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The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis [1]

['Julie Kovářová', 'Institute Of Parasitology', 'Biology Centre Of The Czech Academy Of Sciences', 'České Budějovice', 'Czech Republic', 'Martin Moos', 'Institute Of Entomology', 'Michael P. Barrett', 'Wellcome Centre For Integrative Parasitology', 'University Of Glasgow']

Date: 2024-03

Abstract Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Author summary Trypanosoma brucei is a unicellular parasite causing sleeping sickness in humans and nagana disease in cattle. The parasite invades the bloodstream and cerebrospinal fluid and only recently, it has been shown to infect additional tissues such as skin, adipose tissue, or lungs. While the glucose-based metabolism of the bloodstream form is well understood, the parasite’s metabolism in these secondary tissues has not been sufficiently explored, despite its importance for drug development. One possibility is the use of gluconeogenesis since the mammalian-infective stages can use glycerol as a carbon and ATP source. First, enzymes involved in gluconeogenesis have to be identified, then it can be tested if the pathway is advantageous for the survival of the parasite. We generated mutants in four different enzymes potentially involved in this metabolic pathway. Surprisingly, the flux in gluconeogenesis was maintained in all cell lines tested, implying that another non-canonical enzyme participates in the production of glucose from glycerol in these parasites.

Citation: Kovářová J, Moos M, Barrett MP, Horn D, Zíková A (2024) The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis. PLoS Negl Trop Dis 18(2): e0012007. https://doi.org/10.1371/journal.pntd.0012007 Editor: Walderez O. Dutra, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, BRAZIL Received: August 11, 2023; Accepted: February 16, 2024; Published: February 23, 2024 Copyright: © 2024 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: The data is available from the Figshare database. https://figshare.com/authors/Julie_Kovarova/17814896. Funding: This work was supported by CZ.02.2.69/0.0/0.0/19_074/0016248 LeishWeb to J.K.; by the Ministry of Education, Youth and Sports of the Czech Republic grant RNA for therapy (CZ.02.01.01/00/22_008/0004575), by the European Research Council (ERC, MitoSignal, grant agreement no. 101044951), and GACR 20-14409S to A.Z.; D. H. was funded by an Investigator Award from Wellcome [217105/Z/19/Z]. M.P.B was funded by an MRC Newton grant ‘Bridging epigenetics, metabolism and cell cycle in pathogenic trypanosomatids’ MR/S019650/1. 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 Trypanosoma brucei brucei is the causative agent of Human African Trypanosomiasis, also termed sleeping sickness [1]. The mammalian-infective stage of the parasite is called the bloodstream form (BSF), and it is transmitted between hosts by blood-feeding tsetse flies. In the first stage of the disease, these extracellular parasites divide in the bloodstream of the mammalian host. If left untreated, the trypanosomes invade the central nervous system, manifesting as the second stage of the disease. BSF parasites can also inhabit skin, adipose tissue, lungs and other tissues [1–5]. Previous dogma stated that BSF trypanosomes are absolutely glucose-dependent. However, we [6], and others [7] have shown that they also employ gluconeogenesis (GNG) and can use glycerol as a carbon source. Glycerol utilisation is expected to be most physiologically relevant to parasites that inhabit adipose tissue or skin, but the significance of glycerol as a carbon source remains elusive. Nevertheless, it is clear that the parasite’s metabolism is highly flexible and adaptable, and may differ between the various mammalian forms, which should be considered when developing drugs inhibiting metabolic enzymes. Notably, the adipose tissue forms are less responsive to several trypanocidal drugs [8]. Under standard culture conditions in medium containing glucose, glycolysis provides the majority of cellular ATP and is indispensable to BSF trypanosomes [9]. However, in culture medium containing glycerol, BSF trypanosomes use GNG to produce sugars from non-sugar carbon sources by converting glycerol to glucose 6-phosphate (G6P) [6,7]. GNG primarily uses the same enzymes as glycolysis, but operating in the opposite direction. The key difference between the two pathways is the