Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli

Molecular & Biochemical Parasitology 216 (2017) 21–29

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Research Paper

Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli

MARK

Rocío Rondón-Mercado, Héctor Acosta, Ana J. Cáceres, Wilfredo Quiñones, ⁎ Juan Luis Concepción Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela

A R T I C L E I N F O

A B S T R A C T

Keywords: Trypanosoma rangeli Trypanosoma cruzi Glycosome Intermediary metabolism Glucose consumption

Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The velocity of glucose consumption is about 40% higher than that of procyclic Trypanosoma brucei, and four times faster than by T. cruzi epimastigotes under the same culture conditions.

1. Introduction Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Latin America [1]. In contrast to Trypanosoma brucei and Trypanosoma cruzi, T. rangeli is considered non-pathogenic to mammalian hosts but harmful to insect vectors, especially those of the genus Rhodnius. The parasites cause morphological abnormalities and death of the triatomine nymphs during molting [2,3]. Unlike T. cruzi, T. rangeli is transmitted among mammals through an inoculative route during hematophagy by the triatomine bugs in

which the trypanosomes develop and multiply in the hemolymph and finally invade the salivary glands where they transform into metacyclic trypomastigotes [4–7]. Although this parasite is not pathogenic for human, its deserves to be studied because it shares with T. cruzi (the etiological agent of Chagas' disease) biological characteristics, geographic distribution, vectors, vertebrate hosts and more than 60% of the antigens [8–10]. This overlap makes possible the occurrence of single and/or mixed infections with T. cruzi that can lead to misdiagnosis resulting in false positives, especially when complex mixtures of antigens are used in serodiagnosis. This may endanger human health due to

Abbreviations: ALD, aldolase; ENO, enolase; FH, fumarate hydratase; FRD, fumarate reductase; GALK, galactokinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlcK, glucokinase; GlDH, glutamate dehydrogenase; GPDH, glycerol-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; HK, hexokinase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PBS, phosphate-buffered saline; PEPCK, phosphoenolpyruvate carboxykinase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; PPDK, pyruvate phosphate dikinase ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (J.L. Concepción). http://dx.doi.org/10.1016/j.molbiopara.2017.06.007 Received 10 April 2017; Received in revised form 14 June 2017; Accepted 15 June 2017 Available online 20 June 2017 0166-6851/ © 2017 Elsevier B.V. All rights reserved.

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encoded by genes identified in the T. rangeli genome project [28]. Although in most cases there is not yet proteomic evidence for their expression, we confirmed the presence of predicted glycosome-targeting sequences in the genes’ open-reading frames for most enzymes of which the subcellular localization had been determined by immunofluorescence microscopy of activity assays upon cell fractionation.

inappropriate administration of the toxic drugs used for Chagas’ disease treatment [11]. Literature documenting serological cross-reactivity between T. rangeli and T. cruzi has resulted in a strong controversy because other authors have reported no detectable cross-reactivity when recombinant antigens or species-specific synthetic peptides were used [12]. Recently, some species-specific proteins were identified in T. rangeli trypomastigotes which may provide an effective differential serodiagnosis [13]. One of the peculiarities of T. cruzi and other kinetoplastids is the compartmentalization of some of their key metabolic pathways in peroxisome-like organelles named glycosomes [14–17], where notably the major part of glycolysis takes place. These organelles contain the first six or seven glycolytic enzymes together with some auxiliary pathways involved in the reoxidation of the NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and in the regeneration of the ATP consumed in the activation of the glucose molecule [18,19]. In addition, these glycosomes may contain enzymes of several other processes such as the pentose-phosphate pathway, β-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes [20]. Trypanosomatids are dependent on available carbon sources in their hosts to maintain energy metabolism. In their life cycle, they experience drastic changes between the insect vector and vertebrate host, with regard to nutrients, pH, oxidative stress and temperature, so they undergo various metabolic (e.g. associated with carbon and energy sources) and morphological adaptations. Glucose is a main source of carbon and energy for most developmental stages within the vertebrate host [21]. Hence, the energy metabolism of these parasites is often based primarily on the aerobic fermentation of glucose. This latter process is characterized by the absence of a ‘Pasteur effect’ (inhibitory effect of oxygen on the fermentation process) where most of the carbons of the hexose are excreted as still largely reduced intermediary metabolites such as succinate, pyruvate, acetate, ethanol, L-alanine, or lactate (depending on the species), not only in anaerobiosis, but also under aerobic conditions [21]. The trypanosomatids show, in most cases studied, even a ‘reverse Pasteur effect’, meaning that glucose consumption may be even lower under anaerobic conditions; this odd behaviour is related to the lack of the major controls points on the glycolytic pathway [22]. In the absence of glucose, amino acids (L-proline, also L-threonine and L-glutamine) are often used as the main source of energy, although glycolysis is almost always active except, for example, in the early stages of amastigote development in T. cruzi [23,24]. This is especially true for the stages that develop in the invertebrate vector such as some strains of Trypanosoma spp. and Leishmania spp. It has been observed that, in complex culture media of T. cruzi epimastigotes and promastigotes of Leishmania spp., consumption of amino acids begins after the glucose is exhausted. This was evidenced by the excretion of large amounts of ammonium into the medium [20,21,25–27]. Actually, no studies have been reported yet about the energy and carbon sources preferred for each of the morphological stages of T. rangeli, nor about its intermediary metabolism and the involvement of glycosomes. We present here the first report which shows that epimastigotes of T. rangeli consume only glucose as carbon and energy source. The subcellular distribution of the enzymes responsible for the conversion of glucose to pyruvate has also been determined. Part of glycolysis occurs in glycosomes along with the first oxidative step in the pentose-phosphate pathway, by glucose-6-phosphate dehydrogenase (G6PDH). Other enzymes like glucokinase (GlcK) and galactokinase (GALK) are also expressed as glycosomal proteins. Finally, glycosomes contain auxiliary pathways to glycolysis, needed to maintain the adenine nucleotide and redox balances within the glycosomes, since also found in these organelles were the enzymes phosphoenolpyruvate carboxykinase (PEPCK), malate dehydrogenase (MDH), fumarate reductase (FRD), pyruvate phosphate dikinase (PPDK) and glycerol-3phosphate dehydrogenase (GPDH). All glycolytic and auxiliary enzymes studied in this work are

