue). To detect mNG-tagged THT proteins, samples were sonicated (3 cycles for 3s, 4°C), for VSG-2 and EP1 detection, samples were boiled at 95°C for 10 min. Equivalent of 107 cells was loaded per well. Proteins were transferred from SDS gels onto Hybond ECL nitrocellulose membrane (GE Healthcare) using Trans-Blot Turbo Transfer System (BioRad) at 1.3 A, 25 V, for 10 min. Membranes were blocked in 5% milk in 0.005% PBS-Tween. Incubation with α-mNG antibody (Chromotek) was performed at 1:1,000, 4°C, overnight, α-VSG-2 at 1:10,000 for 1 h at room temperature (RT), α-EP1 (Cedarlane) at 1:1,000 for 1 h at RT, α-EF1 (Millipore) at 1:10,000 for 1 h at RT. Following three washes in PBS-Tween for 10 min, the secondary α-mouse or α-rabbit HRP-coupled antibody (BioRad) incubation was performed at 1:10,000, for 1 h at RT. Following a further three washes in PBS-Tween, the signal was visualised using an ECL kit (GE Healthcare) with a G:BOX chemidoc (Syngene). Microscopy and immunofluorescence microscopy assay For microscopy, cells were washed with 1 x PBS and fixed in methanol-free 3% formaldehyde (Thermo Scientific) for 15 min at RT. Following two washes in PBS, cells were resuspended in 1% bovine serum albumin (Sigma-Aldrich) and allowed to dry on microscopy slides. For direct fluorescence microscopy, slides were immediately mounted with Vectashield with DAPI (Vector Laboratories). For immunofluorescence microscopy, slides were blocked with 50% FBS in PBS for 15 min at RT and, after two washes with PBS, primary α-VSG-2 (1:10,000; [62]) or α-EP1 (1:1,000; Cedarlane) antibodies were applied for 1 h at RT. Following 3 washes in PBS, the secondary antibodies, α-rat IgA-rhodamine (1:1,1000; Sigma-Aldrich) and α-mouse Alexa 568 (1:1,000; Life Technologies), respectively, were applied for 1 h at RT, followed by a further three washes in PBS. Images were captured using an Axiovert 200 epifluorescence microscope and processed using Zen imaging software (Zeiss). 2-14C(U)-deoxyglucose uptake assay The uptake assay was performed as described previously [24] with minor modifications. Briefly, 108 cells were harvested (1000 g, 10 min 4°C), washed twice in ice-cold transport buffer without glucose (33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.55 mM CaCl 2 , 0.07 mM MgSO 4 , 5.8 mM NaH 2 PO 4 , 0.3 mM MgCl 2 , 23 mM NaHCO 3 , pH 7.3) and resuspended to 108 cells/ml in the same buffer. Uptake was initiated by adding 100 μl of cell suspension to 100 μl of transport buffer containing 0.25 μCi 14C-2-deoxyglucose (PerkinElmer) layered over 100 μl of dibutyl phthalate (Sigma-Aldrich). After incubation at 37°C or 4°C for the appropriate time, transport was stopped by centrifugation through the oil layer (16,000 g, 1 min). Microcentrifuge tubes were flash frozen in liquid nitrogen and the bottoms of the tubes, containing the cell pellets, were snipped into scintillation vials. Pellets were solubilised overnight in 150 μl of 1 M NaOH before mixing with 2 ml of scintillation fluid and radioactivity was measured on a scintillation counter (Beckman LS 6500) for 1 min. LC-MS metabolomic analysis For the liquid chromatography–mass spectrometry (LC-MS) metabolomic analysis the sample extraction was performed as described previously [11]. THT1/THT2 knockdown cells were grown in the presence of tetracycline and 5 mM 13C 3 -U-glycerol (Sigma-Aldrich) three days prior to sample preparation. Briefly, 5 x 107 cells were used for each final 200 μl sample. Cells were rapidly cooled in a dry ice/ethanol bath to 4°C, centrifuged at 1,300g, 4°C for 10 min, washed with 1 x PBS, and resuspended in extraction solvent (chloroform:methanol:water, 1:3:1 volume ratio). Following shaking for 1 h at 4°C, samples were centrifuged at 16,000g at 4°C for 10 min and the supernatant was collected and stored at -80°C. The analysis was performed using separation on 150 x 4.6 mm ZIC-pHILIC (Merck) on Dionex UltiMate 3000 RSLC (Thermo Scientific) followed by mass detection on an Orbitrap QExactive mass spectrometer (Thermo Fisher) at Glasgow Polyomics. Analysis was operated in polarity switching mode, using 10 μl injection volume and a flow rate of 300 μl/min. The samples were run alongside 170 authentic standards. The data were processed and analyzed using mzMatch software [63] and mzMatchISO [64]. The analysis was performed in 4 replicates, means of which are indicated, non-labelled samples were run in parallel. Metabolites were identified based on matches with standards or were predicted based on mass and retention time. Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (https://www.ebi.ac.uk/metabolights/index) with the identifier MTBLS706. Fructose-1,6-bisphosphatase activity assay To measure FbPase activity, cells were harvested, washed in PBS, and resuspended in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% Triton X-100, cOmplete Protease Inhibitor Cocktail [Roche]) at 2 x 108 cells/ml. Following 20 min incubation at RT, cell extracts were centrifuged at 14,000 g, 16°C, 10 min, and supernatants were collected and kept on ice. The reaction mixture (20 mM Tris pH 7.8, 10 mM MgCl 2 , 1 mM NADP, 1 μl glucose-6-phosphate isomerase [Sigma-Aldrich], 1 μl glucose-6-phosphate dehydrogenase [Sigma-Aldrich], 100 μl cell extract in H 2 O) was incubated at 30°C for 5 min and 5 mM fructose 1,6-bisphosphate was added immediately prior to reading at 340 nm, 30°C with an UV-1601 spectrophotometer (Shimadzu). VSG glycosylation analysis The VSG isolation was performed as described previously with minor modifications [65]. The cells were cultured in 5 mM 13C 3 -U-glycerol (Sigma-Aldrich) for three days prior to sample preparation, and unlabelled samples were prepared alongside. 108 cells were harvested (1,300 g, 4°C, 10 min), washed twice in ice-cold PBS and resuspended in 300 μl of 10 mM Na 2 HPO 4 pH 8.0 in MS grade water, containing 0.1 mM TLCK (Sigma-Aldrich), 1 μg/ml leupeptin (Sigma-Aldrich), 1 μg/ml apoprotinin (Sigma-Aldrich), and 10 mM PMSF (Sigma-Aldrich). Following 5 min incubation at 37°C, the samples were cooled on ice and centrifuged at 14,000 g, 4°C for 5 min. The samples were applied on chromatography columns containing 400 μl of bead mixture (50:50 volume ratio, Anion Exchange Cellulose DE52 (Whatman) in 10 mM Na 2 HPO 4 pH 8.0 buffer) and eluted with 800 μl of 10 mM Na 2 HPO 4 pH 8.0. The obtained eluate was concentrated and diafiltered against water using Millipore Amicon Ultra-0.5 Centrifugal Filter Devices (Merck) following the manufacturer’s instructions. Carbohydrate compositional analysis was performed by GC-MS. Samples (6–10 μg) were mixed with 2 nmol scyllo-inositol internal standard and dried using vacuum centrifugation. Dried samples were then subjected to methanolysis by adding 50 μl of 0.5 M HCl in dry methanol and incubating at 85°C for 4 h. Methanolysates were re-N-acetylated by the addition of 10 μl pyridine and 10 μl acetic anhydride and incubating at RT for 30 min. The samples were dried under vacuum and derivatised with 15 μl trimethysilylation (TMS) reagent at RT for 30 min. 1 μl aliquots of each sample was injected in GC-MS (Agilent Technologies, 7890B Gas Chromatography system with 5977A MSD) equipped with Agilent J&W HP-5ms GC Column (30 m X 0.25 mm, 0.25 μm) with He carrier gas at 0.5 ml/min. The temperature program used was run over 32.5 min with 95°C (for 1 min) - 140°C (30°C/min) to 265°C at 5°C/min (for 5 min). The mass spectra were collected from linear scanning over m/z 50–650, and quantification was based on the integration of the extracted ion-current chromatograms and empirically determined molar response factors.

Supporting information S1 Fig. THT knockdown in bloodstream and insect stage cells. (A) The protein blot shows native tagged mNGTHT2 in bloodstream form cells following THT1 knockdown for 5 days. (B) The protein blot shows depletion of native tagged mNGTHT2 following THT1/THT2 knockdown in insect stage cells; see Fig 2C for depletion of mNGTHT1 by the same approach in bloodstream form cells. (C) Bloodstream form THT1/THT2 knockdown cells were grown in the presence of tetracycline and glycerol for up to 6 days, and scrutinised by immunofluorescence microscopy. Staining of the cell surface with α-VSG-2, but not α-EP procyclin antibody validated that these cells are not differentiated into PCF. A PCF cell is shown as a control. DNA was counter stained with DAPI; scale bars 5 μm. https://doi.org/10.1371/journal.ppat.1007475.s001 (PDF) S2 Fig. Additional metabolites detected by the LC-MS analysis. The size of the bars represents the total abundance, and coloured parts indicate 13C labelling as depicted in the legend. The samples are from WT cells, WT grown in 13C-glycerol, THT1/THT2 RNAi grown in 12C-glycerol and THT1/THT2 RNAi grown in 13C-glycerol. Natural abundance of 13C is 1%, hence the 1C labelling in ‘un-labelled’ samples. * the identity of these metabolites was confirmed using a match with a standard. https://doi.org/10.1371/journal.ppat.1007475.s002 (PDF) S3 Fig. Measurement of incorporation of 13C glycerol into the mannose and galactose residues of VSG. Total ion chromatograms of the methyl-glycoside TMS derivatives from wild-type (panels A-B) and THT1/THT2 knockdown trypanosomes (panels C-D) grown in the absence and presence of 13C glycerol, respectively. The peaks due to mannose (Man), galactose (Gal) and the scyllo-inositol internal standard (s-I) are indicated. The insets show a detail of the electron impact mass spectra of the main Man peak, illustrating the natural abundance of m/z 206 relative to m/z 204 (panel A) compared to a sample where 13C has been incorporated into the VSG sugar residues (panel D). One representative replicate, n = 3. https://doi.org/10.1371/journal.ppat.1007475.s003 (PDF) S1 Table. LC-MS metabolomics data with 13C 3 -glycerol. The samples are from WT cells, WT grown in 13C-glycerol, THT1/THT2 RNAi grown in 12C-glycerol and THT1/THT2 RNAi grown in 13C-glycerol, n = 4. https://doi.org/10.1371/journal.ppat.1007475.s004 (XLSX)

Acknowledgments We thank A. Fairlamb for advice on trypanosomatid metabolism and E. Rico for advice on Cas9-based editing.