Fig 1.
Proline metabolism of the PCF trypanosomes in the presence of other carbon sources.
Panels A, B, C and D correspond to schematic metabolic representations of PCF trypanosomes incubated in glucose-depleted medium, containing proline (grey arrows) without or with succinate (red arrows), malate (green arrows) and α-ketoglutarate (blue arrows), respectively. End-products excreted from catabolism of proline and the other carbon sources are shown in rectangles with the corresponding colour code and the enzyme numbers under investigation are circled. Enzymatic reactions of proline metabolism that have not been shown to occur in parental PCF are represented by dashed lines. Similarly, the red (B) and blue (D) dashed arrows highlight that these reactions have not been formally demonstrated to occur in the catabolism of succinate and α-ketoglutarate, respectively. In panel C, enzymatic reactions occurring in the reducing direction of the TCA cycle are shown in light green. The production and consumption of ATP and NADH are indicated in bold and the asterisks mean that production of 2-hydroxyglutarate from proline has not been previously described for PCF. It should be noted that, according to the literature, the TCA cycle does not work as a cycle in trypanosomes and only branches are used through anaplerotic reactions [16]. Indicated enzymes are: 1, proline dehydrogenase (PRODH); 2, spontaneous reaction; 3, pyrroline-5 carboxylate dehydrogenase (P5CDH); 4, alanine aminotransferase (AAT); 5, α-ketoglutarate dehydrogenase complex (KDH); 6, succinyl-CoA synthetase (SCoAS); 7, succinate dehydrogenase (SDH, complex II of the respiratory chain); 8, respiratory chain and mitochondrial ATP synthetase (oxidative phosphorylation); 9, mitochondrial NADH-dependent fumarate reductase (FRDm1); 10, mitochondrial fumarase (FHm); 11, mitochondrial malate dehydrogenase (MDHm); 12, citrate synthase (CS); 13, aconitase (ACO); 14, mitochondrial isocitrate dehydrogenase (IDHm); 15, mitochondrial malic enzyme (MEm); 16, pyruvate dehydrogenase complex (PDH); 17, acetyl-CoA thioesterase (ACH); 18, acetate:succinate CoA-transferase (ASCT); 19, unknown enzyme; 20, possibly NADH-dependent glutamate dehydrogenase.
Fig 2.
Kinetic analyses of end-products excretion from [U-13C]-glucose and proline metabolism.
PCF cells were incubated for 6 h in PBS containing 0.5 mM [U-13C]-glucose in the presence (panel B) or not (panel A) of 4 mM non-enriched proline (Pro). Excreted end-products were analyzed in the spent medium every 0.5 h by 1H-NMR spectrometry. The top panels A and B show the amounts of 13C-enriched end-products excreted from [U-13C]-glucose metabolism and the amounts of glucose present in the spent medium. The bottom panels show the amounts of non-enriched end-products produced from the metabolism of the unknown internal carbon source (ICS) (panel A) or from the metabolism of proline plus the unknown ICS (panel B). In panel C, the kinetics of end-products excreted from 0.5 mM [U-13C]-glucose in the presence of 4 mM proline was determined for the Δach/RNAiASCT.i mutant cell line as performed in panel B. Western blot controls with the anti-ASCT and anti-aldolase immune sera are shown below the graph.
Table 1.
Strategies and plasmids used for gene knock down (RNAi), knock out (KO) and/or overexpression in the cell lines analyzed.
Fig 3.
Proton (1H) NMR analyses of end-products excreted from the metabolism of 13C-enriched succinate, alanine, pyruvate and acetate.
