Simple modeling of the PPP creates a glycosomal phosphate “leak”

The existing mathematical model of glycolysis in T. brucei [8] was extended to include the reactions from the pentose phosphate pathway (PPP). In T. brucei, the PPP is localized to both the cytosol and glycosomes, based on subcellular localization studies [15]–[17], proteomic analysis [18], [19] and subcellular targeting-sequence-based predictions [20]. Figure 1 gives an overview of the models considered in this paper, while the stoichiometry and kinetic parameters are described in Table 1 and S1, respectively.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Schematic overview of the models used for simulation.

(A) Detailed scheme of the modeled metabolic pathways. The numbered arrows correspond to reactions from Table 1. Extensions to the original model of glycolysis are indicated by colored shapes. Boundary metabolites are in bold, glycosomal Rib-5-P is a boundary metabolite in model C and D. (B) Schematic overview of the different models, each consisting of a unique combination of the colored modules described in (A) and Table 1. Model C and D can alternatively utilize fructose (model C^fru and D^fru), but this branch is switched off unless specifically mentioned.

https://doi.org/10.1371/journal.pcbi.1003371.g001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Model stoichiometry by modules and their subsequent reactions.

https://doi.org/10.1371/journal.pcbi.1003371.t001

To provide glucose 6-phosphate (Glc-6-P) to the cytosolic glucose 6-phosphate dehydrogenase (G6PDH), a trace amount of cytosolic hexokinase was added in models B–D. An alternative solution, where transport of Glc-6-P over the glycosomal membrane is allowed, gave similar results. Fractionation studies that suggest an exclusively glycosomal localization of hexokinase activity [21] are insufficiently sensitive to discriminate a trace amount of cytosolic hexokinase. Moreover, enzymes targeted to the glycosome can be fully folded in the cytosol, which leaves room for a small, residual activity in this compartment. We therefore decided to explore the model behavior in the presence of cytosolic hexokinase in the following analyses, yet acknowledge that this choice is somewhat arbitrary. A reaction utilizing cytosolic Glc-6-P was added in models B–D, as absence of this reaction resulted in accumulation of Glc-6-P in the cytosol (25% of the models have a cytosolic Glc-6-P concentration over 20 mM, in contrast to a measured steady-state value of 2.6 mM). This reaction primarily represents the use of Glc-6-P in other cytosolic pathways, such as the glycosylation of proteins [22].

The model of glycolysis (Figure 1B, model A) has a conserved sum of phosphorylated metabolites in the glycosome, consisting of ATP, ADP and glycolytic intermediates (Table 2). Extension of the glycolysis model with the PPP (Figure 1B, model B) introduces a drain from this moiety of phosphorylated glycosomal compounds. While the extension with the PPP adds metabolites in the glycosome that are derived from Glc-6-P, these phosphorylated PPP intermediates are prevented from re-entering glycolysis as two key enzymes of the non-oxidative PPP—ribulose-5-phosphate epimerase [23] and transketolase [17], [23]—are not expressed in the bloodstream form of the parasite. This renders ribose 5-phosphate (Rib-5-P) as the end product of the PPP according to the classical scheme, and leads to a lethal drainage of phosphates from the previously conserved moiety (Figure 2). This phosphate “leak” only entails bound phosphate, and not inorganic phosphate, which is independent of the conserved moiety. This leak of phosphates from the conserved moiety of bound phosphates is only observed in the glycosome, as the bound phosphates are not conserved in the cytosol, where there is a net production of ATP via pyruvate kinase. Model B is therefore an obviously non-physiological model and was not investigated further. The phosphate leak is an effect of the stoichiometry of the model and not the enzyme kinetics: the formal proof of this is that there exists no elementary mode (Table S2) for model B with a flux through the PPP.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Phosphate leak in model with PPP.

