The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Trypanosoma brucei proliferates normally even after losing all S-adenosylhomocysteine hydrolase genes.

S-adenosylhomocysteine (SAH) hydrolase is the enzyme responsible for breaking down SAH into adenosine and homocysteine. It has long been believed that a deficiency of this enzyme leads to SAH accumulation, subsequently inhibiting methyltransferases responsible for nucleic acids and proteins, which severely affects cell proliferation. To investigate whether targeting this enzyme could be a viable strategy to combat Trypanosoma brucei, the causative agent of human African trypanosomiasis, we created a null mutant of the SAH hydrolase gene in T. brucei using the Cre/loxP system and conducted a phenotype analysis. Surprisingly, the null mutant, where all five SAH hydrolase gene loci were deleted, exhibited normal proliferation despite the observed SAH accumulation. These findings suggest that inhibiting SAH hydrolase may not be an effective approach to suppressing T. brucei proliferation, making the enzyme a less promising target for antitrypanosome drug development.

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes.

BACKGROUND: Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown. METHODS: Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4',6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation. RESULTS: We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation. CONCLUSIONS: We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.

Erythrocytic, Enzymatic, and Histological Markers of Oxidative Stress in Subacute and Chronic Stage Infections in Wistar Rats (Rattus norvegicus) Infected with Trypanosoma brucei brucei.

Trypanosomiasis is a complex of diseases caused by a haemoprotozoan parasite of medical and veterinary importance. One of the leading factors that cause morbidity and death in trypanosomiasis is oxidative stress. The oxidative stress biomarkers in trypanosomiasis at the subacute and chronic stages of infection were investigated in this study. A total of twenty-four Wistar rats were used; the animals were placed in two groups: group A (subacute and chronic) and group B (control). The weight and body temperature of the experimental animals were determined using a digital weighing balance and thermometer. A hematology analyzer was used to determine the erythrocyte indices. Spectrophotometry was used to estimate enzyme (superoxide dismutase, catalase, and glutathione) activities in the serum, kidney, and liver of experimental animals. Liver, kidney, and spleen were harvested and analyzed for histological changes. The mean body weight of the infected decreased compared to the control (P < 0.05). The mean body temperature of infected individuals increased (35-37°C) compared to the control (P < 0.05). The erythrocyte indices of the infected and control groups indicate a significant decrease (P < 0.05). In erythrocyte indices, only MCHC indicated a nonsignificant decrease (P > 0.05). The SOD of serum shows a significant increase (P < 0.05), and no significant increase SOD (P > 0.05) in kidney and the liver SOD indicates a significant decrease (P < 0.05). The serum, kidney, and liver show a significant increase (P < 0.05) in CAT. The serum GSH from the findings indicates a nonsignificant increase (P > 0.05), and the kidney and liver GSH shows a significant increase (P < 0.05). The correlation analysis for SOD shows nonsignificant negative correlation for serum/kidney, and the serum/liver and kidney/liver show significant positive correlation. The result of CAT shows significant correlations for serum and kidney, serum and liver, and kidney/liver with a positive correlation. The GSH result shows no significant negative correlation for serum/kidney and no significant positive correlation for serum/liver and kidney/liver. The histological damage in the kidney, liver, and spleen was much higher in the chronic stage than in the subacute stage and no tissue damage in the control group. In conclusion, subacute and chronic stage trypanosome infection is associated with changes in hematological indices, antioxidants of the liver, spleen and kidney, and histological architecture.

Drugging the Undruggable Trypanosoma brucei Monothiol Glutaredoxin 1.