enzymatic step between fructose 6-phosphate (F6P), and fructose 1,6-bisphosphate (F1,6bP) when phosphofructokinase (PFK) phosphorylates F6P to F1,6bP in glycolysis, while fructose-1,6-bisphosphatase (FBPase) dephosphorylates F1,6bP to form F6P in GNG (Fig 1A) [10]. In addition, since glycerol is used as the non-sugar substrate, glycerol kinase (GK) becomes a key GNG enzyme [7]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Generation and growth analysis of Δfbp.sbp strains. A–The scheme shows glycolysis, gluconeogenesis, and the pentose phosphate pathway in BSF T. brucei with highlighted FBPase, PFK, and TAL. Missing reactions of transketolase are depicted by grey arrows. B–A scheme for the Cas9 editing method by transient transfection. The templates for an sgRNA and for an antibiotic resistance cassette were transfected simultaneously, which resulted in replacement of both alleles for FBPase in the first step, and for SBPase in the second step. Fig 1B was created in BioRender. C–Validation of the Δfbp.sbp cell line by PCR, shows parts of ORFs for FBPase and SBPase amplified in the parental 2T1T7.Cas9 cell line, but absent from Δfbp.sbp. D—Validation of the Δfbp.sbp cell line by western blot. Only the parental cell line shows signal for FBPase (whole cell lysates) and SBPase (organellar fractions), *—cross-reacting protein, gel loading–fluorescent protein detection on a TGX gel. E–Growth curves of two independent clones of Δfbp.sbp show no defect in the standard HMI-11 medium. Growth curves in the CMM medium show a mild growth defect of the Δfbp.sbp clones and higher variability. https://doi.org/10.1371/journal.pntd.0012007.g001 Previously, we demonstrated the presence of GNG in the BSF parasites by RNA interference (RNAi) silencing of the glucose transporters, where the observed lethal phenotype was rescued by the addition of glycerol to the culture media [6]. As verified by LC-MS metabolomics with 13C-glycerol, glycerol was incorporated into fructose 6-phosphate (F6P) and other metabolites via GNG. The same conclusion was reached by Pineda and colleagues after adapting BSF parasites to glucose-free, glycerol-containing medium [7]. Surprisingly, however, GNG was not abolished after deletion of the FBPase gene indicating an involvement of another, so far unknown, enzyme [6,7]. The role of FBPase in GNG was also studied in the insect procyclic form (PCF) of T. brucei [11]. This stage has a more elaborate mitochondrion in terms of both morphology and metabolism, which can provide additional substrates for GNG. Hence, proline is utilised by PCF as a carbon source, via proline degradation in the mitochondrion, and fed into GNG via phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase [11]. FBPase knock-out (KO) PCF cells are viable when grown in medium containing proline and they incorporate proline-derived metabolites into G6P by GNG. This suggests that, similarly to BSF cells, the activity of FBPase can be compensated by another unknown enzyme. Interestingly, the FBPase KO PCF cells display a mild growth defect, defects in metacyclogenesis and transmission through the tsetse fly [11]. The presence of another enzyme catalyzing the F1,6bP to F6P conversion is intriguing, as in the majority of eukaryotic cells studied to date, this metabolic reaction is performed solely by the activity of FBPase. Therefore, the balance between FBPase and PFK activity is strictly and tightly regulated [10]. FBPase is positively regulated by ATP, and negatively by AMP and fructose 2,6-bisphosphate (F2,6bP). PFK is controlled by the same metabolites in a reciprocal manner, i.e. it is positively regulated by AMP, ADP, and F2,6bP. F2,6bP is an activator of most eukaryotic PFK enzymes, normally produced from F1,6bP by the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase [12]. The finely tuned regulatory mechanism between PFK and FBPase ensures mutual exclusivity of these two reactions, and prevents futile cycling (a situation when both reactions would be running in a cycle, only consuming ATP). Interestingly, T. brucei PFK is much less sensitive to inhibition by ATP than many eukaryotic PFKs, and its activity is not affected by the presence of F2,6bP [13]. Therefore, the well-established PFK regulatory mechanism appears to be missing from T. brucei [14]. Regulation of T. brucei FBPase has not yet been elucidated. Although T. brucei PFK is an ATP-dependent enzyme, its amino acid sequence is a ‘chimera’ between representative pyrophosphate- and ATP-dependent enzymes [15]. PFK isolated from T. brucei is regulated by AMP, which serves as the only allosteric activator, although 10-fold less potent than for the leishmania enzyme [13,15], and by phosphoenolpyruvate acting as an allosteric inhibitor. F1,6bP or F2,6bP did not significantly influence enzyme activity [13]. As predicted by a mathematical model, and experimentally validated, PFK is present in excess in T. brucei and therefore, glycolytic flux is reduced only after depletion of PFK beyond 60% of WT activity. It is unclear, however, whether the effect is direct, or a secondary consequence of decreased activity of other glycolytic enzymes (hexokinase, enolase, pyruvate kinase) [9]. Highly specific allosteric inhibitors of T. brucei PFK have been developed, and shown to be effective in killing BSF parasites in vitro and in infected mice [16]. Fernandes and colleagues [17] reported reverse activity of trypanosomal and mammalian PFKs in vitro, but the physiological relevance is unclear. The reverse activity of three mammalian PFK isoforms is not thought to occur in vivo [18] and although trypanosomal PFK is localised in specialized organelles called glycosomes, which could theoretically form an environment that allows reverse activity, experimental evidence is still lacking. In trypanosomatids, glycosomes are highly adapted peroxisomes, as they share related PEX-dependent protein import machineries. The main role of these specialized organelles is to harbor both glycolytic and gluconeogenic enzymes, parts of the pentose phosphate pathway (PPP), nucleotide metabolism, and other associated pathways [19]. The arrangement and function of the PPP is not fully resolved in T.brucei BSF because transketolase is not expressed in this stage, but transaldolase is, resulting in an incomplete non-oxidative branch of the PPP (Fig 1A) [20, 21]. The glycosomal membrane is ‘semi-permeable’, thought to allow free passage of small molecules (up to 340 Da based on a mathematical model of trypanosome metabolism) through open channels, but to be impermeable to larger molecules [22,23]. Hence, cofactors such as ADP/ATP or NAD+/NADH are balanced inside glycosomes, and glycosomal localisation of the glycolytic enzymes has important implications for their regulation [24]. We and others have shown previously that deletion of the FBPase gene does not eliminate the enzymatic activity responsible for dephosphorylation of F1,6bP [6,7], indicating that other enzymes capable of this activity are also present. Notably, a gene encoding sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme typically involved in the Calvin cycle of plants, is present in the genome of T. brucei. Due to a relatively high sequence identity (26%) to FBPase, we considered it a possible candidate for possessing FBPase activity. We also explored two other scenarios in which the reverse PFK activity [17] or a pathway involving transaldolase (TAL) [25] could bypass FBPase in GNG. To our surprise, double knock-out cell lines for FBPase and SBPase (Δfbp.sbp), triple knock out cell lines for FBPas, SBPase and TAL (Δfbp.sbp.tal) and PFK RNAi cell lines in a background of Δfbp.sbp, retained GNG flux, suggesting an unknown enzyme performing the FBPase reaction. Nevertheless, we present several unique metabolomic datasets that provide new insights into non-canonical GNG and PPP in BSF trypanosomes.

Materials and methods T. brucei cell culture and cell line construction Bloodstream form Trypanosoma brucei brucei Lister 427 cells were cultured in HMI-11 medium [26] supplemented with 10% FCS (BioSera) at 37° C and 5% CO 2 . The fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase knock-out cell line (Δfbp.sbp) was generated by CRISPR/Cas9 gene editing in the 2T1T7-Cas9 cell line [27]. In order to delete FBP (Tb927.9.8720), a template for sgRNA transcription was synthesised by end-filling PCR with primers FBPgRNA_F and sgRNA_R, and a repair template for DNA integration was synthesised with primers FBP.NPT50_5 and FBP.NPT50_3. Cas9 expression was induced with tetracycline at 1 μg/ml (InvivoGen) 24 h prior to transfection with an Amaxa nucleofector (Lonza) using programme Z-001. First, the Δfbp cell line was generated, and after validation of successful replacement of both FBP alleles with a G418 resistance cassette, a subsequent transfection was performed to replace the SBP (Tb927.2.5800) alleles with a puromycin resistance cassette (primer pairs SBPgRNA_F and sgRNA_R for sgRNA, and SBP.PAC50_5 and SBP.PAC50_3 for the repair template with puromycin resistance). Likewise, Δtal (TAL is encoded by Tb927.8.5600) was generated in the parental and Δfbp.sbp cell lines, using primer pairs AZ1550 and sgRNA_R for sgRNA, and AZ1551 and AZ1552 for the repair template with phleomycin resistance. The primers used are listed in Table 1. Hygromycin was used at 5 μg/ml (InvivoGen), blasticidin at 5 μg/ml (InvivoGen), G418 at 2.5 μg/ml (Invivogen), and puromycin at 0.1 μg/ml (InvivoGen). PPT PowerPoint slide