Time course simulation of model B, in which the reactions of the glycosomal PPP are switched on at t = 0 by increasing their V_max value from zero to the value given in Table S1. Glc_e is 5 mM and k_TOX = 2 µl·min⁻¹·mg protein⁻¹. Solid lines indicate medians, shaded areas show interquartile ranges. Fluxes (J) are plotted on the left y-axis and are indicative of glucose uptake (GlcT_{plasma membrane}), glycerol (GK) and pyruvate production (PyrT) and the two branches of pentose phosphate pathways (G6PDH_c/g). The sum of bound phosphates in the glycosome (ΣP_g), as exists in the model of glycolysis (Table 2, moiety 5), is plotted on the right y-axis. Within 25 minutes, all bound phosphates within the glycosome are depleted and all metabolic fluxes subsequently drop to zero.

https://doi.org/10.1371/journal.pcbi.1003371.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Conserved moieties in the four models of Figure 1.

https://doi.org/10.1371/journal.pcbi.1003371.t002

Exploring solutions to the phosphate leak

There is uncertainty as to how this glycosomal phosphate leak is prevented in the parasite. Two types of solution can be considered: (i) maintaining the conserved moiety of phosphorylated metabolites by including additional reactions in the glycosome, or (ii) transferring phosphorylated metabolites over the glycosomal membrane. No a priori knowledge exists on balancing of bound phosphates in the glycosome that would convincingly support either solution over the other.

First type of solution: Maintaining the conserved moiety

For the first type of solution, a stoichiometric analysis of all known and predicted glycosomal enzymes [18]–[20] was performed to indicate what additional reactions in the glycosome can recover the bound-phosphate lost via Rib-5-P (Tables S3, S4, Figure S1). Only ribokinase, which has a C-terminal PTS-1 sequence that localizes it to the glycosome, is able to support a steady-state flux through the PPP without the requirement of additional reactions that are not known or predicted to be localized to the glycosome. The addition of hypothetical reactions in the glycosome can provide alternative solutions to the phosphate leak, however, the ribokinase solution is representative of this class of solutions where the bound phosphate is balanced within the glycosome and the moiety of bound phosphates is conserved. This solution requires ribokinase to operate in the direction of ribose and ATP production, which is not favored thermodynamically in standard conditions. With an equilibrium constant of 0.0036 (in the direction of ribose formation, [24]) ribokinase would require an almost 300-fold accumulation of Rib-5-P over ribose in the glycosome, indicating that ribose in the glycosome would be in the µM range and Rib-5-P in the mM range. This ratio can be further improved by a low ATP/ADP ratio. A similar solution, however, has already been exploited with regard to glycerol kinase at the onset of anaerobiosis. In response to loss of the mitochondrial alternative oxidase reaction under these conditions, trypanosomal glycerol kinase runs in the direction of glycerol and ATP production, as glycerol 3-phosphate accumulates and a low ATP/ADP ratio is maintained in the glycosome [25].

Model B was therefore extended with a glycosomal ribokinase, generating model C (Figure 1), which represents a solution where the glycosomal conserved moiety of bound phosphates is restored (Table 2, moiety 9). In order to provide kinetic parameters for model C, the enzyme kinetics of heterologously expressed T. brucei ribokinase were characterized and used to update the model (see Table S1 for determined kinetic parameters). Dynamic simulations of the extended model subsequently indicated the feasibility of ribokinase as a solution, since it allows metabolic fluxes through all branches (Figure S2 and Table S2).

When ribokinase is indeed responsible of maintaining the phosphate balance in the glycosomes, its presence is expected to be essential for parasite survival. To investigate this hypothesis, a genetic mutant (RK^RNAi) was generated to knockdown the levels of ribokinase transcript by RNA interference. However, no growth phenotype was observed (Figure S3). Attempts to generate a gene knockout of ribokinase, surprisingly, failed in spite of numerous rounds of transfection. A conditional knockout was generated with inducible expression of an exogenous copy of the T. brucei ribokinase gene. While non-induced growth resulted in depletion of ribokinase, again no growth phenotype could be observed, even if the ectopic copy was switched off. It was concluded that ribokinase activity is essential but that residual levels in RK^RNAi and leaky expression in the conditional knockout were sufficient to fulfill this essential role.