2. Materials and methods 2.1. Parasites Epimastigotes of the T. rangeli Dog82 strain (isolated from blood of a dog) were cultured at 28 °C in LIT medium (liver infusion-tryptose) supplemented with 10% (v/v) inactivated fetal bovine serum as previously described [29]. Parasites were harvested in the exponential phase of growth, at an optical density at 600 nm (OD600) of 0.4. 2.2. Antibodies Polyclonal anti-hexokinase (HK) [30], anti-GlcK [31] anti-phosphoglycerate kinase (PGK) [32], anti-PPDK [33], anti-enolase (ENO) [34] and anti-gGALK-1 [35] were raised in rabbits against recombinant enzymes from T. cruzi, whereas anti-GAPDH, anti-aldolase (ALD) [36], anti-GPDH and anti-FRD, all prepared against recombinant enzymes from T. brucei, were kindly provided by Dr Michels (Brussels) and Dr Bringaud (Bordeaux), respectively. The mouse polyclonal anti-PEPCK [37] was raised using the recombinant Trypanosoma evansi enzyme. Monoclonal mouse anti-human tubulin (Sigma, USA), goat anti-rabbit IgG conjugated with either peroxidase (Sigma) or Cy3 (Amersham Biosciences), and goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) (Sigma) were purchased. 2.3. Glucose and ammonium determination during growth of T. rangeliepimastigotes T. rangeli epimastigotes were grown in LIT medium supplemented or not with 25 mM glucose. Medium was inoculated with 5 × 106 parasites/ml and their growth was followed by OD600 measurement of samples collected every 24 h until the culture reached the stationary growth phase. All samples were centrifuged at 5000 x g for 10 min to harvest parasites from the culture medium. Subsequently, pellets were washed four times with buffer (140 mM NaCl, 11 mM KCl and 75 mM Tris, pH 7.4) and stored at −80 °C for further HK and NAD+-dependent + L-glutamate dehydrogenase (NAD -GlDH) enzymatic activity determinations. Supernatants were used for determination of pH, and concentrations of glucose and ammonium in the culture medium. The glucose concentration was determined using a commercial peroxidaseglucose oxidase assay system (Wiener, Chile). Ammonium was determined using α-ketoglutaric acid in the presence of NAD+-GlDH with a commercial system (Sigma), following the instructions of the manufacturer. For growth of parasites while maintaining the pH constant, cultures were collected every 48 h, centrifuged at 5000 x g and the parasites resuspended in fresh LIT medium under sterile conditions. 2.4. Subcellular fractionation and glycosome purification A homogenate of T. rangeli epimastigotes was obtained by grinding washed cells with silicon carbide (200 mesh), and the homogenate was fractionated by differential and isopycnic centrifugation [38]. For subcellular fractionation of enzymes by partial permeabilization of membranes by digitonin, parasites were collected in the exponential phase of growth (3 × 107 cells/ml) and treated as described previously [39]. Briefly, all fractions (pellets and supernatants) were used for enzymatic activity assays and immunoblotting. 22

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In addition, the exponential growth phase is followed by a stationary phase with a minimal growth rate (over four times lower than in the exponential phase), although at the start of this transition only 50% of the glucose has been consumed. Interestingly, the stationary phase culture maintained the same rate of glucose consumption. On the other hand, unlike T. cruzi and other kinetoplastids [45], T. rangeli seemed not able to sustain further growth by consumption of the amino acids present in the culture medium. This is in agreement with the glutamate dehydrogenase (NAD+-GlDH) specific activity, which remains constant at a low level throughout the cultivation (Fig. 1B and D). Moreover, we observed that the glucose consumption was accompanied by a decrease in pH of the medium from 7.4 to 6.6, when the parasites entered the stationary phase at day 3 (Fig. 1C). An experiment in which the culture was replenished with fresh medium every 48 h, thus restoring the initial pH, showed that under these conditions the parasites were able to continue their growth, reaching twice of the cellular density compared to cultures of which the medium was not changed (Fig. 1E).

2.5. Immunofluorescence microscopy T. rangeli epimastigotes were washed with isosmotic phosphatebuffer saline (PBS) (39 mM Na2HPO4, 10 mM NaH2PO4, 137 mM NaCl, 22 mM KCl, pH 7.4) and processed in two different ways: (i) Cells were fixed with 4% of formaldehyde in PBS, washed with the same buffer, adhered to poly-(L-lysine)-coated coverslips and permeabilized with 0.2% (v/v) Triton X-100 and (ii) Cells were directly fixed to poly-(Llysine)-coated coverslips and air-dried, but not permeabilized. In both cases the slips with fixed cells were blocked with PBS containing 3% bovine serum albumin (BSA) and 50 mM ammonium chloride and washed again with PBS. Then, cells were incubated with respective primary antibodies for 1 h in PBS containing 1% BSA, rinsed with PBS and incubated for 1 h with goat anti-mouse IgG conjugated with FITC and goat anti-rabbit IgG conjugated with Cy3 (Sigma) and subsequently with DAPI (Sigma) for 30 min, according to the protocol described previously [34]. Finally cover slips were mounted with 90% glycerol in PBS and 5% (w/v) 1,4-diazabicyclo[2.2.2]octane (DABCO) and analyzed with a Nikon eclipse 80i microscope.

