The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.     

trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).

Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

A sialidase mutant displaying trans-sialidase activity

Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.     

Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi

Trypanosomes are unable to synthesize the monosaccharide sialic acid, but some African trypanosomes and the American Trypanosoma cruzi can incorporate sialic acid derived from the host. To do so, T. cruzi expresses a trans-sialidase, an enzyme that catalyzes the transfer of sialic acid from host glycoconjugates to mucin-like molecules located on the parasite surface membrane. The importance of the process is indicated by the fact that T. cruzi has hundreds of genes encoding trans-sialidase, trans-sialidase-like proteins and mucin core proteins. Sequence divergence of members of these families has resulted in some molecules having functions unrelated to the acquisition of sialic acid. In this article, Alberto Frasch reviews the structure and possible function of the proteins making up these families.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin

African trypanosomes have a tightly coordinated cell cycle to effect efficient segregation of their single organelles, the nucleus, flagellum, and kinetoplast. To investigate cell cycle control in trypanosomes, a mitotic cyclin gene (CYC6) has been identified in Trypanosoma brucei. We show that CYC6 forms an active kinase complex with CRK3, the trypanosome CDK1 homologue, in vivo. Using RNA interference, we demonstrate that absence of CYC6 mRNA results in a mitotic block and growth arrest in both the insect procyclic and mammalian bloodstream forms. In the procyclic form, CYC6 RNA interference generates anucleate cells with a single kinetoplast, whereas in bloodstream form trypanosomes, cells with one nucleus and multiple kinetoplasts are observed. Fluorescence-activated cell sorting analysis shows that bloodstream but not procyclic trypanosomes are able to reinitiate nuclear S phase in the absence of mitosis. Taken together, these data show that procyclic trypanosomes can undergo cytokinesis without completion of mitosis, whereas a mitotic block in bloodstream form trypanosomes inhibits cytokinesis but not kinetoplast replication and segregation nor an additional round of nuclear DNA synthesis. This indicates that there are fundamental differences in cell cycle controls between life cycle forms of T. brucei and that key cell cycle checkpoints present in higher eukaryotes are absent from trypanosomes.     

Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis

The single flagellum of the protozoan parasite Trypanosoma brucei is attached along the length of the cell body by a complex structure that requires the FLA1 protein. We show here that inhibition of FLA1 expression by RNA interference in procyclic trypanosomes causes flagellar detachment and prevents cytokinesis. Despite being unable to divide, these cells undergo mitosis and develop a multinucleated phenotype. The Trypanosoma cruzi FLA1 homolog, GP72, is unable to complement either the flagellar detachment or cytokinesis defects in procyclic T. brucei that have been depleted of FLA1 by RNA interference. Instead, GP72 itself caused flagellar detachment when expressed in T. brucei. In contrast to T. brucei cells depleted of FLA1, procyclic T. brucei expressing GP72 continued to divide despite having detached flagella, demonstrating that flagellar attachment is not absolutely necessary for cytokinesis. We have also identified a FLA1-related gene (FLA2) whose sequence is similar but not identical to FLA1. Inhibition of FLA1 and FLA2 expression in bloodstream T. brucei caused flagellar detachment and blocked cytokinesis but did not inhibit mitosis. These experiments demonstrate that the FLA proteins are essential and suggest that in procyclic T. brucei, the FLA1 protein has separable functions in flagellar attachment and cytokinesis.     

A sialidase activity in the midgut of the insect Triatoma infestans is responsible for the low levels of sialic acid in Trypanosoma cruzi growing in the insect vector

Trypanosoma cruzi expresses a unique trans-sialidase that isresponsible for the transfer of sialic acid from host glycoproteinsand glycolipids to mucin-like glycoprotein acceptors on theparasite surface. The enzyme and the sialic acid acceptors arepresent in the mammalian forms of the parasite and in the parasiteforms that grow in axenic cultures, which correspond to thedevelopmental stages found in the insect vectors. Here we showthat parasite forms growing in the vector Triatoma infestansexpress trans-sialidase in the hind gut portions of the insectHowever, the sialic acid acceptors are poorly sialylated dueto the low concentration of sialic acid donors in the gut lumenof T.infestans, which feeds exclusively on blood that is richin sialic acid donors. These low levels of sialic acid donorsare due to a novel sialidase activity present mainly in theanterior midgut with high specificity for     

Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.     

Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.     

trans-Sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction.     

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.     

Identification and characterization of three peroxins--PEX6, PEX10 and PEX12--involved in glycosome biogenesis in Trypanosoma brucei

Protozoan Kinetoplastida such as the pathogenic trypanosomes compartmentalize several important metabolic systems, including the glycolytic pathway, in peroxisome-like organelles designated glycosomes. Genes for three proteins involved in glycosome biogenesis of Trypanosoma brucei were identified. A preliminary analysis of these proteins, the peroxins PEX6, PEX10 and PEX12, was performed. Cellular depletion of these peroxins by RNA interference affected growth of both mammalian bloodstream-form and insect-form (procyclic) trypanosomes. The bloodstream forms, which rely entirely on glycolysis for their ATP supply, were more rapidly killed. Both by immunofluorescence studies of intact procyclic T. brucei cells and subcellular fractionation experiments involving differential permeabilization of plasma and organellar membranes it was shown that RNAi-dependent knockdown of the expression of each of these peroxins resulted in the partial mis-localization of different types of glycosomal matrix enzymes to the cytoplasm: proteins with consensus motifs such as the C-terminal type 1 peroxisomal targeting signal PTS1 or the N-terminal signal PTS2 and a protein for which the sorting information is present in a polypeptide-internal fragment not containing an identifiable consensus sequence.     

Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei

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.     

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.     

Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid     

A function for a specific zinc metalloprotease of African trypanosomes

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.     

Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei

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).