Additionally, the requirement of a low ribose/Rib-5-P ratio to catalyze the formation of ribose by ribokinase predicts that ribose should be toxic to the parasites (Figure S4), in a similar way to glycerol that can prevent glycerol kinase from working in the reverse direction. However, incubation of parasites with ribose up to 30 mM for over 72 hours was non-lethal. From these results and the genetic mutants, it was concluded that ribokinase is unlikely the sole glycosomal enzyme responsible for maintaining the phosphate balance. Other kinases, such as arginine kinase [26] and phosphoenolpyruvate carboxykinase (PEPCK), are also present within the glycosome and bound phosphate homeostasis could involve a large and redundant network of contributing metabolic pathways. While uncertainty remains on which enzymes would be involved in preventing the phosphate leak by restoring the conserved moiety of phosphorylated metabolites, model C represents a generic representation of such a solution. Continued analysis of this model can, therefore, still provide insight into the effects of such a solution even in the absence of a specific individual enzyme.

Second type of solution: Transferring phosphates over the glycosomal membrane

An alternative solution to the phosphate leak requires transferring bound-phosphates over the glycosomal membrane. This can be achieved by the presence of a single ATP:ADP antiporter operative across the glycosomal membrane, analogous to the yeast peroxisomal adenine nucleotide transporter [27]. Model D was generated by the addition of an ATP:ADP antiporter to model B (Figure 1), and stoichiometric analysis of model D showed the absence of a conserved moiety of phosphorylated glycosomal metabolites (Table 2).

Dynamic analysis of model D showed that the absence of the conservation constraint on phosphorylated metabolites in the glycosome introduced a risk of high accumulation of these metabolites. The autocatalytic design of glycolysis means that surplus ATP produced in the later steps of glycolysis can fuel the first reactions in a positive feedback loop. This “turbo design” [28] is controlled in most organisms by additional negative feedback regulation, e.g. inhibition of PFK by PEP [29]. Trypanosomes lack such regulation, but dynamic modeling supported by direct experimentation revealed that the glycosomal localization of part of glycolysis prevents the turbo effect in trypanosomes [30], [31]. Glucose is predicted to be lethal for glycosome deficient bloodstream forms [32], while procyclic forms, which can grow on both glucose and proline, could be rescued by the removal of sugars from the medium [32], or by the ablation of hexokinase [33]. Simulations of model D indicate that the presence of an ATP:ADP antiporter mimics the absence of a glycosome, with a high risk of sugar-phosphate accumulation (Figure 3). While the presence of the PPP can partly rescue a model with the ATP:ADP antiporter from accumulation of sugar-phosphates (Figure 3B–C, compare model A+AAT and D), a high risk of accumulation remains when the antiporter is too active (Figure 3D–E). It has been shown experimentally in procyclic trypanosomes that such accumulation occurs only when enzymes are mislocalized, and this is detrimental for the cells [31]. Other types of transporters, including recently described pores in the glycosomal membrane [34], facilitating a net transport of phosphorylated compounds into the glycosome gave similar results, while the absence of known genes encoding these transporters impedes further testing of this hypothesis by genetic manipulation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. ATP:ADP antiporter mimics turbo-state.

(A) Overview of the models used in this figure. Model A and D are from Figure 1, model A–glyc is model A without glycosomal localization, as described in [31], model A+AAT is model A with an ATP:ADP antiporter. (B–C) Steady-state concentrations of glycosomal Glc-6-P and Fru-1,6-BP are depicted in the various models. (D) Increasing the activity of the ATP:ADP antiporter (V_{max,ATP:ADP antiporter}) in model D leads to a high risk of accumulation of hexose phosphates. The green line indicates the concentration of Fru-1,6-BP in the original model of glycolysis (17.2 mM, panel C, model A). Glc_e in this simulation is 25 mM. (E) Time course simulation of model D at 25 mM Glc_e and various values for the V_{max,ATP:ADP antiporter} parameter. Plotted is the concentration of glycosomal phosphates (ΣP similar as in Figure 2, moiety 5 in Table 2). ATP:ADP antiporter activity values below 1 nmol·min⁻¹·mg protein⁻¹ result in depletion of glycosomal phosphates (cf. Figure 2). k_TOX = 2 µl·min⁻¹·mg protein⁻¹ in all models. Solid lines indicate medians, shaded areas and error bars show interquartile ranges, as derived from the uncertainty modeling.