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TIFF original image Download: Table 1. Sequences of DNA primers used in the study. https://doi.org/10.1371/journal.pntd.0012007.t001 For depletion of PFK (Tb927.3.3270), the RNAi plasmid p2T7-177 [28] was used, digested with BamHI and HindIII. A PFK PCR product was amplified using primer pair AZ1339 and AZ1340, digested with the same restriction enzymes and ligated with the plasmid. The resulting p2T7-177-PFK construct was linearized with NotI prior to electroporation of 2T1T7-Cas9 and Δfbp.sbp cell lines in parallel. Clones obtained after phleomycin selection (at 2.5 μg/ml) were tested for a growth defect after tetracycline induction at 1 μg/ml, and validated by qRT-PCR. The TyPFK cell line was also created by Cas9 editing in the 2T1T7-Cas9 cell line. Following 24 h of Cas9 induction, electroporation was performed with a sgRNA template (primer pair AZ1388 and sgRNA_R), and PURO.TyPFK cassette (primer pair AZ1281 and AZ1282). Clones were selected with puromycin at 0.1 μg/ml and integration validated by PCR (primer pair AZ1403 and AZ1404) and western blot. qRT-PCR Total RNA was extracted from 1–2 x 108 cells using the RNeasy kit (Qiagen). DNA was removed using Turbo DNase (Applichem) at 37° C for 30 min, which was subsequently treated with DNase inactivation reagent (Ambion) for 5 min at RT. Following ethanol precipitation, cDNA was synthesised from 2 μg of RNA using TaqMan Reverse Transcription Reagent (Applied Biosystems) and random hexamer primers. Real-time PCR amplification was performed using LightCycler 480 SYBR Green I Master (Roche) and LightCycler 480 thermocycler (Roche). Primers used for the PFK target and reference genes are listed in Table 2. PPT PowerPoint slide

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TIFF original image Download: Table 2. Sequences of DNA primers used for qRT-PCR. https://doi.org/10.1371/journal.pntd.0012007.t002 Subcellular fractionation and western blotting In order to separate organellar fractions containing glycosomes from the cytosol, cells were subjected to digitonin-based subcellular fractionation and the obtained samples used for western blots. Briefly, 1 x 108 cells were harvested, washed in 1 x PBS and resuspended in 500 μl of SoTE buffer (0.6 M sorbitol, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5). A further 500 μl of SoTE buffer containing 0.03% digitonin (Sigma-Aldrich) was added, samples were incubated on ice for 5 min and subsequently centrifuged at 4,500 g, 4° C for 3 min. The obtained supernatant was used as cytosolic fraction, and pellets resuspended in an equivalent volume of 1 x PBS and used as organellar fractions. 40 μl of samples were loaded for western blots. For whole cell lysates, the equivalent of 1 x 107 cells was used. For western blots (WB) 4–12% NuPAGE polyacrylamide gels (Invitrogen) and 1 x SDS running buffer (25 mM Tris, 192 mM glycine, 1% SDS) were used. Subsequently, proteins were transferred to a PVDF membrane (Pierce) in transfer buffer (39 mM glycine, 48 mM Tris, 20% methanol) at 90 V for 90 min at 4°C. Following 30 min blocking in 5% milk (Serva) in PBS-Tween (0.05%), primary antibody was incubated in milk solution overnight at 4°C. Following 3 x 10 min wash in PBS-Tween, secondary antibody was incubated in milk solution for 1 h at RT. Following 3 x wash in PBS-T, signal was visualised using Western ECL Substrate (BioRad). The following antibodies were used: α-FBP at 1:500, α-SBP at 1:500 (both kind gifts from Frédéric Bringaud), anti-Ty 1:1,000 (ThermoFisher), α-APRT at 1:500 was used as a marker for cytosolic fraction, and α-hexokinase at 1:2,000 as an organellar marker. Secondary α-mouse (BioRad) and α-rabbit (BioRad) antibodies conjugated to HRP were used at 1:2,000. Metabolomics For the experiment with Δfbp.sbp in 13C-glycerol, cells were grown in