Trypanosoma brucei is a species of kinetoplastid causing sleeping sickness in humans and nagana in cows and horses. One of the peculiarities of this species of parasites is represented by their redox metabolism. One of the proteins involved in this redox machinery is the monothiol glutaredoxin 1 (1CGrx1) which is characterized by a unique disordered N-terminal extension exclusively conserved in trypanosomatids and other organisms. This region modulates the binding profile of the glutathione/trypanothione binding site, one of the functional regions of 1CGrx1. No endogenous ligands are known to bind this protein which does not present well-shaped binding sites, making it target particularly challenging to target. With the aim of targeting this peculiar system, we carried out two different screenings: (i) a fragment-based lead discovery campaign directed to the N-terminal as well as to the canonical binding site of 1CGrx1; (ii) a structure-based virtual screening directed to the 1CGrx1 canonical binding site. Here we report a small molecule that binds at the glutathione binding site in which the binding mode of the molecule was deeply investigated by Nuclear Magnetic Resonance (NMR). This compound represents an important step in the attempt to develop a novel strategy to interfere with the peculiar Trypanosoma Brucei redox system, making it possible to shed light on the perturbation of this biochemical machinery and eventually to novel therapeutic possibilities.

Identification of megacerotonic acid and a quinazoline derivative from Universal Natural Product Database as potential inhibitors of Trypanosoma brucei brucei alternative oxidase: molecular docking, molecular dynamic simulation and MM/PBSA analysis.

African trypanosomiasis is caused by Trypanosoma brucei subspecies and available drugs against it, are unsatisfactory due to poor pharmacokinetic properties. Trypanosomal Alternative Oxidase (TAO) is an attractive target for anti-trypanosome rational drug discovery because it is essential for parasite-specific ATP generation and absent in the mammalian host. In this study, 360 filtered ligands from the Universal Natural Product Database were virtually screened and docked on T. brucei brucei TAO (PDB-ID 3VVA). From the virtual screening, 10 ligands with binding energy from -10.6 to -9.0 kcal/mol were selected as hits and further subjected pharmacokinetic and toxicity analyses where all of them passed Lipinski's rule of five. Also, the compounds were non-mutagenic, non-tumorigenic and could cross the blood brain barrier. The two topmost hits (UNPD29179; megacerotonic acid and UNPD41551; a quinazoline derivative) interacted with `four glutamates (Glu123, Glu162, Glu213 and Glu266) close to di-iron (2 iron elements) at the catalytic site of the enzyme. Subsequently, 100 ns MD simulations of the two topmost hits were performed using GROMACS where high RMSD values of 0.75 nm (TAO-UNPD29179) and 0.52 nm (TAO- UNPD41551), low residues fluctuations and consistent values of radius of gyration were observed. Moreover, Solvent Accessible Surface Area showed a consistent value of 160 nm2 for both complexes while TAO-UNPD29179 had higher number of hydrogen bonds than the TAO-UNPD41551. Similarly, MM/PBSA calculations indicated that UNPD29179 had higher free binding energy with TAO than UNPD41551. The data suggest that megacerotonic acid and a quinazoline derivative could be potential inhibitors of TAO with improved pharmacokinetic properties.Communicated by Ramaswamy H. Sarma.

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions.

BACKGROUND: Spliced leader trans splicing is the addition of a short, capped sequence to the 5' end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5' trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target. METHODOLOGY AND PRINCIPAL FINDINGS: We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. CONCLUSIONS: Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor.

A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID).

The protein translocase of the mitochondrial inner membrane in Trypanosoma brucei, TbTIM17, forms a modular complex in association with several other trypanosome-specific proteins. To identify transiently interacting proximal partner(s) of TbTim17, we used Biotinylation Identification (BioID) by expressing a modified biotin ligase-TbTim17 (BirA∗-TbTim17) fusion protein in T. brucei. BirA∗-TbTim17 was targeted to mitochondria and assembled in the TbTIM complex. In the presence of biotin, BirA∗-TbTim17 biotinylated several mitochondrial proteins. Interestingly, TbHsp84/TbTRAP1, a mitochondrial Hsp90 homolog, was identified as the highest enriched biotinylated proteins. We validated that interaction and colocalization of TbTim17 and TbHsp84 in T. brucei mitochondria by coimmunoprecipitation analysis and confocal microscopy, respectively. TbTim17 association with TbTRAP1 increased several folds during denaturation/renaturation of mitochondrial proteins in vitro, suggesting TbTRAP1 acts as a chaperone for TbTim17 refolding. We demonstrated that knockdown of TbTRAP1 reduced cell growth and decreased the levels of the TbTIM17, TbTim62, and mitochondrial (m)Hsp70 complexes. However, ATPase, VDAC, and Atom69 complexes were minimally affected. Additionally, the steady state levels of TbTim17, TbTim62, and mHsp70 were reduced significantly, but Atom69, ATPase ß, and RBP16 were mostly unaltered due to TbTRAP1 knockdown. Quantitative proteomics analysis also showed significant reduction of TbTim62 along with a few other mitochondrial proteins due to TbTRAP1 knockdown. Finally, TbTRAP1 depletion did not hamper the import of the ectopically expressed TbTim17-2xMyc into mitochondria but reduced its assembly into the TbTIM17 complex, indicating TbTRAP1 is critical for assembly of TbTim17. This is the first report showing the role of TRAP1 in the TIM complex assembly in eukaryotes.