https://doi.org/10.1371/journal.pcbi.1003371.g003

The two hypothesized solutions to the phosphate leak are both theoretically able to restore a glycolytic and PPP flux. Although current available knowledge is insufficient to explicitly exclude one of the two types of solutions, further analysis of the two models can still give us valuable insights on for instance 6PGDH inhibition, while not ignoring the uncertainty involved. We demonstrate this below.

Predictive behavior using the extended models of trypanosome glucose metabolism

Dynamic simulations using both models, containing either of the two theoretical solutions to the phosphate-leak problem, showed that oxidative stress regulates the flux through the PPP (Figure 4A), in line with measured increases in the flux through the PPP in the related species T. cruzi upon induced oxidative stress [35]. Low oxidative stress (k_TOX = 2 µL•min⁻¹•mg protein⁻¹) represents a healthy proliferating trypanosome and results in a low PPP flux, supported by the experimental observation that almost all consumed glucose is excreted as either pyruvate or glycerol [36], [37]. A sudden burst of oxidative stress was simulated by almost completely (99%) oxidizing the pools of trypanothione and NADPH and subsequently allowing the model to reach steady state. The parasite was able to restore its redox balance in about one minute (Figure 4B). This experiment simulates an extreme level of oxidative stress, but if in reality the pools never become 99% oxidized, the PPP can respond even faster.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Simulations of oxidative stress in model C.

(A) The steady state flux through the cytosolic pentose phosphate pathway in model C as a function of the oxidative stress by varying the kinetic constant k_TOX. (B) Fluxes through the cytosolic PPP enzymes as a function of time upon sudden oxidative stress. During the whole time-course, k_TOX = 2 µl·min⁻¹ · mg protein⁻¹. The system is removed from steady state at t = 0, by setting 99% of the NAD(P)H and trypanothione pools to the oxidized form. Shown is the relaxation of the cytosolic PPP fluxes. Solid lines indicate medians, shaded areas show interquartile ranges. Near identical results were obtained for model D (Figure S5).

https://doi.org/10.1371/journal.pcbi.1003371.g004

It has previously been proposed that inhibition of the third enzyme of the PPP, 6-phosphogluconate dehydrogenase (6PGDH), should be lethal to trypanosomes [38]. In Drosophila [39], [40], yeast [41] and mammalian cancer cells [42], lethality of 6PGDH inhibition is based on the inhibition of glycolysis by the accumulation of 6-phosphogluconate (6-PG). Inhibition of 6PGDH results in accumulation of its substrate 6-PG, and as a potent inhibitor of phosphoglucose isomerase (PGI) [43], 6-PG subsequently inhibits the conversion from glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate. With Glc-6-P then instead directed towards the PPP, 6-PG accumulates to even higher levels, ultimately inhibiting the ATP producing glycolytic pathway. In Drosophila and yeast, this lethal phenotype can be rescued by additional loss of G6PDH, completely shutting down the PPP and as such preventing accumulation of 6-PG [40], [41]. Growth on alternative hexoses such as fructose, bypassing the PGI reaction and as such preventing inhibition of glycolysis, was shown to rescue 6PGDH deficient cancer cells [42]. A similar route to lethality of 6PGDH inhibition via PGI inhibition was hypothesized for T. brucei [10], but no experimental proof exists to corroborate this hypothesis. Alternatively, the reduced PPP flux in 6PGDH inhibited trypanosomes potentially renders the parasites more susceptible to oxidative stress. To analyze these two hypotheses, the activity of 6PGDH was investigated in both models C and D.