3.2. Subcellular localization of enzymes of carbon metabolism

2.6. Enzymatic assays and data analysis

In order to determine the subcellular localization of enzymes of the glycolytic, pentose-phosphate and auxiliary pathways, an isopycnic ultracentrifugation of an epimastigote homogenate was performed (Fig. 2A). The activity of these enzymes was measured and immuneprobed in the different samples obtained after the density-based subcellular fractionation. We found that most of the HK activity was systematically recovered in fractions with a density of 1.23–1.24 g .cm−3, characteristic of glycosomes [29,39,46,47]. In contrast, the PGI, G6PDH and MDH activities were recovered in two fractions with densities of 1.23–1.24 g .cm−3 (glycosomes) and 1.03–1.08 g cm−3, the latter being characteristic of the cytosol. Other activities such as those of ENO and PGK were recovered completely at 1.03–1.08 g .cm−3, indicative of their exclusive cytosolic localization. This result is in agreement with the signal obtained by western blot analysis of the samples obtained in the same fractionation (Fig. 2B). On the other hand, the immunodetection of auxiliary enzymes such as PPDK, PEPCK, GPDH and GALK also showed a glycosomal localization (Fig. 2B). IDH was used as a marker enzyme that is known to be present in the cytosol and mitochondrion but not glycosomes of T. cruzi. A progressive permeabilization of different epimastigote membranes was performed by increasing amounts of digitonin, and the release of several enzymatic activities from the cells was followed (Fig. 3A). The first enzyme to be released was ENO, reported as a cytosolic enzyme in all kinetoplastids [34,48,49]; this enzyme was completely released by addition of about 0.06 mg digitonin. mg protein−1. The release of the total PGK activity coincided with ENO, confirming its cytosolic location. In contrast, the complete release of the classical glycosomal marker HK [30,50] required 0.15 mg digitonin. mg protein−1. On the other hand, approximately 70% of the PGI activity was released at a similar digitonin concentration as ENO, whereas the remaining 30% followed the same pattern as HK, confirming a dual location, cytosolic and glycosomal. This result was corroborated with their immunodetection in the protein blots made from supernatants obtained by centrifugation of digitonin treated cells (Fig. 3B). Other enzymes such as PPDK, PEPCK, FRD, GPDH, G6PDH, GALK and GlcK also showed a glycosomal location. A third method to confirm the subcellular localization of the enzymes studied was performed by immuno-fluorescence microscopy analysis. Epimastigotes of T. rangeli were stained simultaneously with the monoclonal mouse anti-PPDK (as a glycosomal marker) and rabbit serum antibodies for different enzymes. In T. rangeli PPDK, previously reported as a specific protein of trypanosomatid glycosomes [5,39], showed a punctate fluorescence pattern consistent with spherical structures in the cytoplasm of the epimastigotes (Fig. 4A). An identical staining pattern was observed with antisera specific for ALD, FRD, PEPCK, GPDH, GlcK, G6PDH, GALK and HK (Fig. 4A). Superposition of

All enzymes were assayed spectrophotometry by coupling the respective reactions to a NAD(P)+/NAD(P)H-dependent system and measured at 340 nm and at room temperature, in the presence of 0.1% (v/v) Triton X-100 and 150 mM NaCl, in order to eliminate possible latency artifacts. Hexokinase (HK, EC 2.7.1.), phosphoglucose isomerase (PGI, EC 5.3.1.9), enolase (ENO, EC 4.2.1.11), pyruvate phosphate dikinase (PPDK, EC 2.7.9.1), malate dehydrogenase (MDH, EC 1.1.1.37) and galactokinase (GALK, EC 2.7.1.6) were assayed as described [30,29,34,39,40,35]. All other enzyme activities were assayed as described in [41]: phosphofructokinase (PFK, EC 2.7.1.11), pyruvate kinase (PK, EC 2.7.1.40), phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.4.9), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and isocitrate dehydrogenase (IDH, EC 1.1.1.41 or 1.1.1.42). 2.7. Protein determination, SDS-PAGE and western blotting Proteins were quantitatively assayed by the Lowry method as modified previously [42] with BSA as standard. SDS-PAGE was performed in 12% polyacrylamide gels as described [43]. Proteins separated by SDS-PAGE were transferred to a PVDF membrane (Amersham Bioscience) and western blotting was performed as described elsewhere [44]. The membranes were incubated with respective polyclonal antisera, washed with PBS, incubated with secondary antibody peroxidaseconjugated goat anti-rabbit IgG (Sigma) diluted 1:4000. Immunoblots were revealed using diaminobenzidine 1 mg .ml−1 (Sigma) and 0.2% (v/v) H2O2. 3. Results 3.1. Dependence of T. rangeli growth on glucose availability and medium pH Most of the current knowledge about the carbon sources used by the different developmental stages of trypanosomatid family members has been learned from studies with T. brucei, T. cruzi and Leishmania spp. In order to elucidate if the epimastigote form of T. rangeli uses the same carbon sources as its relatives, this developmental stage was grown in modified LIT medium supplemented or not with glucose. This involved both partially, to 8.3 mM glucose-depleted LIT medium (“low glucose”) and the same medium to which 25 mM of glucose was added (“high glucose”). The growth curve in Fig. 1A shows that epimastigotes multiply exponentially during approximately 48 h, concomitantly with a decrease in the levels of glucose in the medium, corresponding to a consumption rate of 16.5 × 10−8 nmol .min−1 .parasite−1 (Table 1). 23

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Fig. 1. Growth of the epimastigotes of T. rangeli in LIT medium. Parasites growth with (●) and without (○) glucose. Parasites were counted in a Neubauer chamber, after being treated with formaldehyde 1% (v/v) (A). Variation of glucose (●) and ammonium (○) in the LIT medium along the culture time (B). Variation of pH the LIT medium, parasites grown with (●) and without (○) glucose (C). Parasite grown at constant pH. Variation of the specific activity (SA) of hexokinase (HK) (●) and glutamate dehydrogenase (GlDH) (○) along the growth of parasites in LIT medium (D). The arrows indicate the days where the cells were collected and passed to fresh medium (E). The data given in the figure (panels A–C) represent the mean ± the standard deviation of samples from three independent experiments.