In panel A, PCF trypanosomes were incubated for 6 h in PBS containing 4 mM [U-13C]-succinate, [U-13C]-alanine, [U-13C]-pyruvate or [U-13C]-acetate alone or in combination with 4 mM glucose (+Glc) or proline (+Pro) before analysis of the spent medium by 1H-NMR spectrometry. As reference, the same experiment was performed with 4 mM proline (Pro), 4 mM glucose (Glc) or 4 mM glucose with 4 mM [U-13C]-proline (13C-Pro). The amounts of each end-product excreted are documented in S1 Table. Panel B shows equivalent 1H-NMR spectrometry experiments performed on the parental (WT), RNAiSDH.i, RNAiPDH-E2.i and Δkdh-e2 cell lines incubated with 4 mM [U-13C]-proline and 4 mM succinate. Because of high background, 13C-enriched 2-hydroxyglutarate produced from [U-13C]-proline cannot be quantified, however, it is detectable in the Δkdh-e2 cell line as indicated by an asterisk (*). Traces of fumarate produced from these carbon sources are not shown in the figure. Abbreviations: A, acetate; Al, alanine; H, 2-hydroxyglutarate; K, α-ketoglutarate; M, malate; P, pyruvate; S, succinate; nd, not detectable; *, detectable but not quantifiable. The efficiency of RNAi-mediated downregulation of SDH and PDH-E2 expression in the tetracycline-induced (.i) or non induced (.ni) cell line, as well as in the parental cell line (WT), was determined by SDH activity assays (panel C) and Western blotting with the anti-PDH-E2 and anti-enolase (control) immune sera (panel D). Panel E shows a PCR analysis of genomic DNA isolated from the parental (WT) and Δkdh-e2 cell line. Lanes 1 to 6 of the gel picture correspond to different PCR products described in the right panel. As expected, PCR amplification of the KDH-E2 gene (lanes 1–2) was only observed in the parental cell line, while PAC and BSD PCR-products were observed only in the Δkdh-e2 mutant (lanes 3–4 and 5–6, respectively).
Fig 4.
Succinate, malate and α-ketoglutarate stimulate growth of the PCF.
Panel A shows growth of the PCF trypanosomes in the glucose-depleted SDM79 medium containing 0.2 mM, 2 mM (low-proline) or 12 mM (high-proline) proline, 2 mM glucose/2 mM proline (low-glucose) or 12 mM glucose/2 mM proline (high-glucose) in the presence of added 10 μM to 100 mM succinate, malate, α-ketoglutarate or proline, using the Alamar Blue assay. Incubation was started at 2 x 106 cell density and the Alamar Blue assay was performed after 48 h at 27°C. The dashed line indicates the concentrations of succinate, malate, α-ketoglutarate or proline (10 mM) used in panel B, which shows growth curves of the PCF in low-proline, high-proline and low-glucose conditions in the presence or not of 10 mM of each metabolite. Cells were maintained in the exponential growth phase (between 106 and 107 cells/ml), and cumulative cell numbers reflect normalization for dilution during cultivation. In panel C, the PCF trypanosomes were incubated for 6 h in PBS containing 4 mM succinate (S), malate (M) or α-ketoglutarate (K), in the presence or absence of 4 mM [U-13C]-proline (P) or [U-13C]-glucose (G), before analysis of the spent medium by 1H-NMR spectrometry. As a control, the cells were also incubated with 4 mM [U-13C]-proline (P) or [U-13C]-glucose (G) alone. The amounts of end-products excreted from the metabolism of proline (black), glucose (grey), succinate (red), malate (green) and α-ketoglutarate (blue) are expressed as μmol excreted/h/mg of protein.
Fig 5.
1H-NMR analyses of end-products excreted from the metabolism of malate and α-ketoglutarate.
