Structural and molecular basis of cross-seeding barriers in amyloids.

Neurodegenerative disorders are frequently associated with ß-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical ß-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents.

Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins.

NLR proteins are intracellular receptors constituting a conserved component of the innate immune system of cellular organisms. In fungi, NLRs are characterized by high diversity of architectures and presence of amyloid signaling. Here, we explore the diverse world of effector and signaling domains of fungal NLRs using state-of-the-art bioinformatic methods including MMseqs2 for fast clustering, probabilistic context-free grammars for sequence analysis, and AlphaFold2 deep neural networks for structure prediction. In addition to substantially improving the overall annotation, especially in basidiomycetes, the study identifies novel domains and reveals the structural similarity of MLKL-related HeLo- and Goodbye-like domains forming the most abundant superfamily of fungal NLR effectors. Moreover, compared to previous studies, we found several times more amyloid motif instances, including novel families, and validated aggregating and prion-forming properties of the most abundant of them in vitro and in vivo. Also, through an extensive in silico search, the NLR-associated amyloid signaling was identified in basidiomycetes. The emerging picture highlights similarities and differences in the NLR architectures and amyloid signaling in ascomycetes, basidiomycetes and other branches of life.

Identification of trans-sialidases as a common mediator of endothelial cell activation by African trypanosomes.

Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an "anti-disease vaccine", a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.

Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection.

Trypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite.

Sialidases play a key role in infection and anaemia in Trypanosoma congolense animal trypanosomiasis.

Animal African trypanosomiasis is a major constraint to livestock productivity and has an important impact on millions of people in developing African countries. This parasitic disease, caused mainly by Trypanosoma congolense, results in severe anaemia leading to animal death. In order to characterize potential targets for an anti-disease vaccine, we investigated a multigenic trans-sialidase family (TcoTS) in T. congolense. Sialidase and trans-sialidase activities were quantified for the first time, as well as the tightly regulated TcoTS expression pattern throughout the life cycle. Active enzymes were expressed in bloodstream form parasites and released into the blood during infection. Using genetic tools, we demonstrated a significant correlation between TcoTS silencing and impairment of virulence during experimental infection with T. congolense. Reduced TcoTS expression affected infectivity, parasitaemia and pathogenesis development. Immunization-challenge experiments using recombinant TcoTS highlighted their potential protective use in an anti-disease vaccine.

Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense.

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.

Partial Prion Cross-Seeding between Fungal and Mammalian Amyloid Signaling Motifs.

In filamentous fungi, NLR-based signalosomes activate downstream membrane-targeting cell death-inducing proteins by a mechanism of amyloid templating. In the species Podospora anserina, two such signalosomes, NWD2/HET-S and FNT1/HELLF, have been described. An analogous system involving a distinct amyloid signaling motif, termed PP, was also identified in the genome of the species Chaetomium globosum and studied using heterologous expression in Podospora anserina The PP motif bears resemblance to the RIP homotypic interaction motif (RHIM) and to RHIM-like motifs controlling necroptosis in mammals and innate immunity in flies. We identify here a third NLR signalosome in Podospora anserina comprising a PP motif and organized as a two-gene cluster encoding an NLR and an HELL domain cell death execution protein termed HELLP. We show that the PP motif region of HELLP forms a prion we term [π] and that [π] prions trigger the cell death-inducing activity of full-length HELLP. We detect no prion cross-seeding between HET-S, HELLF, and HELLP amyloid motifs. In addition, we find that, like PP motifs, RHIMs from human RIP1 and RIP3 kinases are able to form prions in Podospora and that [π] and [Rhim] prions partially cross-seed. Our study shows that Podospora anserina displays three independent cell death-inducing amyloid signalosomes. Based on the described functional similarity between RHIM and PP, it appears likely that these amyloid motifs constitute evolutionarily related cell death signaling modules.IMPORTANCE Amyloids are ß-sheet-rich protein polymers that can be pathological or display a variety of biological roles. In filamentous fungi, specific immune receptors activate programmed cell death execution proteins through a process of amyloid templating akin to prion propagation. Among these fungal amyloid signaling sequences, the PP motif stands out because it shows similarity to the RHIM, an amyloid sequence controlling necroptotic cell death in mammals. We characterized an amyloid signaling system comprising a PP motif in the model species Podospora anserina, thus bringing to three the number of independent amyloid signaling cell death pathways described in that species. We then showed that human RHIMs not only propagate as prions in P. anserina but also partially cross-seed with fungal PP prions. These results indicate that, in addition to showing sequence similarity, the PP and RHIM motifs are at least partially functionally related, supporting a model of long-term evolutionary conservation of amyloid signaling mechanisms from fungi to mammals.