A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission.

Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns.

The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for more than 200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3'-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in nondepleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans-spliced, polyadenylated RNAs containing only downstream 3'-untranslated region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and DRBD18 was found in both the nucleus and the cytoplasm. We therefore suggest that in the nucleus, DRBD18 might bind to polypyrimidine tracts in 3'-UTRs of mRNA precursors. Such binding might both prevent recognition of mRNA-internal polypyrimidine tracts by splicing factors, and promote export of the processed bound mRNAs to the cytosol.

A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling.

A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed.

Physiologic Targets and Modes of Action for CBL0137, a Lead for Human African Trypanosomiasis Drug Development.

CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei Herein, we use a four-step strategy to 1) identify physiologic targets and 2) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase) and DNA-binding proteins [universal minicircle sequence binding protein 2, replication protein A1 (RPA1), replication protein A2 (RPA2)]. In chemical biology studies, CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiologic targets of the drug. CBL0137 inhibited 1) nucleus mitosis, 2) nuclear DNA replication, and 3) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNA interference (RNAi) against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiologic targets of CBL0137 can be deployed with different drugs in other biologic systems. SIGNIFICANCE STATEMENT: To distinguish drug-binding proteins from physiologic targets in the African trypanosome, we devised and executed a multidisciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.

New peptidomimetic rhodesain inhibitors with improved selectivity towards human cathepsins.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k2nd = 883 • 103 M-1 s-1) with single-digit nanomolar binding affinity (Ki = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis.

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active.

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based pro-drug activation.

The essential cysteines in the CIPC motif of the thioredoxin-like Trypanosoma brucei MICOS subunit TbMic20 do not form an intramolecular disulfide bridge in vivo.

The mitochondrial protein import machinery of trypanosomatids is highly divergent from that of the well-studied models such as baker's yeast. A notable example is that the central catalyst of the mitochondrial intermembrane space import and assembly pathway (MIA), named Mia40, is missing in trypanosomatids. Mia40 works in a two-step process. First it recognizes by direct binding reduced MIA substrate proteins and then catalyzes their oxidative folding to produce intramolecular disulfide bridges. It was recently proposed that a thioredoxin-like subunit of the trypanosomal mitochondrial contact site and cristae organizing system (MICOS) called TbMic20 may be the Mia40 replacement. Our study performed on procyclic stage of the parasite revealed that each of the two cysteines in TbMic20's active site is essential for the stability of MIA substrate proteins although they do not form a disulfide bridge in vivo. The two cysteines of Mia40's active site form an intramolecular disulfide bridge at steady state, which is a prerequisite for its oxidative folding of MIA substrates. Thus, we conclude that TbMic20 is unlikely to represent a bona fide Mia40 replacement and plays a still unresolved role in the stability and/or import of MIA substrates in trypanosomatids. Despite this, the effect of TbMic20 depletion and mutation indicates that the trypanosomal MICOS complex still plays a vital role in the maturation and/or stability of proteins imported by the MIA pathway.

The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

The protozoan parasite Trypanosoma brucei is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of T. brucei is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest.

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro.

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).