Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability

Abstract

Background: The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/principal findings: A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/significance: Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the "acetate shuttle" that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.

Figure 1. Schematic representation of acetate production from glucose and threonine in BSF.

Black arrows indicate enzymatic steps of glucose and threonine metabolism and the double line arrow represents de novo biosynthesis of fatty acids. Excreted end-products of metabolism of glucose and threonine are boxed. The thick arrows illustrate the high glycolytic flux leading to production of pyruvate, which accounts for 85.1% of the excreted end-products from glucose metabolism. Abbreviations: AcCoA, acetyl-CoA; AOB, amino oxobutyrate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; MAL, malate; PEP, phosphoenolpyruvate; PYR, pyruvate; SCoA, succinyl-CoA. Indicated enzymes are: 1, pyruvate dehydrogenase complex (PDH); 2, acetate∶succinate CoA-transferase (ASCT); 3, acetyl-CoA thioesterase (ACH); 4, succinyl-CoA synthetase (SCoAS); 5, threonine 3-dehydrogenase (TDH); 6, 2-amino-3-ketobutyrate coenzyme A ligase (AKCT); 7, acetyl-CoA synthetase (AMP-dependent enzyme, AceCS).

Figure 2. Expression and immunolocalization of enzymes involved in acetate metabolism.

In Panel A, expression of AceCS, ASCT, ACH, PDH-E2 and TDH was analyzed by western blotting with specific immune sera on total protein lysates (5×10⁶ cells per lane) of wild-type 427 monomorphic bloodstream forms (LS-BSF) and procyclic cells (PF). The anti-hps60 antibodies were used as loading controls. The positions of the molecular weight markers are indicated in kDa on the right margin of each panel. The PDH and TDH activities (milliunits/mg of protein) were determined in both forms of the parasite. The “±” signs indicate SD of at least 3 independent experiments. In panel B, bloodstream cells were stained with mouse anti-AceCS (Alexa 488 channel), mouse anti-PDH-E1α (Alexa 488 channel) or rabbit anti-TDH (Alexa 488 channel) with MitoTracker as mitochondrial control. Differential interference contrast (DIC) of cells is shown to the left of each panel. Scale bar, 1 µm.

Figure 3. AceCS is essential for growth and lipid biosynthesis.

Panel A shows growth curves of the parental 427 90-13 strain (WT) and the ^RNAiAceCS mutant cell line incubated in the presence (.i) or in the absence (.ni) of 10 µg/mL tetracycline. Cells were maintained in the exponential growth phase (between 2×10⁵ and 2×10⁶ cells/mL) and cumulative cell numbers reflect normalization for dilution during cultivation. The cross indicates that all cells were dead. It is to be noted that addition of 10 µg/mL tetracycline did not affect growth of the parental cell line (see Fig. 6A). Panel B shows [1-¹⁴C]-acetate incorporation into lipids of WT and tetracycline-induced (one and two days) and uninduced ^RNAiAceCS cells. ¹⁴C-labeled fatty acid methyl esters were separated by HPTLC after transesterification and analyzed as described in the Experimental Procedures section. The values were normalized with the amounts of total esters measured in each sample. Error bars indicate mean ± SD of 3 independent experiments.

Figure 4. ¹H-NMR analysis of excreted end-products from glucose by LS-BSF and PF.

Metabolic end-products (pyruvate, Pyr; acetate, Ace; alanine, Ala and succinate, Suc) excreted by the wild-type 427 monomorphic BSF (A) and PF cells (B) from 4 mM glucose (Glc) were determined by ¹H-NMR. The experiment was performed in 2.5 mL of PBS with 2.5×10⁷ LS-BSF cells (A) and 5×10⁷ PF cells (B). Each spectrum corresponds to one representative experiment from a set of at least 3. A part of each spectrum ranging from 1.1 ppm to 4 ppm is shown.

Figure 5. ¹H-NMR analysis of excreted end-products from glucose and threonine metabolism.

Metabolic end-products (pyruvate and acetate) excreted by the wild-type BSF cell line (A–B), the tetracycline-induced ^RNAiPDH.i mutant (C), the Δtdh mutant (D) and the tetracycline-induced Δtdh/^RNAiPDH.i mutant (E) from D-[U-¹³C]-glucose and/or threonine was determined by ¹H-NMR. The cells were incubated in PBS containing 4 mM D-[U-¹³C]-glucose with (_GT) or without (_G) 4 mM threonine. Each spectrum corresponds to one representative experiment from a set of at least 3. A part of each spectrum ranging from 1.6 ppm to 2.6 ppm is shown. The resonances were assigned as indicated below the spectra: A₁₂, acetate; A₁₃, ¹³C-enriched acetate; P₁₂, pyruvate; P₁₃, ¹³C-enriched pyruvate.

Figure 6. Analysis of mutant cell lines.

Panels A and C–E show growth curve of the parental 427 90-13 BSF cells (WT) and mutant cell lines incubated in the IMDM medium. The mutant cell lines are ^RNAiPDH (A and D), Δtdh (C) and Δtdh/^RNAiPDH (E) incubated in the presence (.i, +tet) or in the absence (.ni) of tetracycline. In panel D, the ^RNAiPDH.i cell line was incubated in threonine-depleted medium supplemented with 0 to 150 µM threonine. The cells were maintained in the exponential growth phase (between 2×10⁵ and 2×10⁶ cells/mL) and cumulative cell numbers reflect normalization for dilution during cultivation. The insets in panels A and C–E show western blot analyses of the parental (WT) and mutant cell lines with the immune sera indicated in the right margin. The lower inset in panel C shows the TDH activity (milliunits/mg of protein) measured in the WT and Δtdh cell lines (ND stands for not detectable). Panel B shows a PCR analysis of genomic DNA isolated from the parental WT and Δtdh cell lines. Amplifications were performed with primers based on sequences that flank the 5′UTR and 3′UTR fragments used to target the TDH gene depletion (black boxes) and internal sequences of the puromycin (PAC, PCR products 3 and 5) or blasticidin (BSD, PCR products 4 and 6) resistance genes. As controls, we also used primers corresponding to the 5′UTR (PCR product 1), the TDH, PAC and BLE genes (PCR products 2, 7 and 8, respectively). As expected, PCR amplification of the 5′UTR was observed for both cell lines (lane 1), while the TDH gene was PCR-amplified only in the WT cells (lane 2) and PCR products with marker-derived primers were observed only in the Δtdh cell line (lanes 3–8).