4. Discussion

Table 1 Glucose consumption rates of trypanosomatids. Organism

Stage

Glucose consumption rate (nmol .min1 .parasite-1)

Glucose consumption rate (nmol .min-1 .mg-1)

T. T. T. T.

Epimastigote Epimastigote Procyclic Bloodstream

16.5 × 10−8 3.9 × 10-8 12 × 10−8 160 × 10−8

10.6 3.12 12.0 160.0

rangeli cruzia bruceib bruceic

During their life cycle, most trypanosomatid species alternate between a vertebrate host and an insect vector. As a result, these protists find environments that differ significantly in the amount and type of carbon and energy sources, as well as the availability of oxygen. Carbohydrates among them glucose, are the principal source of energy and carbon used in at least some stages of the life cycle by all trypanosomatids studied to date; however, when the sugars are exhausted the trypanosomatids will start to use other nutrients, often amino acids and fatty acids [21]. In contrast, T. rangeli epimastigotes (grown in LIT medium) use glucose as sole source of energy and were unable to proliferate in medium without glucose, where the alternative potential primary source of carbon and energy are amino acids. This is in agreement with the absence of ammonium production resulting from amino acid degradation in both culture conditions tested, partial-glucose depleted and glucose supplemented media. Accordingly, no increase in the specific activity of NAD+-dependent glutamate dehydrogenase (a marker enzyme of amino-acid degradation) was observed throughout the time of the cultivation (both during growth and in the stationary phase). These results contrast with those reported for T. cruzi, T. brucei, L. mexicana and C. fasciculata [52] where ammonium excretion and the specific activity of NAD+-dependent glutamate dehydrogenase strongly increase when glucose is exhausted. Moreover, it was observed that the pH decreased gradually to values close to 6.6 in glucose-containing medium, even though this sugar was

Consumption rates shown for other trypanosomatids were taken from (a) [32], (b) Haanstra (personal communication) and (c) [66]. The data were transformed from nmol. min−1. parasite−1 to nmol. min−1. mg−1 or vice versa using as conversion factors that 1 mg of protein is equal to 1 × 108 T. brucei cells (bloodstream-form), 8 × 107 T. cruzi cells (epimastigote) and 6 × 107 T. rangeli cells (epimastigote).

the images in panels B and C resulted in a yellow spherical pattern indicative of PPDK and the afore mentioned enzymes colocalizing to the glycosomes (Fig. 4A, Panel D). Another analysis was performed using epimastigotes of T. rangeli stained with monoclonal mouse anti-tubulin and rabbit serum anti-ENO and anti-PGK. The pattern observed in this case confirmed the cytosolic localization of these enzymes (Fig. 4B). The glycosomal localization of the following enzymes was in agreement with the detection of a potential peroxisomal-targeting signal in the proteins as predicted from their sequences in the T. rangeli genome database: HK, GALK, GlcK, PEPCK, PPDK, MDHg, FRD, PGI, ALD, G6PDH (not shown). 24

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Fig. 2. Distribution profile of a homogenate of T. rangeli epimastigotes after isopycnic centrifugation. Densities of the fractions varied from about 1.05 to 1.27 g. cm−3. (A) The determination of enzymatic activities was performed at least twice for each fraction as described in Materials and methods. (B) Each fraction (indicated by numbers) of the gradient was analyzed by western blot probed with polyclonal antibodies as explained in Materials and methods. HK, hexokinase; PGK, phosphoglycerate kinase; ENO, enolase; PPDK, pyruvate phosphate dikinase; PEPCK, phosphoenolpyruvate carboxykinase; GPDH, glycerol-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; GALK, galactokinase.

remained above the critical value while, importantly, the glucose was never completely consumed. The notion that the growth arrest is caused by the low pH and not due to a limiting nutrient or accumulation of a toxic by product is further supported by the observation that T. cruzi epimastigotes, cultured under exactly the same conditions, can grow to a density more than three times higher than T. rangeli, thanks to the switch to amino-acid catabolism that, through the ammonium production, prevents further decrease of the culture pH [32]. Surprisingly, the rate of glucose consumption, as well as a further acidification of the medium continued, even when the parasites were not growing anymore, thus in stationary phase. We speculate that the rate of glucose consumption in the stationary phase is used for maintenance such as the production of ATP. This would provide the energy for, among others, the operation of H+- and ion (e.g. Na+/K+)-pumping ATPases to

only consumed partially. When 50% of the glucose was consumed and the pH reached the value indicated, the growth rate of the parasites was severely slowed down and the parasites entered in an early stationary phase. This pH decrease should be attributed to the acidic metabolites (monocarboxylic and dicarboxylic acids such as acetate and succinate) which are excreted together with alanine) of glucose catabolism, as previously reported for this Trypanosoma species by RodríguezGonzález et al. [53]. Succinate production is consistent with the presence of a glycosomal auxiliary branch comprising PEPCK-MDH-fumarase-FRD, similar to what has been reported for T. cruzi epimastigotes and procyclic form T. brucei [39,54]. The growth inhibitory effect of the pH was confirmed by replacing the culture medium every 48 h (each time when the pH decreased to below 6.6). Under this condition the parasites could restore cell growth once again, for as long as the pH 25

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Fig. 3. Release of some enzymes from T. rangeli by digitonin treatment. The enzymes were released from whole epimastigotes upon addition of digitonin at increasing concentrations. (A) The enzyme activities were determined in the supernatant of the centrifuged cells after their incubation for 20 min in the presence of the indicated proportion of digitonin. HK (●), MDH (□), PGI (▲), ENO (◊), PGK (□). (B) Western blot analysis of the supernatants using the respective polyclonal antibodies as probes. HK, hexokinase; MDH, malate dehydrogenase; PGI, phosphoglucose isomerase; ENO, enolase; PGK, phosphoglycerate kinase; PPDK, pyruvate phosphate dikinase; PEPCK, phosphoenolpyruvate carboxykinase; FRD, fumarate reductase; GPDH, glycerol-3phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; GALK, galactokinase; GlcK, glucokinase.

