Abstract
The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.
Highlights
Several vertebrate hosts and a hematophagous insect vector that allows their transmission between vertebrate hosts
Recent advances in understanding about trypanosomatid catabolism have focused on procyclic T. brucei, which has significant overlap with other species regarding its metabolism, but for which RNA interference (RNAi)2 has been extensively developed
Succinate represents ϳ70% of the excreted end products of D-glucose metabolism. It is produced in the glycosomes and the mitochondrion by two NADH-dependent fumarate reductase isoforms, FRDg and FRDm1, respectively [10, 12, 13]
Summary
Trypanosome and Cell Cultures—The procyclic form of T. brucei EATRO1125 was cultured at 27 °C in SDM79 medium containing 10% (v/v) heat-inactivated fetal calf serum and 3.5 mg/ml hemin [21] or in a glucose-depleted medium derived from SDM79, called SDM80 [18]. The resulting plasmid (pLew-SDH-SAS) contains a sense and antisense version of the targeted gene fragment, separated by a 96-bp fragment, under the control of the PARP promoter linked to a prokaryotic tetracycline operator. To inhibit expression of the mitochondrial F0/F1-ATP synthase, the sense and antisense versions of a fragment (bp 471–934) of the -subunit gene of the F1 complex (ATP⑀-F1; Tb927.3.1380), separated by a 58-bp fragment, were cloned into the BamHI and HindIII restriction sites of the p2T7Ti-177 vector. Trypanosome Transfection, Adaptation to SDM80 Medium, and RNAi Induction—The EATRO1125 procyclic form cell line (EATRO1125.T7T), constitutively expressing the T7 RNA polymerase gene and the tetracycline repressor under the control of a T7 RNA polymerase promoter for tetracycline-inducible expression [23], was transformed with each of the plasmids. The selected cell lines were adapted to glucose-rich SDM80glu medium and glucose-depleted SDM80 medium containing the same concentration of the three antibiotics. The reproducibility and accuracy of the method were assessed using several mixtures of 13C-enriched amino acids and lactate with known fractional enrichments; the relative errors in the 13C enrichment determinations were Ͻ5%
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