Identification of NLR-associated Amyloid Signaling Motifs in Bacterial Genomes.

In filamentous fungi, amyloid signaling sequences allow Nod-like receptors (NLRs) to activate downstream cell-death inducing proteins with HeLo and HeLo-like (HELL) domains and amyloid RHIM and RHIM-related motifs control immune defense pathways in mammals and flies. Herein, we show bioinformatically that analogous amyloid signaling motifs exist in bacteria. These short motifs are found at the N terminus of NLRs and at the C terminus of proteins with a domain we term BELL. The corresponding NLR and BELL proteins are encoded by adjacent genes. We identify 10 families of such bacterial amyloid signaling sequences (BASS), one of which (BASS3) is homologous to RHIM and a fungal amyloid motif termed PP. BASS motifs occur nearly exclusively in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of seven of these motifs (from the most common BASS1 family and the RHIM-related BASS3 family) and find that these sequences form fibrils in vitro. Using a fungal in vivo model, we show that all tested BASS-motifs form prions and that the NLR-side motifs seed prion-formation of the corresponding BELL-side motif. We find that BASS3 motifs show partial prion cross-seeding with mammalian RHIM and fungal PP-motifs and that proline mutations on key positions of the BASS3 core motif, conserved in RHIM and PP-motifs, abolish prion formation. This work expands the paradigm of prion amyloid signaling to multicellular prokaryotes and suggests a long-term evolutionary conservation of these motifs from bacteria, to fungi and animals.

Molecular and biochemical characterization of a cathepsin B-like protease family unique to Trypanosoma congolense.

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.

Combinations of Spok genes create multiple meiotic drivers in Podospora.

Meiotic drive is the preferential transmission of a particular allele during sexual reproduction. The phenomenon is observed as spore killing in multiple fungi. In natural populations of Podospora anserina, seven spore killer types (Psks) have been identified through classical genetic analyses. Here we show that the Spok gene family underlies the Psks. The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within a population. We identify two novel Spok homologs located within a large (74-167 kbp) region (the Spok block) that resides in different chromosomal locations in different strains. We confirm that the SPOK protein performs both killing and resistance functions and show that these activities are dependent on distinct domains, a predicted nuclease and kinase domain. Genomic and phylogenetic analyses across ascomycetes suggest that the Spok genes disperse through cross-species transfer, and evolve by duplication and diversification within lineages.

Energy metabolism of trypanosomatids: adaptation to available carbon sources.

Some development stages of the trypanosomatid protozoan parasites are well adapted to in vitro culture. They can be maintained in rich medium containing large excess of glucose and amino acids, which they use as carbon sources for ATP production. Under these growth conditions, carbon sources are converted into partially oxidized end products by so-called aerobic fermentation. Surprisingly, some species, such as the Trypanosoma brucei, Trypanosoma cruzi and Crithidia insect stages, prefer consuming glucose to amino acids, although their natural habitat is L-proline-rich. This review focuses on recent progress in understanding glucose and l-proline metabolism of insect stages, how these metabolic processes are regulated, and the rationale of the aerobic fermentation strategies developed by these parasites.

The susceptibility of Trypanosoma congolense and Trypanosoma brucei to isometamidium chloride and its synthetic impurities.