much lower than the enzyme activities in the T. brucei bloodstream form (Table 2), except for MDH, in agreement with the fact that these latter cells produce predominantly pyruvate and hardly any succinate. The glycosomes of T. rangeli epimastigotes are able to take up glucose, catabolize it to 1,3-bisphosphoglycerate that will be exported from the organelles. This catabolism of glucose results, within the glycosomes, in the formation of reduced cofactor NADH in the GAPDHcatalyzed reaction, and the consumption of ATP by HK and PFK. Impermeability of the glycosomal membrane to NAD(H) and ATP is consistent with either the presence of the auxiliary enzymes PEPCK, MDH, FRD and PPDK inside glycosomes and/or the export of glycerol 3phosphate (G3P) and import of dihydroxyacetone phosphate (DHAP) to reoxidize the NADH by the auxiliary branch or to transfer its electrons to the mitochondrion via the G3PDH-dependent G3P/DHAP shuttle, respectively (Fig. 5). Additionally, ATP can be regenerated within the glycosomes during conversion of PEP to malate or pyruvate, also by the

maintain cytosolic pH and ion homeostasis (to compensate for unavoidable leakage) as is required in both growing and quiescent cells. Such a H+-ATPase would be similar to that observed in T. cruzi and Leishmania [55,56]. It is likely that other ion/H+-translocating pumps will also contribute to maintaining homeostasis. For example, a putative proton-translocating inorganic pyrophosphatase (H+-PPase) has been identified in epimastigote forms of T. rangeli [57]. H+-PPases have been reported to occur in the acidocalcisomal membranes of a number of pathogenic trypanosomatids [58], and genes encoding these proteins have been cloned and sequenced in some kinetoplastids [59,60]. The glucose consumption rate of T. rangeli epimastigotes is 4 times superior to that in T. cruzi epimastigotes, in a similar range to the rate measured in procyclic form of T. brucei, but 10 times lower than in the bloodstream form of T. brucei (Table 1). These values are in agreement with the specific activities measured for glycolytic enzymes of T. rangeli in this work; they are very similar to those of T. cruzi epimastigotes, but 26

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Fig. 4. (A) Subcellular localization of Trypanosoma rangeli glycolytic enzymes by immunofluorescence. Parasites were permabilized with 0.1% Triton X-100 and processed as described in Materials and methods. Five images of each parasite are shown per line. Panel a- (DIC)/DAPI merge; Panel b- green fluorescence showing labeling with primary PPDK antibody; Panel c- red fluorescence showing labeling with primary glycolytic enzyme antibody; Panel d- merge of b & c panels. (B) Glycolytic enzymes in the cytosol. Panel a- DIC/DAPI merge; Panel b- green fluorescence showing labeling with primary tubulin antibody; Panel c- red fluorescence showing labeling with primary glycolytic enzyme (PGK or ENO) antibody; Panel d- merge of b & cpanels.

Table 2 Specific activities of enzymes. Enzyme

Hexokinase (HK) Phosphoglycerate kinase (PGK) Enolase (ENO) Pyruvate kinase (PYK) Phosphoenolpyruvate carboxykinase (PEPCK) Pyruvate phosphate dikinase (PPDK) Malate dehydrogenase (MDH) Glucose-6-phosphate dehydrogenase (G6PDH)

Specific Activity (μmol. min−1. mg−1) T. rangeli

T. cruzi

T. brucei (p)

T. brucei (b)

0.120 0.300 0.097 0.005 0.004

0.0701 0.0402 0.020 0.008 0.0033

0.2074 0.5704 0.1695 0.0174 0.6706

1.9294 1.3584 0.7685 1.0204 NR

0.002

0.0053

0.0177

NR

0.600 0.008

0.670 0.006

0.2128 0.0129

0.0668 0.0179

Abbreviations: (p)procyclic form; (b) bloodstream form. References: 1. [30]; 2. [67]; 3. [39]; 4. [68]; 5. [69]; 6. [70]; 7. [51]; 8. [16]; 9. [71]; NR: not reported. (p)procyclic form; (b) bloodstream form.

[39,61]. This system comprises the ATP-producing and CO2-fixing PEPCK in the branch also containing MDH, fumarate hydratase (FH) and FRD [39,53]. Similarly, a PPi-dependent PPDK converts PEP, PPi and AMP to pyruvate, Pi and ATP. An additional role assigned to this enzyme in T. cruzi is that its PPi-hydrolyzing activity prevent accumulation to toxic levels of PPi produced by the biosynthetic processes that take place in the glycosomes [39]. The PEPCK and PPDK branches are each able to regenerate one molecule of ATP per molecule of PEP. In addition, recently a gene for adenylate kinase (ADK) in T. rangeli has been reported which presents 99% similarity with its counterpart in T. cruzi [28] and codes for an amino-acid sequence with a SKL tripeptide in its C-terminal region suggestive of a glycosomal localization. This enzyme inside of the glycosomes will serve to maintain the equilibrium of the adenine nucleotides. Meanwhile, a cytosolic localization of PGK as found in T. rangeli has also been reported for the procyclic form of T. brucei, promastigotes and amastigotes of Leishmania species [62,63], epimastigotes of T. cruzi [17] and bloodstream form of T. congolense [64]. However, a difference is that all these other trypanosomatids have also a glycosomal PGK, although often most PGK activity is in the cytosol. T. rangeli shows also similarity with bloodstream-form T. brucei in the sense that it only grows at the expense of glucose as source for ATP. However, it differs from T. brucei in the localization of PGK. This enzyme was found in T. rangeli entirely in the cytosol, unlike in bloodstream-form T. brucei where even a low level of expression of PGK activity in the cytosol inhibits parasite growth [65], indicating that correct localization of this glycolytic enzyme is important. Due to the presence of PGK in the cytosol, the ATP balance inside glycosomes of T. rangeli can only be maintained by the activity of PEPCK and or PPDK. It is noteworthy that the presence of a single PGK in the cytosol of the epimastigotes implies that it has to function in both the glycolytic and gluconeogenic pathways, depending on the availability of glucose. Thus, when the glucose concentration is adequate, the role of PGK is glycolytic, providing ATP in the cytosol. However, when the vector spends days without eating, glucose is absent and the PGK is expected to participate in gluconeogenesis, when the trypanosome consumes lipids and/or amino acids.