6PGDH inhibition in both models resulted in the accumulation of 6-PG as anticipated (Figure 5A–B). Both models gave very similar results, as the only major difference is that glycosomal 6-PG in model C is restricted to 45 mM due to the constraint of the conserved moiety of phosphorylated metabolites. Highly oxidative conditions resulted in a cytosolic 6-PG accumulation that started at lower 6PGDH inhibitions, as the cytosolic PPP flux is activated to provide the NADPH required by trypanothione reductase. Only when the 6PGDH activity was decreased by more than 95% was glycolysis reduced to a negligible flux. To observe the effects of accumulated 6-PG on PGI in isolation from other effects, model A with a 6-PG sensitive PGI was simulated at different 6-PG levels. A 50% reduction in glycolytic flux is lethal to the parasite [9], but this level of inhibition was reached only at 6-PG levels of around 500 mM (Figure 5C). In model C, the conserved moiety of bound phosphates restrict the glycosomal 6-PG level to only 45 mM (Figure 5A), while in model D such high accumulation of glycosomal 6-PG occurs in combination with far more extreme accumulation of cytosolic 6-PG (Figure 5B). Neither model therefore supports the PGI inhibition hypothesis as the explanation of the lethality 6PGDH inhibition in T. brucei.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. Simulations of 6PGDH inhibition and 6-PG accumulation.

(A–B) The effects of inhibition of 6PGDH on 6-PG concentrations and metabolic fluxes were simulated by reducing V_max,6PGDH in model C and D at high oxidative stress (k_TOX = 200 µl·min⁻¹·mg protein⁻¹). Simulations at low oxidative stress (k_TOX = 2 µl·min⁻¹·mg protein⁻¹) are shown in Figure S6. ATP production flux as steady-state flux through PFK is indicated in red, while trypanothione reductase steady-state flux is indicated in yellow, both plotted on the left y-axis. Steady-state concentration of cytosolic (blue) and glycosomal (green) 6-phosphogluconate are plotted on the right y-axis. Shaded areas indicate interquartile ranges. (C) Steady-state flux through glycolysis as a function of the glycosomal 6-PG concentration in model A. A glycosomal 6-PG concentration of around 500 mM reduces the glycolytic flux by 50%.

https://doi.org/10.1371/journal.pcbi.1003371.g005

The second hypothesis initially proposed posits that 6PGDH inhibition is lethal due to the reduced flux through the PPP and the associated increased sensitivity for oxidative stress. In both models, the simulation of 6PGDH inhibition demonstrates how the cell's ability to cope with oxidative stress is strongly affected, as the flux through trypanothione reductase is inhibited when the activity of 6PGDH is reduced by more than 50% (Figure 5A–B). A third hypothesis introduced in model C reveals that the accumulation of glycosomal 6-PG depletes other bound phosphates within the glycosome, thus inhibiting the glycolytic flux. Sensitivity to oxidative stress, and the depletion of bound phosphates from the conserved moiety, could therefore both explain loss of viability due to ablation of 6PGDH. While accumulation of the toxic lactone precursor of 6-PG through product inhibition of the lactonase enzyme is another possibility, the high equilibrium constant of this reaction renders it insensitive to its product [44]. Finally, a reduced production of the nucleotide precursor Rib-5-P by the PPP could explain the 6PGDH inhibition lethality.

RNA interference confirms that 6PGDH is essential, but lethality is not due to inhibition of glycolysis

The predictions from the modeling were tested experimentally. A genetic mutant in which the 6PGDH transcript is knocked down by RNA interference (6PGDH^RNAi) yielded reduced levels and activities of 6PGDH (Figure 6A) and subsequent cell death, confirming the severe effects of 6PGDH inhibition observed during model simulations. Antibodies against 6PGDH also revealed this enzyme to be distributed to both cytosol and to a lesser degree the glycosome (Figure 6B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. Ablation of 6PGDH.