Panel A shows 1H-NMR spectrometry analyses of the exometabolome produced by the parental (WT), RNAiPDH-E2.i, RNAiFRDg/m1.i, RNAiFHc/m.i, Δaco and Δkdh-e2 cell lines incubated with 4 mM [U-13C]-proline (13C-Pro) and 4 mM malate (Mal). The insets show Western blot analyses with the immune sera indicated in the right margin of the parental (WT), the tetracycline-induced (.i) and non-induced (.ni) RNAiFRDg/m1 and RNAiFHc/m cell lines, and the Δaco cell line. Panel B is equivalent to panel A with the parental (WT), RNAiSDH.i and Δkdh-e2 cell lines incubated in the presence of [U-13C]-proline alone (13C-Pro) or with α-ketoglutarate (13C-Pro + αKG). Because of high background, 13C-enriched 2-hydroxyglutarate produced from [U-13C]-proline cannot be quantified, however, asterisks (*) mean that it is detectable. Since the amounts of 2-hydroxyglutarate produced from non-enriched proline are quantifiable, the values are indicated with grey columns when applicable. The excreted amounts are indicated in the truncated columns (nmol/h/mg of protein). The AAT and MDH (control) enzymatic activities of the parental (WT) and the tetracycline-induced (.i) and non-induced (.ni) RNAiAAT cell lines are shown in panel C. In panel D, the WT and RNAiAAT.i cells were incubated for 6 h in PBS containing 4 mM proline (Pro) with or without 4 mM [U-13C]-alanine (13C-Ala), before analysis of the spent medium by 1H-NMR spectrometry. The amounts of end-products excreted from the metabolism of proline (black) and alanine (pink) are expressed as μmol excreted/h/mg of protein. Panel E is equivalent to panel B, except that the RNAiAAT.i is analyzed. Abbreviations: A, acetate; Al, alanine; F, fumarate; G, glutamate; H, 2-hydroxyglutarate; K, α-ketoglutarate; nd, not detectable; M, Malate; O, malate + pyruvate + acetate + alanine; S, succinate; *, detected but not quantifiable.
Fig 6.
Growth of the RNAiPRODH.i mutant is rescued by α-ketoglutarate.
The efficiency of RNAi-mediated downregulation of PRODH expression in the tetracycline-induced (.i) or non induced (.ni) RNAiPRODH cell line, as well as in the parental cell line (WT) was determined by the PRODH activity assay (panel A) and 1H-NMR quantification of end-products excreted from metabolism of proline and glucose in two independent experiments (panel B). Panel C shows growth curves of the RNAiPRODH.i cell line in glucose-depleted medium containing 2 mM proline, in the presence (colored circles) or absence (open circles) of 10 mM α-ketoglutarate, malate, succinate or proline. Cells were maintained in the exponential growth phase and cumulative cell numbers reflect normalization for dilution during cultivation. The effect of 10 mM succinate, malate, α-ketoglutarate or proline on growth of the parental and RNAiPRODH.i cell lines in glucose-depleted medium containing 2 mM proline, using the Alamar Blue assay described in Fig 4, is shown in panel D.
Fig 7.
α-ketoglutarate is toxic for the Δkdh-e2, RNAiSCoAS.i and RNAiSDH.i cell lines.
Panel A compares the effect of succinate, malate, α-ketoglutarate or proline (10 mM) on growth of the parental and mutant cell lines in glucose-depleted medium containing 2 mM proline, using the Alamar Blue assay described in Fig 4. Panel B shows growth curves of the RNAiAAT, Δkdh-e2, RNAiSDH and RNAiSCoAS cell lines incubated in glucose-depleted medium containing 2 mM proline, in the presence (colored circles) or absence (open circles) of α-ketoglutarate, malate, succinate or proline (10 mM) (.i, tetracycline-induced cells;.ni, non induced cells). Cells were maintained in the exponential growth phase and cumulative cell numbers reflect normalization for dilution during cultivation. Panel C shows Western blot analyses with the immune sera indicated in the right margin of the parental (WT), Δidhm and tetracycline-induced (.i) and non-induced (.ni) RNAiSCoAS, Δidhm/RNAiSCoAS and Δkdh-e2/RNAiIDHm cell lines.
Fig 8.
Large concentration of carbon sources are required for the growth of epimastigote-like cells.
Panels A and C show Western blot analyses with the indicated immune sera (BARP, brucei alanine rich proteins; TAO, terminal alternative oxidase) of the OERBP6 cell line upon tetracycline induction and growth in low-proline conditions (2 mM) after addition (panel C) or not (panel A) of 10 mM α-ketoglutarate or 10 mM proline. HSP60 was used as loading control. Panel B shows growth curves of the tetracycline-induced (.i) and non-induced (.ni) OERBP6 cell line, in the presence of 2 mM proline, with or without addition of 10 mM α-ketoglutarate, succinate, malate, or proline. The curves are representative replicates of three different experiments.