auxiliary branch. The presence of these auxiliary routes thus contributes to maintaining the intraglycosomal redox and ATP/ADP balances in a similar manner to that reported for other kinetoplastids

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Fig. 5. A model for the intermediary metabolism of glucose in glycosomes of T. rangeli epimastigotes. All reactions indicated by a continuous arrow in the model have been demonstrated in this work. Metabolites abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3BPGA, 1,3-bisphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; OXAC, oxaloacetate; MAL, malate; Fum, fumarate; Succ, succinate; ALA, alanine; Pyr, pyruvate. Enzymes: 1. hexokinase; 2. phosphoglucose isomerase; 3. phosphofructokinase; 4. aldolase; 5. glycerol-3-phosphate dehydrogenase; 6. phosphoglycerate kinase; 7. enolase; 8. pyruvate kinase; 9. phosphoenolpyruvate carboxykinase; 10. malate dehydrogenase; 11. fumarate reductase; 12. pyruvate phosphate dikinase; 13. glucose-6-phosphate dehydrogenase; 14. galactokinase; 15. glucokinase. 16. adenylate kinase. PPP, pentose-phosphate pathway; ISP, Isselbacher salvage pathway. [13] G. Wagner, L. Eiko Yamanaka, H. Moura, D. Denardin, A.D. Schlindwein, P. Hermes, et al., The Trypanosoma rangeli tripomastigote surfaceome reveals novel proteins and targets for specific diagnosis, J Proteomics 82 (2013) 52–63. [14] F.R. Opperdoes, P. Borst, Localization of nine glycolytic enzymes in a microbodylike organelle in Trypanosoma brucei: the glycosome, FEBS Lett. 80 (1977) 360–364. [15] J. McLaughlin, The presence of alpha-glycerophosphate dehydrogenase (NAD+linked) and adenylate kinase as core and integral membrane enzymes respectively in the glycosomes of Trypanosoma rhodesiense, Mol. Biochem. Parasitol. 14 (1985) 219–230. [16] D.T. Hart, O. Misset, S.W. Edwards, F.R. Opperdoes, A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes, Mol. Biochem. Parasitol. 12 (1984) 25–35. [17] M.B. Taylor, W.E. Gutteridge, Trypanosoma cruzi: subcellular distribution of glycolytic and some related enzymes of epimastigotes, Exp. Parasitol. 63 (1987) 84–97. [18] F.R. Opperdoes, Topogenesis of glycolytic enzymes in Trypanosoma brucei, Biochem. Soc. Symp. 53 (1987) 123–129. [19] P.A. Michels, V. Hannaert, F. Bringaud, Metabolic aspects of glycosomes in Trypanosomatidae − new data and views, Parasitol Today 16 (2000) 482–489. [20] P.A. Michels, F. Bringaud, M. Herman, V. Hannaert, Metabolic functions of glycosomes in trypanosomatids, Biochim. Biophys. Acta 1763 (2006) 1463–1477. [21] F. Bringaud, L. Rivière, V. Coustou, Energy metabolism of trypanosomatids: adaptation to available carbon sources, Mol. Biochem. Parasitol. 149 (2006) 1–9. [22] J.J. Cazzulo, Aerobic fermentation of glucose by trypanosomatids, FASEB J. 6 (1992) 3153–3161. [23] A.M. Silber, R.R. Tonelli, C.G. Lopes, N. Cunha-e-Silva, A.C. Torrecilhas, R.I. Schumacher, W. Colli, M.J. Alves, Glucose uptake in the mammalian stages of Trypanosoma cruzi, Mol. Biochem. Parasitol. 168 (2009) 102–108. [24] Y. Li, S. Shah-Simpson, K. Okrah, A.T. Belew, J. Choi, K.L. Caradonna, et al., Transcriptome remodeling in Trypanosoma cruzi and human cells during intracellular infection, PLoS Pathog. 512 (4) (2016) e1005511. [25] J.A. Urbina, Intermediary metabolism of Trypanosoma cruzi, Parasitol Today 3 (1994) 107–110. [26] S.W. van Weelden, B. Fast, A. Vogt, P. van der Meer, J. Saas, J.J. van Hellemond, et al., Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation, J. Biol. Chem. 278 (2003) 12854–12863. [27] S.W. van Weelden, J.J. van Hellemond, F.R. Opperdoes, A.G. Tielens, New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle, J. Biol. Chem. 280 (2005) 12451–12460. [28] P.H. Stoco, G. Wagner, C. Talavera-Lopez, A. Gerber, A. Zaha, C.E. Thompson, et al., Genome of the avirulent human-infective trypanosome Trypanosoma rangeli, PLoS Negl Trop Dis 8 (2014) e3176. [29] J.L. Concepción, B. Chataing, M. Dubourdieu, Purification and properties of phosphoglucose isomerases of Trypanosoma cruzi, Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 122 (1999) 211–222. [30] A.J. Cáceres, R. Portillo, H. Acosta, D. Rosales, W. Quiñones, L. Avilán, L. Salazar,