the standard HMI-11 medium supplemented with 5 mM 13C-U-glycerol (Cambridge Isotope Laboratories). For the experiment with Δfbp.sbp/RNAiPFK, Δtal, and Δfbp.sbp.tal, HMI-11 medium was prepared from components according to the recipe [26], but glucose was omitted and instead 5 mM 13C 3 -U-glycerol (Cambridge Isotope Laboratories) was supplied. Cells were grown in this medium for 24 h (PFK cell lines) or 48 h (TAL cell lines) prior to sample extraction. Samples for the metabolomic experiments were prepared by the same extraction protocol as reported previously [29]. Briefly, 5 x 107 cells were used per 100 μl sample, which were first rapidly cooled to 4° C in a dry ice–ethanol bath. Following a wash with 1 x PBS, cell pellets were resuspended in 100 μl of chloroform:methanol:water (1:3:1) suspension and incubated with shaking at 4° C for 1 h in order to achieve full extraction into the solvent. Subsequently, samples were centrifuged (12,000 g, 10 min, 4° C), supernatants were collected and stored at -80° C until analysis. The metabolomic methods used were described in detail elswere [30]. Briefly, an Orbitrap Q Exactive Plus mass spectrometer coupled to an LC Dionex Ultimate 3000 (Thermo Fisher Scientific, San Jose, CA, USA) was used for metabolite profiling. LC condition: column SeQuant ZIC-pHILIC 150 mm x 4.6 mm i.d., 5 μm, (Merck KGaA, Darmstadt, Germany); flow rate of 450 μl/min; injection volume of 5 μl; column temperature of 35°C; mobile phase A = acetonitrile and B = 20 mmol/l aqueous ammonium carbonate (pH = 9.2; adjusted with NH 4 OH); gradient: 0 min, 20% B; 20 min, 80% B; 20.1 min, 95% B; 23.3 min, 95% B; 23.4 min, 20% B; 30.0 min 20% B. The Q-Exactive settings were: mass range 70–1050 Daltons; 70 000 resolution; electrospray ion source operated in the positive and negative modes. The analysis of the Δfbp.sbp cell line in HMI-11 medium was performed at Glasgow Polyomics, using separation on 150 x 4.6 mm ZIC-pHILIC (Merck) on a Dionex UltiMate 3000 RSLC system (Thermo Scientific) followed by mass detection on Orbitrap QExactive (Thermo Fisher Scientific) mass spectrometer (Thermo Fisher). Analysis was operated in polarity switching mode, using 10 μl injection volume and a flow rate of 300 μl/min. The analyses was performed in four replicates, and a set of standards was run in parallel. Metabolite identification was based on matches with standards where possible or otherwise predicted based on mass and retention time. Data were analysed using mzMatch [31] and mzMatch.ISO [32], Xcalibur software, version 4.0 (Thermo Fisher Scientific, San Jose, CA, USA), and an in-house developed Metabolite Mapper platform. The raw data is publically available from the Figshare depository (https://figshare.com/authors/Julie_Kovarova/17814896). FBPase assay 2 x 107 cells were used per sample. They were centrifuged (1,300 g, 10 min), washed with 1 x PBS and resuspended in 100 μl of SoTE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.15% Triton X-100, protease inhibitor cocktail (Roche)). After 20 min incubation at RT, samples were centrifuged (14,000 g, 10 min, 16° C) and supernatant collected. The reaction mixture containing 20 mM Tris pH 7.8, 10 mM MgCl 2 , 1 mM NADP, 1 U PGI (Sigma-Aldrich), 1 U G6PDH, 100 μl cell extract was incubated at 30° C for 5 min prior to activity measurement. The reaction was triggered by addition of 5 mM F1,6bP (Sigma-Aldrich) immediately prior to measurement of NADPH production at 340 nm for 5 min at 30° C using a UV-1601 spectrophotometer (Shimadzu). Immunofluorescence assay (IFA) For the immunofluorescence assay, cells were fixed in 7.4% formaldehyde in 1 x PBS for 15 min, and subsequently washed three times with 1 x PBS. For permeabilisation, 0.1% Triton X-100 (AppliChem) in 1 x PBS was applied for 10 min, and subsequently washed 3 x with 1 x PBS. Following blocking in 5.5% FBS in 1 x PBS-Tween (0.05% Tween) and 2 x wash with 1 x PBS, the primary antibodies were applied (α-Ty at 1:100 (ThermoFisher) and α-FBP at 1:1,000) for 1 h at RT. Following three washes with 1 x PBS-T and two washes with 1 x PBS, the secondary antibodies were applied (Alexa Fluor 647 α-mouse (Life Technologies) at 1:2,000 and Alexa Fluor 488 α-rabbit (Life Technologies) at 1:2,000) for 1 h at RT. After an additional three washes with 1 x PBS-T and two washes with PBS, ProLong Gold Antifade mounting solution (Invitrogen) was applied. Imaging was performed using an Axioplan microscope (Zeiss).

