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.

Correction: 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.

[This corrects the article DOI: 10.1371/journal.ppat.1011522.].

Nucleosome landscape reflects phenotypic differences in Trypanosoma cruzi life forms.

Trypanosoma cruzi alternates between replicative and nonreplicative life forms, accompanied by a shift in global transcription levels and by changes in the nuclear architecture, the chromatin proteome and histone posttranslational modifications. To gain further insights into the epigenetic regulation that accompanies life form changes, we performed genome-wide high-resolution nucleosome mapping using two T. cruzi life forms (epimastigotes and cellular trypomastigotes). By combining a powerful pipeline that allowed us to faithfully compare nucleosome positioning and occupancy, more than 125 thousand nucleosomes were mapped, and approximately 20% of them differed between replicative and nonreplicative forms. The nonreplicative forms have less dynamic nucleosomes, possibly reflecting their lower global transcription levels and DNA replication arrest. However, dynamic nucleosomes are enriched at nonreplicative regulatory transcription initiation regions and at multigenic family members, which are associated with infective-stage and virulence factors. Strikingly, dynamic nucleosome regions are associated with GO terms related to nuclear division, translation, gene regulation and metabolism and, notably, associated with transcripts with different expression levels among life forms. Finally, the nucleosome landscape reflects the steady-state transcription expression: more abundant genes have a more deeply nucleosome-depleted region at putative 5' splice sites, likely associated with trans-splicing efficiency. Taken together, our results indicate that chromatin architecture, defined primarily by nucleosome positioning and occupancy, reflects the phenotypic differences found among T. cruzi life forms despite the lack of a canonical transcriptional control context.

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline.

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

The Trypanosoma cruzi TcrNT2 Nucleoside Transporter Is a Conduit for the Uptake of 5-F-2'-Deoxyuridine and Tubercidin Analogues.

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi, the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a Km of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9ΔNT1 and Cas9ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

Carbohydrate metabolism in trypanosomatids: New insights revealing novel complexity, diversity and species-unique features.

The human pathogenic trypanosomatid species collectively called the "TriTryp parasites" - Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. - have complex life cycles, with each of these parasitic protists residing in a different niche during their successive developmental stages where they encounter diverse nutrients. Consequently, they adapt their metabolic network accordingly. Yet, throughout the life cycles, carbohydrate metabolism - involving the glycolytic, gluconeogenic and pentose-phosphate pathways - always plays a central role in the biology of these parasites, whether the available carbon and free energy sources are saccharides, amino acids or lipids. In this paper, we provide an updated review of the carbohydrate metabolism of the TriTryps, highlighting new data about this metabolic network, the interconnection of its pathways and the compartmentalisation of its enzymes within glycosomes, cytosol and mitochondrion. Differences in the expression of the branches of the metabolic network between the successive life-cycle stages of each of these parasitic trypanosomatids are discussed, as well as differences between them. Recent structural and kinetic studies have revealed unique regulatory mechanisms for some of the network's key enzymes with important species-specific variations. Furthermore, reports of multiple post-translational modifications of trypanosomal glycolytic enzymes suggest that additional mechanisms for stage- and/or environmental cues that regulate activity are operational in the parasites. The detailed comparison of the carbohydrate metabolism of the TriTryps has thus revealed multiple differences and a greater complexity, including for the reduced metabolic network in bloodstream-form T. brucei, than previously appreciated. Although these parasites are related, share many cytological and metabolic features and are grouped within a single taxonomic family, the differences highlighted in this review reflect their separate evolutionary tracks from a common ancestor to the extant organisms. These differences are indicative of their adaptation to the different insect vectors and niches occupied in their mammalian hosts.

Responsible Science Assessment: downplaying indexes, boosting quality.

Scientists are facing enormous pressures posed by growing scientific communities and stagnant/reduced funding. In this scenario, mechanisms of knowledge achievement and management, as well as how recruitment, progression and evaluation are carried out should be reevaluated. We argue here that knowledge has become a profitable commodity and, as a consequence, excessive academic quantification, individual output assessment problems and abusive editorial market strategies have reached unsustainable levels. We propose to reinforce existing guidelines and to establish new ones to overcome these issues. Our proposal, the Initiative for Responsible Scientific Assessment (IRSA), has the main goal to strengthen and expand previous movements in the scientific community to promote higher quality research assessment, focused on better Science.

Computational approaches for drug discovery against trypanosomatid-caused diseases.

During three decades, only about 20 new drugs have been developed for malaria, tuberculosis and all neglected tropical diseases (NTDs). This critical situation was reached because NTDs represent only 10% of health research investments; however, they comprise about 90% of the global disease burden. Computational simulations applied in virtual screening (VS) strategies are very efficient tools to identify pharmacologically active compounds or new indications for drugs already administered for other diseases. One of the advantages of this approach is the low time-consuming and low-budget first stage, which filters for testing experimentally a group of candidate compounds with high chances of binding to the target and present trypanocidal activity. In this work, we review the most common VS strategies that have been used for the identification of new drugs with special emphasis on those applied to trypanosomiasis and leishmaniasis. Computational simulations based on the selected protein targets or their ligands are explained, including the method selection criteria, examples of successful VS campaigns applied to NTDs, a list of validated molecular targets for drug development and repositioned drugs for trypanosomatid-caused diseases. Thereby, here we present the state-of-the-art of VS and drug repurposing to conclude pointing out the future perspectives in the field.

Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity.

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector.

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

Role of Δ1-pyrroline-5-carboxylate dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi.

Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which produce reducing equivalents as follows: the conversion of proline to Δ(1)-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ(1)-pyrroline-5-carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusion chromatography, two-dimensional gel electrophoresis, and small angle x-ray scattering analysis of TcP5CDH revealed an oligomeric state composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity, confirming the enzyme's functional role; and the biochemical parameters (Km, kcat, and kcat/Km) of the recombinant TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the up-regulation (6-fold change) of TcP5CDH during the infective stages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that this enzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi.

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level.

Cystathionine γ-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp.

Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine ß-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 µM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.

Natural products as a source for treating neglected parasitic diseases.

Infectious diseases caused by parasites are a major threat for the entire mankind, especially in the tropics. More than 1 billion people world-wide are directly exposed to tropical parasites such as the causative agents of trypanosomiasis, leishmaniasis, schistosomiasis, lymphatic filariasis and onchocerciasis, which represent a major health problem, particularly in impecunious areas. Unlike most antibiotics, there is no "general" antiparasitic drug available. Here, the selection of antiparasitic drugs varies between different organisms. Some of the currently available drugs are chemically de novo synthesized, however, the majority of drugs are derived from natural sources such as plants which have subsequently been chemically modified to warrant higher potency against these human pathogens. In this review article we will provide an overview of the current status of plant derived pharmaceuticals and their chemical modifications to target parasite-specific peculiarities in order to interfere with their proliferation in the human host.

Protein kinases on carbon metabolism: potential targets for alternative chemotherapies against toxoplasmosis.

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a global disease that significantly impacts human health. The clinical manifestations are mainly observed in immunocompromised patients, including ocular damage and neuronal alterations leading to psychiatric disorders. The congenital infection leads to miscarriage or severe alterations in the development of newborns. The conventional treatment is limited to the acute phase of illness, without effects in latent parasites; consequently, a cure is not available yet. Furthermore, considerable toxic effects and long-term therapy contribute to high treatment abandonment rates. The investigation of exclusive parasite pathways would provide new drug targets for more effective therapies, eliminating or reducing the side effects of conventional pharmacological approaches. Protein kinases (PKs) have emerged as promising targets for developing specific inhibitors with high selectivity and efficiency against diseases. Studies in T. gondii have indicated the presence of exclusive PKs without homologs in human cells, which could become important targets for developing new drugs. Knockout of specific kinases linked to energy metabolism have shown to impair the parasite development, reinforcing the essentiality of these enzymes in parasite metabolism. In addition, the specificities found in the PKs that regulate the energy metabolism in this parasite could bring new perspectives for safer and more efficient therapies for treating toxoplasmosis. Therefore, this review provides an overview of the limitations for reaching an efficient treatment and explores the role of PKs in regulating carbon metabolism in Toxoplasma, discussing their potential as targets for more applied and efficient pharmacological approaches.

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci.

BACKGROUND: Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. RESULTS: Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. CONCLUSION: Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

CD4+ CD25+ Foxp3+ regulatory T cells, dendritic cells, and circulating cytokines in uncomplicated malaria: do different parasite species elicit similar host responses?

Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4(+) CD25(+) Foxp3(+) Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123(+)), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-α) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and long-lasting protective immunity to malaria.

Trypanosome prereplication machinery contains a single functional orc1/cdc6 protein, which is typical of archaea.

In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

Crystal structure of histidine ammonia-lyase from Trypanosoma cruzi.

Chagas disease is one of seventeen neglected tropical diseases according to the World Health Organization (WHO). The histidine-glutamate metabolic pathway is an oxidative route that has shown to be relevant for the bioenergetics in Trypanosoma cruzi, the etiological agent for Chagas disease. Histidine ammonia-lyase participates in the first stage of the histidine catabolism, catalyzing the conversion of l-histidine into urocanate. This work presents the three-dimensional (3D) structure of Trypanosoma cruzi histidine ammonia-lyase enzyme (TcHAL) and some comparisons of it to homologous structures. The enzyme was expressed, purified and assayed for crystallization, what allowed the obtainment of crystals of sufficient quality to collect X-ray diffraction data up to 2.55 Å resolution. After refinement, some structural analyses indicated that the structure does not contain the active site protection domain, in opposition to previously known 3D structures from plants and fungi phenylalanine ammonia-lyase, therefore, it is the first structure of eukaryotic ammonia-lyases that lacks this domain.