Acknowledgements This work was financially supported by the ‘Fondo Nacional de Ciencia, Tecnología Innovación’ (FONACIT) in Project MC-2007001425 (to J.L. Concepción). The authors would like to thank Dr Paul Michels (The University of Edinburgh) for stimulating discussions and critical reading of the manuscript. References [1] A. D’Alessandro, Biology of Trypanosoma (Herpetosoma) rangeli Tejera, in: W.H.R. Lumsden, D.A. Evans (Eds.), Biology of the Kinetoplastida, London Academic, London, 1976, pp. 327–403. [2] N. Añez, Studies on Trypanosoma rangeli Tejera, 1920. VII–Its effect on the survival of infected triatomine bugs, Mem. Inst. Oswaldo Cruz 79 (1984) 249–255. [3] E.J. Tobie, Biological factors influencing transmission of Trypanosoma rangeli by Rhodnius prolixus, J. Parasitol. 51 (1965) 837–841. [4] C.C. Hoare, Trypanosomes of Mammals, A Zoological Monograph, Blackwell, Oxford, Edinburgh, 1972. [5] A. D’Alessandro, N.G. Saravia, Trypanosoma rangeli, in: J.P. Kreier, E.J.R. Baker (Eds.), Parasitic Protozoa, 2nd edition, Academic Press, London, 1992, pp. 1–54. [6] F. Guhl, G.A. Vallejo, Trypanosoma (Herpetosoma) rangeli tejera 1920: an updated review, Mem. Inst. Oswaldo Cruz 98 (2003) 435–442. [7] P. Azambuja, E.S. Garcia, Trypanosoma rangeli interactions within the vector Rhodnius prolixus–a mini review, Mem. Inst. Oswaldo Cruz 100 (2005) 567–572. [8] J.R. Coura, O. Fernandes, M. Arboleda, T.V. Barrett, N. Carrara, W. Degrave, et al., Human infection by Trypanosoma rangeli in the Brazilian Amazon, Trans. R. Soc. Trop. Med. Hyg. 90 (1996) 278–279. [9] A. Cuba, Review of the biologic and diagnostic aspects of Trypanosoma (Herpetosoma) rangeli, Rev. Soc. Bras. Med. Trop. 31 (1998) 207–220. [10] J.E. Calzada, V. Pineda, J.D. Garisto, F. Samudio, A.M. Santamaria, A. Saldana, Human trypanosomiasis in the eastern region of the Panama Province: new endemic areas for Chagas disease, Am. J. Trop. Med. Hyg. 82 (2010) 580–582. [11] M.A. de Sousa, T. da Silva Fonseca, B.N. Dos Santos, S.M. Dos Santos Pereira, C. Carvalhal, A.M. Hasslocher Moreno, Trypanosoma rangeli tejera, 1920, in chronic chagas' disease patients under ambulatory care at the evandro chagas clinical research institute (IPEC-Fiocruz, Brazil), Parasitol. Res. 103 (2008) 697–703. [12] Z.C. Caballero, O.E. Sousa, W.P. Marques, A. Saez-Alquezar, E.S. Umezawa, Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp, Clin. Vaccine Immunol. 14 (2007) 1045–1049.

28

Molecular & Biochemical Parasitology 216 (2017) 21–29

R. Rondón-Mercado et al.

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41] [42] [43] [44] [45]

[46]

[47] [48] [49] [50]

[51]

[52]

M. Dubourdieu, P.A.M. Michels, J.L. Concepción, Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi, Mol. Biochem. Parasitol. 126 (2003) 251–262. A.J. Cáceres, W. Quiñones, M. Gualdrón, A. Cordeiro, L. Avilán, P.A. Michels, et al., Molecular and biochemical characterization of novel glucokinases from Trypanosoma cruzi and Leishmania spp, Mol. Biochem. Parasitol. 156 (2007) 235–245. X. Barros-Álvarez, A.J. Cáceres, P.A. Michels, J.L. Concepción, W. Quiñones, The phosphoglycerate kinase isoenzymes have distinct roles in the regulation of carbohydrate metabolism in Trypanosoma cruzi, Exp. Parasitol. 143 (2014) 39–47. E. González-Marcano, A. Mijares, W. Quiñones, A. Cáceres, J.L. Concepción, Posttranslational modification of the pyruvate phosphate dikinase from Trypanosoma cruzi, Parasitol. Int. 63 (2014) 80–86. W. Quiñones, P. Peña, M. Domingo-Sananes, A. Cáceres, P.A. Michels, L. Avilan, et al., Leishmania mexicana: molecular cloning and characterization of enolase, Exp. Parasitol. 116 (2007) 241–251. Á.E. Lobo-Rojas, E.B. González-Marcano, E.A. Valera-Vera, H.R. Acosta, W.A. Quiñones, R.J. Burchmore, et al., Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization, Parasitol. Int. 65 (2016) 472–482. A.J. Cáceres, P.A. Michels, V. Hannaert, Genetic validation of aldolase and glyceraldehyde-3-phosphate dehydrogenase as drug targets in Trypanosoma brucei, Mol. Biochem. Parasitol. 169 (2010) 50–54. L.A. Rivero, J.L. Concepción, E. Quintero-Troconis, W. Quiñones, P.A. Michels, H. Acosta, Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase, Exp. Parasitol. 165 (2016) 7–15. J.L. Concepcion, D. Gonzalez-Pacanowska, J.A. Urbina, 3-Hydroxy-3-methyl-glutaryl-CoA reductase in Trypanosoma (Schizotrypanum) cruzi: subcellular localization and kinetic properties, Arch. Biochem. Biophys. 352 (1998) 114–120. H. Acosta, M. Dubourdieu, W. Quiñones, A. Cáceres, F. Bringaud, J.L. Concepción, Pyruvate phosphate dikinase and pyrophosphate metabolism in the glycosome of Trypanosoma cruzi epimastigotes, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 138 (2004) 347–356. A. Aranda, D. Maugeri, A.D. Uttaro, F. Opperdoes, J.J. Cazzulo, C. Nowicki, The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms, Int. J. Parasitol. 36 (2006) 295–307. H.U. Bergmeyer, Methods of Enzymatic Analysis, Academic Press, New York, 1983. G.R. Schacterle, R.L. Pollack, A simplified method for the quantitative assay of small amounts of protein in biologic material, Anal. Biochem. 51 (1973) 654–655. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. F. Ausubel, R. Brent, R. Kingston, D. Moore, J. Seidman, J. Smith, et al., Current Protocols in Molecular Biology, Wiley, New York, 1989. F.J. Adroher, A. Osuna, J.A. Lupiáñez, Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase, Mol. Cell. Biochem. 94 (1990) 71–82. R.F. Steiger, F.R. Opperdoes, J. Bontemps, Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases, Eur. J. Biochem. 105 (1980) 163–175. F.R. Opperdoes, D. Cottem, Involvement of the glycosome of Trypanosoma brucei in carbon dioxide fixation, FEBS Lett. 143 (1982) 60–64. N. Visser, F.R. Opperdoes, Glycolysis in Trypanosoma brucei, Eur. J. Biochem. 103 (1980) 623–632. J.J. Cazzulo, Intermediate metabolism in Trypanosoma cruzi, J. Bioenerg. Biomembr. 26 (1994) 157–165. O. Misset, F.R. Opperdoes, Simultaneous purification of hexokinase, class-I fructosebisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei, Eur. J. Biochem. 144 (1984) 475–483. F. Bringaud, D. Baltz, T. Baltz, Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 7963–7968. J.J. Cazzulo, B.M. Franke de Cazzulo, J.C. Engel, J.J. Cannata, End products and