Discussion We took advantage of Cas9-based gene editing to delete multiple genes and in an attempt to abolish GNG in BSF T. brucei. However, our data show that the combined deletion of FBPase, SBPase, and TAL had little impact on gluconeogenic activity. To the best of our knowledge, this is the first case of deletion of three diploid gene loci simultaneously in BSF T. brucei. Although depletion of PFK was possible only to a limited extent, our data show that the reverse activity of this enzyme is most likely not involved in GNG, as suggested previously [17]. Current understanding of trypanosome metabolism does not provide an alternative explanation for how GNG operates, and which enzymes contribute to GNG flux. In our previous work and that of others, it was demonstrated that deletion of the canonical FBPase gene in T. brucei does not deplete FBPase activity, i.e. conversion of F1,6bP to F6P [6,7]. This is in contrast to closely related Leishmania parasites, where FBPase deletion disrupted GNG and caused a severe phenotype in mammalian infective amastigotes [34]. Since SBPase is not present in the genome of Leishmania, this enzyme was a promising candidate for the observed FBPase activity. The enzyme has 26% sequence identity to FBPase, and its catalytic activity is predicted to be very similar, using a sugar phosphate backbone extended by one carbon. Additionally, SBPase from yeast was demonstrated to posess ‘FBPase’ activity [35]. The most probable origin of SBPase in trypanosomes is acquisition by horizontal gene transfer [36]. The streamlined protocol for Cas9-based gene editing allowed us to generate a double gene knock-out combined with a knock-down. Knock-out for both FBPase and SBPase genes were readily obtained by sequential transfections. The Δfbp.sbp cell line had no growth defect under nutrient-rich culture conditions. However, there was a mild and variable growth defect in CMM. The high variability in growth rate could be caused by different mechanisms of adaptation to nutrient-restricted conditions. The metabolomic experiments with Δfbp.sbp revealed that SBPase bears its canonical enzymatic activity (converting S1,7bP to S7P), since its substrate, S1,7bP was highly accumulated in the knockout cell line. A concomitant increase in S7P is most likely explained by non-enzymatic loss of a single phosphate from the accumulated S1,7bP. Similarly, accumulation of S1P and S1,7bP was observed previously in an SBPase yeast deletion mutant [37]. SBPase was also identified in Toxoplasma gondii, and its deletion resulted in a similar phenotype to what we observed here, both in metabolomics and in decreased infectivity [38]. Fernandes and colleagues [17] showed reverse PFK activity (equal to canonical FBPase) in vitro and proposed that the glycosomal microenvironment might create conditions for reverse PFK activity. However, our data do not support this view because, although depletion of PFK by RNAi was not complete (decrease in PFK expression to 20% in parental and to 60% in the Δfbp.sbp background), we could detect a major decrease in glycolytic flux but not in GNG flux. However, if reactions in both directions occur simultaneously, we would not be able to detect a minor change in GNG flux. More efficient PFK knockdown by RNAi is unlikely to be achievable in the Δfbp.sbp double knock-out mutant, because the metabolic flux is flexible and the enzymes can compensate for each other to some extent, which is impossible in the double knock-out background. Metabolomics showed surprisingly few changes in the levels of detected metabolites in the PFK knockdown cell lines. Nevertheless, we noted a decrease in glycolytic flux, but not in GNG flux. The directionality of the PFK activity is dependent on concentrations of substrates and products and the negative free energy (ΔG) of the PFK reaction is highly favorable for the forward reaction. Significant perturbations in substrate concentrations including ADP/ATP ratio and changes in glycosomal pH might allow the PFK reversal [17]. Nevertheless, in our metabolomic datasets we did not detect any prominent changes in ADP, ATP, F6P, and F1,6bP levels, for instance one of the largest changes is the 2-fold accumulation of F1,6bP in Δfbp.sbp/RNAiPFK compared with WT (S3 Fig). Altogether, our data do not support participation of PFK in GNG. However, it should be noted that we measured metabolites extracted from whole