(A) Effect of 6PGDH ablation on the growth rate. A non-induced 6PGDH^RNAi culture, grown in glucose-containing HMI-9 was split at 0 h, +tet is induced with tetracycline, while −tet is the non-induced control. (B) Specific activities of 6PGDH in induced and control 6PGDH^RNAi parasites. (C) Western blot showing predominant co-localization of 6PGDH with the glycosomal marker aldolase (fraction S), while a faint band can also be observed in the cytosolic fraction P with the marker enolase. (D) Cell densities during growth on different substrates. At t = 0 h, a 6PGDH^RNAi culture grown on glucose was split at 1·10⁵ ml⁻¹ to HMI-9 with either glucose or fructose, and in the absence and presence of tetracycline. Plotted cell densities are cumulative, as −tet cultures were split at 48 h to 1·10⁵ ml⁻¹. A higher starting cell density was used to allow the parasites to adapt to the change in carbon source. Growth on fructose is slower than on glucose, but is unable to rescue the induced cultures.

https://doi.org/10.1371/journal.pcbi.1003371.g006

The 6PGDH activity measurements indicate that the growth of the parasites is affected when the 6PGDH activity is reduced by less than 50% in the first 24 hours. Even after 72 hours, 15% of the 6PGDH activity remains. This suggests that the first and third hypotheses, i.e. PGI inhibition and the depletion of the bound phosphate moiety, are unlikely. 6PGDH^RNAi parasites could not be rescued by the addition of 0.5 mM ribose, as an alternative pathway to Rib-5-P, suggesting that the availability of Rib-5-P for nucleotide biosynthesis was not responsible for the lethal phenotype.

To test whether the proposed feedback loop related to inhibition of PGI by accumulating 6-PG we tested how the 6PGDH^RNAi trypanosomes behaved when grown in fructose, a glycolytic substrate [11], [37], [45] that can enter the pathway after the PGI step, which was previously shown to rescue growth of 6PGDH deficient cancer cells [42].

Model C and D were both extended using the fructose transporter and its subsequent utilizing reactions (Figure 1), using parameter values from the literature (Table S1). The rate equation of hexokinase was adapted to describe the substrate competition of glucose and fructose. Steady-state calculation of the fructose versions of the models (model C^fru and D^fru) predict that growth of parasites is supported on fructose, although the lethal effect of 6PGDH inhibition could not be averted by using this substrate (Figure S7). This again indicates that the lethal effect of 6PGDH loss in trypanosomes is not due to a catastrophic positive feedback loop induced through PGI inhibition.

In vitro, 6PGDH^RNAi had a slightly slower growth when glucose in the medium was substituted with fructose (Figure 6C). However, in common with model predictions, growth on fructose was unable to rescue the lethal phenotype of 6PGDH ablation in the 6PGDH^RNAi cell line (Figure 6C). The fact that G6PDH is also essential for bloodstream form T. brucei [11], whereas loss of this enzyme can revert lethality associated with loss of 6PGDH in Drosophila [40] and yeast [41], further indicates that it is loss of the oxidative branch of the PPP per se in T. brucei that is responsible for death, rather than the glycolysis inhibitory loop proposed in other eukaryotes.

Stoichiometry

The computational model was constructed in a modular way. The reactions included in each module are listed in Table 1 and graphically represented in Figure 1. The starting point is a module describing glycolysis in bloodstream form T. brucei [7], [8]. A few modifications in the kinetic equations are listed below. There are two PPP modules, each consisting of an almost identical set of reactions; one is localized entirely in the glycosomal matrix and the other in the cytosol. To allow flux through the glycosomal PPP the model has been further extended with either a ribokinase module or an ATP:ADP antiporter across the glycosomal membrane. The rationale behind these extensions is discussed in the Results section.

Ribose 5-phosphate and ribose are modeled as end-products of the PPP, depending on model version and compartment. As no published intracellular concentrations are available for ribose 5-phosphate and ribose, their cytosolic and glycosomal concentrations were fixed at 0.01 mM unless stated otherwise.

