Fumarate Is an Essential Intermediary Metabolite Produced by the Procyclic Trypanosoma brucei

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

Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum

“…T. cruzi intact cells upon incubation with [1-13 C]glucose show 13 C enrichment at the C-2 position of succinate (36). A functional fumarate reductase has been identi- fied, with knockdown of this enzyme leading to the accumulation of fumarate in the spent medium of T. cruzi cells (37).…”

Contribution of Pyruvate Phosphate Dikinase in the Maintenance of the Glycosomal ATP/ADP Balance in the Trypanosoma brucei Procyclic Form

“…Here, the same plasmid constructs were used to delete the PEPCK alleles in the ⌬ppdk::TetR-HYG/⌬ppdk::T7RNAPOL-NEO (⌬ppdk) mutant cell line, in which both PPDK alleles have been replaced by TetR-HYG and T7RNAPOL-NEO genes, respectively (10,30). The resulting cell line, ⌬ppdk::TetR-HYG/⌬ppdk::T7RNAPOL-NEO ⌬pepck::BSD/⌬pepck::PAC (⌬ppdk/⌬pepck), was generated by transfection and selection of drug-resistant clones as previously reported (31). The first and second PEPCK alleles were replaced by BSD-and PAC-resistant genes, respectively.…”

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase

“…In human parasites, such as kinetoplastids, the mitochondrial and cytosolic enzymes are members of the class I fumarases and are encoded by two separate genes (Coustou et al, 2006;Feliciano et al, 2012). In humans, both mitochondrial and cytosolic class II fumarases are encoded by the fumC gene and their subcellular localization is determined by post-translational processes (Yogev et al, 2011).…”