cells, whereas metabolite levels and especially ADP/ATP ratios may be significantly shifted within the glycosomal subcompartment compared to that of the whole cell. The application of technically challenging methods is required to determine the metabolic microenvironment of glycosomes to gain a deeper understanding of the involvement of individual enzymes in GNG. The PPP intermediates detected by metabolomics can be separated into two groups. The first represents the oxidative PPP and metabolites derived originally from glucose, where less labelling from glycerol was detected (pentose phosphates, Fig 3C). The second group, representing the non-oxidative PPP, contains S7P or O8P, which have significant proportions labelled (Figs 3C and 5D). Since BSF T. brucei does not express transketolase [20,21], the metabolites cannot be produced in the canonical non-oxidative PPP. This suggests that in a low concentration of glucose, as used here, glucose is used preferentially to feed the oxidative PPP, whereas glycerol is used to feed glycolysis and to synthesize large sugar phosphates such as S7P. Although O8P has previously been shown to be synthesized in vitro from F6P and R5P by TAL [29], there appears to be a separate route for synthesis in the parasites, as evidenced by the presence of this metabolite in TAL-depleted cell lines. In both WT and Δfbp.sbp.tal, much higher quantities of O8P were detected in the presence of glucose than in the same cell lines grown in glycerol-based medium (S4 Fig). Our datasets provide new insights into the role of TAL and the whole PPP in BSF, however, further studies will be required to resolve the arrangement of the pathway in this life stage. A novel metabolic pathway that includes the activities of SBPase, TAL, and fructose-1,6-bisphosphate aldolase was proposed [25]. If active, this pathway conforms to the labelling patterns detected in S7P (and O8P), and also with S1,7bP production catalysed by aldolase, as reported previously [37]. Most importantly, this would present an alternative pathway for the production of F6P from glycerol. Nevertheless, our experiments with the Δtal and Δfbp.sbp.tal mutants show that carbons from glycerol are still incorporated into F1,6bP, F6P, and other metabolites, demonstrating continued flux though GNG, and precluding the existence of the alternative pathway. It is also unclear how the flux between glycolysis and GNG, i.e. between PFK and FBPase activity, is regulated. Potentially, the forward or reverse flux could be controlled by upstream kinases, i.e. hexokinase in glycolysis and glycerol kinase in GNG, due to competition for ATP, as reported recently [39]. Another possibility would be an exclusive compartmentalisation of these enzymes, but our immunofluorescence analysis shows that PFK and FBPase co-localise under all conditions tested. These results suggest that some futile cycling does take place, as substantial 13C 3 -labelling of hexose phosphates (Fig 3B) occurs. However, since FBPase is not a key GNG enzyme, we suggest that regulation is achieved by a distinct enzyme that possesses FBPase activity but whose identity is yet to be established. When glucose is limiting, bloodstream form trypanosomes adapt by activating alternative metabolic pathways. Labelling patterns in succinate and citrate indicate flux in mitochondrial metabolism. Since only a small proportion of labelling in citrate was detected (72–89% of citrate was unlabelled), incorporation of additional substrates, such as glutamine or other amino acids likely occurs, as reported previously [40]. If glycerol is available to trypanosomes, it is used as a carbon source, being incorporated into sugar phosphates under all of conditions tested here. Deletion or depletion of four different enzymes failed to substantially diminish GNG flux in BSF trypanosomes. Taken together, our results indicate that (an)other enzyme(s), currently unrecognized by in silico searchers, is/are responsible for the activity. The question as to whether GNG is essential for BSF T. brucei cannot be readily addressed until these key enzyme(s) are identified.

Acknowledgments We thank Prof. Frederic Bringaud (University of Bordeaux) and Prof. Paul Michels (University of Edinburgh) for the kind gift of antibodies α-FBPase, α-SBPase, and α-PFK.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0012007

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