Kinetic equations

Kinetic parameters

The kinetic parameters in the rate equations mentioned above were predominantly derived from literature. If no parameters of the T. brucei enzyme were available, parameters from related species were used. If necessary, K_eq values were corrected to pH 7. Kinetic parameters used in the fixed parameter model are listed in Table S1. Distributions of parameters used in uncertainty modeling are given in Text S1.

Metabolite concentrations

The extension of the model with the glycosomal pentose phosphate pathway led to an extension of the conserved moiety of phosphorylated glycosomal metabolites as compared to earlier model versions [4]. For a precise overview of conserved moieties in each model version see Table 2. The new conserved sum was chosen such that extension of the glycolysis model with the glycosomal PPP and ribokinase results in similar steady state concentrations of glycolytic metabolites in both models. The conserved sum of NADPH in the cytosol and glycosome was arbitrarily chosen as 2 mM, while the conserved sum of trypanothione was chosen as 0.38 mM [12]. In the models including fructose transport, cytosolic Fru-6-P was fixed to the measured steady-state value of 0.9 mM, as additional reactions in the cytosol that are not included in the model would consume Fru-6-P. Inorganic phosphate (Pi) is not explicitly included in the model, as it is assumed to be present at saturating levels and regulated independently of glycolysis.

Calculations and simulations

The model was initially constructed and simulations were performed in PySCeS 0.7.8 [49] or COPASI 4.8 [50]. SBML versions of the models are supplied as Dataset S1. The models in this paper have additionally been modeled while taking uncertainty into account as described in [8]. Each simulation exists of 1000 individual runs, where in each run all parameter values are sampled simultaneously. The 6PGDH V_max scans of Figures 5 and S5, S6, S7, S8 show 250 simulations, as the longer time-limit and accumulating metabolites greatly increased the time for the simulation to finish (a week on a desktop PC for model D). The parameter distributions are described in more detail in Text S1, while the parameter sets are provided as Dataset S2, S3.

Expression recombinant ribokinase

The T. brucei ribokinase gene (GenBank ID:3664382) was amplified from Lister 427 strain T. brucei genomic DNA and cloned into plasmid pET28a(+) (Novagen) to add an N-terminal His-tag. The protein was overexpressed in E. coli BL21(DE3), purified by Ni²⁺ chelate chromatography and dialyzed in 50 mM Tris-HCl pH 7.0, 100 mM NaCl, 20% glycerol. Purity was assessed by SDS-PAGE and Coomassie blue staining.

Enzyme activity assays

Activity of ribokinase was assayed at 25°C in buffer containing 10 mM KH₂PO₄, 15 mM NaCl, 85 mM KCl and 10 mM MgCl₂ at pH 7. Ribokinase activity in the direction of ribose 5-phosphate production was assayed spectrophotometrically according to [51], with 5 mM ribose, 5 mM phosphoenolpyruvate, 5 mM ATP, 0.2 mM NADH, 5 u PK and 5 u LDH. Ribokinase activity in the direction of ribose production was assayed spectrophotometrically with 5 mM ribose 5-phosphate, 5 mM glucose, 5 mM ADP, 0.2 mM NADP, 5 u HXK and 5 u G6PDH.

Parasites and genetic mutants

Bloodstream form Lister 427 strain T. brucei parasites were routinely cultured in HMI-9 [52] at 37°C and 5% CO₂. For growth of 6PGDH^RNAi on an alternative hexose source, glucose in HMI-9 was substituted with fructose at the same concentration. Antibiotic concentrations for genetic mutants were: 0.5 µg mL⁻¹ phleomycin and 2.5 µg mL⁻¹ hygromycin for RK^RNAi; 0.5 µg mL⁻¹ phleomycin, 5 µg mL⁻¹ G418 and 10 µg mL⁻¹ hygromycin for 6PGDH^RNAi; 0.5 µg mL⁻¹ puromycin for ΔRK::PAC/RK; 2.5 µg mL⁻¹ hygromycin for ΔRK::HYG/RK and 0.5 µg mL⁻¹ puromycin, 2.5 µg mL⁻¹ hygromycin, 0.5 µg mL⁻¹ phleomycin and 2 µg mL⁻¹ blasticidin for ΔRK::HYG/PAC::RK^tet. Sensitivity of T. brucei to ribose was assayed with alamarBlue as described previously [53].