Since the 1950s, the chemotherapy of animal African trypanosomosis in cattle has essentially relied on only two compounds: isometamidium chloride (ISM), a phenanthridine, and diminazene aceturate, an aromatic diamidine. The commercial formulations of ISM, including Veridium(®) and Samorin(®), are a mixture of different compounds: ISM is the major component, mixed with the red isomer, blue isomer and disubstituted compound. To investigate the pharmacological effects of these individual compounds ISM, the blue and red isomers and the disubstituted compound were synthesised and purified by HPLC. The activity of each compound was analysed both in vitro, and in mice in vivo. For the in vitro analysis, a drug sensitivity assay was developed in 96-well tissue culture plates to determine the effective concentration which killed 50% of trypanosome population within 48 h of drug exposure (IC50). All compounds tested in vitro possessed trypanocidal activity, and purified ISM was the most active. Veridium(®) and Samorin(®) had similar IC50 values to purified ISM for both Trypanosoma congolense and Trypanosoma brucei brucei. The disubstituted compound had the highest IC50 values whereas intermediate IC50 values were obtained for the blue and red isomers. In vivo, single-dose tests were used to evaluate the trypanocidal and prophylactic activity against T. congolense. Interestingly, the prophylactic effect two months post treatment was as efficient with ISM, Veridium(®), Samorin(®) and the disubstituted compound at the highest dose of 1mg/kg whereas the red and blue isomers both showed much lower prophylactic activity. This study on T. congolense implies that it is necessary to limit the quantity of the blue and red isomers in the commercial mixture. Finally, the in vitro sensitivity assay may be useful for screening new trypanocides but also for the testing and detection of resistant trypanosome isolates.

Trypanosoma vivax GM6 antigen: a candidate antigen for diagnosis of African animal trypanosomosis in cattle.

BACKGROUND: Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km(2) of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. METHODOLOGY/PRINCIPLE FINDINGS: the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). CONCLUSION/SIGNIFICANCE: this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.

Trypanosoma brucei brucei oligopeptidase B null mutants display increased prolyl oligopeptidase-like activity.

African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. Nagana, the cattle form of the disease, is caused by Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei brucei. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an "anti-disease" approach. One such pathogenic factor is oligopeptidase B (TbOPB), a trypanosome peptidase that hydrolyses Arg/Lys containing peptides smaller than 30 amino acid residues and is suspected to be involved in the hormonal deregulation associated with the disease. To better understand the role TbOPB plays in parasite physiology and host pathogenesis, oligopeptidase B null mutant parasites (Δopb) were generated in the T. b. brucei Lister 427 strain. Δopb Trypanosoma brucei parasites grew at a significantly faster rate in vitro, and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that TbOPB is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatine gel analysis of Δopb null mutants showed an increase in discrete cysteine peptidase activities when compared to wild type strains. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by TbOPB gene deletion. A significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed, but no concomitant increase in TbPOP protein levels, suggesting that a POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants.

Complete in vitro life cycle of Trypanosoma congolense: development of genetic tools.

BACKGROUND: Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses. METHODOLOGY/PRINCIPAL FINDINGS: We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement. CONCLUSIONS/SIGNIFICANCE: We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.

Murine Models for Trypanosoma brucei gambiense disease progression--from silent to chronic infections and early brain tropism.

BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates. METHODOLOGY/PRINCIPAL FINDINGS: T. b. gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms. Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties, immune response and capacity for brain invasion. The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia, an invasion of the central nervous system (CNS), a trypanosome specific-antibody response and death of the animals within 6-8 months. The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection, followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR, and the presence of a specific antibody response. The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout, but infection was detectable by PCR during the whole course of infection. Additionally, specific antibodies were barely detectable when mice were infected with a low number of this group of parasites. In both sub-chronic and chronic infections, most of the mice survived more than one year without major clinical symptoms despite an early dissemination and growth of the parasites in different organs including the CNS, as demonstrated by bioluminescent imaging. CONCLUSIONS/SIGNIFICANCE: Whereas trypanosome characterisation assigned all these isolates to the homogeneous Group I of T. b. gambiense, they clearly induce very different infections in mice thus mimicking the broad clinical diversity observed in HAT due to T. b. gambiense. Therefore, these murine models will be very useful for the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs.

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.

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).

Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose.

Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.