Abstract
The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).
Highlights
The tricarboxylic acid cycle is not used as a bona fide cycle, i.e. to convert acetyl-CoA into carbon dioxide, all the enzymes of the cycle are expressed in the procyclic form grown in glucose-rich medium [27]
We previously characterized two NADH-dependent fumarate reductase isoforms involved in succinate production from glucose metabolism, a glycosomal enzyme (FRDg) and a mitochondrial enzyme (FRDm1) [30, 32]
FHm is located in the mitochondrion and probably provides fumarate to FRDm1, which reduces it into excreted succinate
Summary
Trypanosome and Glycosome Preparation—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 mlϪ1 hemin [33]. PCR-amplified 531- or 588-bp fragments containing the antisense FHc or FHm sequences (458 or 492 bp of targeted sequence, plus 40 or 72 bp used as a spacer to form the loop between the annealing sense and antisense sequences, respectively) and the restriction sites added to the primers were inserted in the HindIII and BamHI restriction sites of the pLew100 plasmid. We used the same approach to generate the chimeric antisense molecule, which is composed of the FHc-AS fragment containing a 72-bp extension as compared with the FHc-S fragment (spacer between the sense and antisense chimeric sequences) followed by the FHm-AS fragment (458 bp) and flanked by the HindIII and XhoI (5Ј-end) and BamHI (3Ј-end) restriction sites. The HindIII/XhoI-digested chimeric sense fragment was inserted, upstream of the chimeric antisense sequence, in the HindIII and XhoI restriction sites of the recombinant pLew100 plasmid.
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