The ribokinase knockdown cell line RK^RNAi was generated using the pRPa^iSL vector/2T1 cell line system [54], while the 6-phosphogluconate dehydrogenase (Tb427tmp.211.3180) knockdown cell line 6PGDH^RNAi was generated using the p2T7 vector/1313 cell line system [55]. The generation of the ribokinase knockout cell line ΔRK::HYG/PAC was attempted using constructs containing 600 bp 5′ and 3′ UTRs adjacent to the ribokinase gene, cloned into vector pTBT [56], flanking either a hygromycin phosphotransferase or puromycin N-acetyl-transferase gene as antibiotic resistance markers. The tetracycline-inducible expression of ribokinase in the conditional knockout ΔRK::HYG/PAC::RK^tet was generated by cloning the ribokinase coding sequence in pHD1336 and using cell line 449 (kind gifts by Prof Christine Clayton, Heidelberg). Parasites were transfected by Amaxa nucleofection (Lonza) [57] using Tb-BSF buffer [58], and plated out by limiting serial dilution.

Western blotting

Polyclonal antibodies against T. brucei ribokinase were raised in rabbits by injection of purified recombinant T. brucei ribokinase, purified by protein A-chromatography, and an antibody titer of >1∶192,000 was measured by ELISA (GenicBio, Shanghai, China). The anti-serum was further purified by immunoaffinity chromatography. Recombinant T. brucei ribokinase was coupled to a mixture of Affi-Gel 10 and 15 (Bio-Rad). Antibodies were eluted from the column with 3.5 M MgCl₂. Polyclonal antibodies against the T. brucei 6PGDH were raised in rats by infection of purified recombinant T. brucei 6PGDH.

For protein samples, 5·10⁶ cells were harvested by centrifugation and washed with PBS. Samples were separated on NuPAGE 4–12% Bis-Tris precast gels (Life Technologies) for 45 min at 200 V and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham) at 100 V for 2 h. Membranes were blocked overnight at 4°C with 5% milk in PBS-T (PBS with 0.05% Tween-20), washed three times with PBS-T, and probed with the purified polyclonal rabbit anti-T. brucei ribokinase antibody, diluted 1∶2000 in 1% milk in PBS-T, for 4 h at room temperature. After three additional washes with PBS-T, the membrane was incubated with the secondary antibody (goat anti-rabbit IgG peroxidase conjugate, Calbiochem) 1∶2000 in 1% milk in PBS-T for 2 h at room temperature. After five washes with PBS-T, horseradish peroxidase activity was measured with SuperSignal HRP substrate (Novagen).

Subcellular localization of 6PGDH in trypanosomes was performed by modification of the digitonin fractionation method described in [59]. Specifically, 5·10⁷ procyclic cells were washed with STEN buffer (250 mM sucrose; 25 mM Tris-HCl, pH 7.5; 1 mM EDTA and 150 mM NaCl) and resuspended in 300 µL STEN supplemented with protease inhibitors (Roche). The cells were differentially permeabilized with 40 µg digitonin per 100 µg cellular protein (2.5·10⁷ cells) for 2 hours on ice and centrifuged at 16,000 g for two minutes. After TCA precipitation, equal amounts (equivalent of 4·10⁶ cells) of the supernatant (S) and pellet (P) were analyzed by SDS-PAGE and blotted with rat anti-Tb6PGDH. A goat anti-rabbit IgG peroxidase was used for visualization as described for ribokinase. Enolase and aldolase were used as cytosolic and glycosomal markers, respectively, and antibodies against these proteins were kindly provided by Prof. Paul Michels (University of Louvain, Belgium).