[53]

[54]

[55]

[56] [57]

[58] [59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70] [71]

29

enzyme levels of aerobic glucose fermentation in trypanosomatids, Mol. Biochem. Parasitol. 16 (1985) 329–343. I. Rodríguez-González, C. Marín, A.B. Hitos, M.J. Rosales, R. Gutierrez-Sánchez, M. Sánchez-Moreno, Biochemical characterization of new strains of Trypanosoma cruzi and T. rangeli isolates from Peru and Mexico, Parasitol. Res. 94 (2004) 294–300. S. Besteiro, M. Biran, N. Biteau, V. Coustou, T. Baltz, P. Canioni, F. Bringaud, Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase, J. Biol. Chem. 277 (2002) 38001–38012. N. Van der Heyden, R. Docampo, Significant differences between procyclic and bloodstream forms of Trypanosoma brucei in the maintenance of their plasma membrane potential, J. Eukaryot. Microbiol. 49 (2002) 407–413. D. Grigore, J.C. Meade, A COOH-terminal domain regulates the activity of Leishmania proton pumps LDH1A and LDH1B, Int. J. Parasitol. 36 (2006) 381–393. A.L. Fonseca-de-Souza, A.L. Freitas-Mesquita, L.P. Vieira, D. Majerowicz, N. DaflonYunes, et .al. Identification and characterization of an ecto-pyrophosphatase activity in intact epimastigotes of Trypanosoma rangeli, PLoS One 9 (2014) e106852. D.A. Scott, R. Docampo, Characterization of isolated acidocalcisomes of Trypanosoma cruzi, J. Biol. Chem. 275 (2000) 24215–24221. J.E. Hill, D.A. Scott, S. Luo, R. Docampo, Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi, Biochem. J. 351 (2000) 281–288. G. Lemercier, S. Dutoya, S. Luo, F.A. Ruiz, C.O. Rodrigues, et al., A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei, J. Biol. Chem. 277 (2002) 37369–37376. K. Deramchia, P. Morand, M. Biran, Y. Millerioux, M. Mazet, M. Wargnies, et al., Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form, J. Biol. Chem. 289 (2014) 17365–17378. C.A. Adjé, F.R. Opperdoes, P.A.M. Michels, Organization, sequence and stage-specific expression of the phosphoglycerate kinase genes of Leishmania mexicana mexicana, Mol. Biochem. Parasitol. 90 (1997) 155–168. G. McKoy, M. Badal, Q. Prescott, H. Lux, D.T. Hart, Characterization of PGK genes in Leishmania major: evidence for the absence of a third PGK gene or isoenzyme, Mol. Biochem. Parasitol. 90 (1997) 169–181. H.L. Parker, T. Hill, K. Alexander, N.B. Murphy, W.R. Fish, M. Parsons, Three genes and two isozymes: gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense, Mol. Biochem. Parasitol. 69 (1995) 269–279. J. Blattner, S. Helfert, P. Michels, C.E. Clayton, Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 11596–11600. J.R. Haanstra, A. van Tuijl, J. van Dam, W. van Winden, A.G. Tielens, J.J. van Hellemond, B.M. Bakker, Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes, Int. J. Parasitol. 42 (7) (2012) 667–673. J.L. Concepción, C.A. Adjé, W. Quiñones, N. Chevalier, M. Dubourdieu, P.A. Michels, The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi, Mol. Biochem. Parasitol. 118 (2001) 111–121. J.R. Haanstra, A. van Tuijl, P. Kessler, W. Reijnders, P.A. Michels, H.V. Westerhoff, M. Parsons, B.M. Bakker, Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 17718–17723. V. Hannaert, M.A. Albert, D.J. Rigden, M.T. da Silva Giotto, O. Thiemann, et al., Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase, Eur. J. Biochem. 270 (2003) 3205–3213. M. Hunt, P. Köhler, Purification and characterization of phosphoenolpyruvate carboxykinase from Trypanosoma brucei, Biochim. Biophys. Acta 1249 (1995) 15–22. C.N. Cronín, D.P. Nolan, H.P. Voorheis, The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei, FEBS Lett. 244 (1989) 26–30.