Search

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non‐membrane‐bound filaments adjacent to the nucleus. In the present work, X‐ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi‐hexameric, left‐handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X‐ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left‐handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is mainly obtained from extracellular medium by transport proteins, in this work we studied the regulation of the T. cruzi proline transporter TcAAAP069. Proline uptake and intracellular concentration presented oscillations during epimastigote growth phases, increasing during the early exponential phase (322 pmol/min) and decreasing to undetectable levels during the late exponential phase. Transporter expression rate correlated with proline uptake, and its subcellular localization alternated from both, the plasma membrane and close to the flagellar pocket, when the transport is higher, to only the flagellar pocket region, when the transport decreased until proline uptake and TcAAAP069 protein became undetectable at the end of the growth curve. Interestingly, when parasites were treated with conditioned medium or were concentrated to artificially increase the culture density, the proline transport was completely abolished resembling the effects observed in late exponential phase. These data highlight for the first time the existence of a density-associated regulation of relevant physiological processes such as proline metabolism. [ABSTRACT FROM AUTHOR]

Highlights: [•] We studied the effect of pentamidine against Trypanosoma cruzi both in vitro and in vivo. [•] Pentamidine decreases the viability of isolated trypomastigotes of T. cruzi. [•] Pentamidine decreases the parasite burden in T. cruzi-infected cells. [•] Pentamidine improves the disease outcome in T. cruzi-infected mice. [•] Pentamidine inhibits the polyamine transport in isolated parasites. [Copyright &y& Elsevier]

Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma cruzi arginine kinase is a key enzyme in cell energy management and is also involved in pH and nutritional stress response mechanisms. T. cruzi epimastigotes treated with hydrogen peroxide presented a time-dependent increase in arginine kinase expression, up to 10-fold, when compared with untreated parasites. Among other oxidative stress-generating compounds tested, only nifurtimox produced more than 2-fold increase in arginine kinase expression. Moreover, parasites overexpressing arginine kinase showed significantly increased survival capability during hydrogen peroxide exposure. These findings suggest the participation of arginine kinase in oxidative stress response systems. [Copyright &y& Elsevier]

Abstract: Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas'' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi. [Copyright &y& Elsevier]

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids. [ABSTRACT FROM AUTHOR]

Adenylate kinases (ADK) are key enzymes involved in cell energy management. Trypanosomatids present the highest number of variants in a single cell in comparison with the rest of the living organisms. In this work, we characterized two flagellar ADKs from Trypanosoma cruzi, called TcADK1 and TcADK4, which are also located in the cell cytosol. Interestingly, TcADK1 presents a stage-specific expression. This variant was detected in epimastigotes cells, and was completely absent in trypomastigotes and amastigotes, while TcADK4 is present in the major life cycle stages of T. cruzi. Both variants are also regulated, in opposite ways, along the parasite growth curve suggesting that their expression depends on the intra- and extracellular conditions. Both, TcADK1 and TcADK4 present N-terminal extension that could be responsible for their subcellular localization. The presence of ADK variants in the flagellum would be critical for the provision of energy in a process of high ATP consumption such as cell motility. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is the causative agent of Chagas disease. There are only two approved treatments, both of them unsuitable for the chronic phase, therefore the development of new drugs is a priority. Trypanosoma cruzi arginine kinase (TcAK) is a promising drug target since it is absent in humans and it is involved in cellular stress responses. In a previous study, possible TcAK inhibitors were identified through computer simulations resulting the best compounds capsaicin and cyanidin derivatives. Here, we evaluate the effect of capsaicin on TcAK activity and its trypanocidal effect. Although capsaicin produced a weak enzyme inhibition, it had a strong trypanocidal effect on epimastigotes and trypomastigotes (IC50 = 6.26 µM and 0.26 µM, respectively) being 20-fold more active on trypomastigotes than mammalian cells. Capsaicin was also active on the intracellular cycle reducing by half the burst of trypomastigotes at approximately 2 µM. Considering the difference between the concentrations at which parasite death and TcAK inhibition occur, other possible targets were predicted. Capsaicin is a selective trypanocidal agent active in nanomolar concentrations, with an IC50 57-fold lower than benznidazole, the drug currently used for treating Chagas disease. [ABSTRACT FROM AUTHOR]

Background: Crystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069. Methodology/Principal findings: CV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and DM28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain. Conclusions/Significance: Loratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs. Author summary: Chagas disease, caused by the parasite Trypanosoma cruzi, affects 7 million people worldwide. Despite there are two drugs available since 50 years ago, the therapy presents severe side effects and is not effective in the chronic phase of the disease when most of the patients are diagnosed. Crystal violet (CV) was utilized as additive in blood banks to prevent transfusion-transmitted Chagas disease. Proline is involved in many pathways, like infection establishment and life cycle progression. In this work we first demonstrate that CV has the proline permease TcAAAP069 as one of its molecular targets. Then we search in a database of already-approved drugs for compounds that were structurally related to CV under the premise "similar structure, similar activity". We identified three drugs that inhibit proline transport and present at least the same trypanocidal effect than benznidazole, the current treatment for Chagas disease. Finally we observed a synergistic effect with the multidrug combined treatment. Drug discovery is an expensive and time-consuming process and Chagas disease is associated with poverty. The discovery of new indications to old drugs, called drug repurposing, can facilitate a rapid and more profitable therapy application since preclinical trials and pharmacokinetic studies are already available. [ABSTRACT FROM AUTHOR]

[Display omitted] Trypanosoma cruzi is the causative agent of Chagas' disease, an endemic and neglected disease. The treatment is limited to only two drugs, benznidazole (BZL) and nifurtimox (NFX), introduced more than fifty years ago and no new advances have been made since then. Nucleoside diphosphate kinases (NDPK) are key metabolic enzymes which have gained interest as drug targets of pathogen organisms. Taking advantage of the computer-assisted drug repurposing approaches, in the present work we initiate a search of potential T. cruzi nucleoside diphosphate kinase 1 (TcNDPK1) inhibitors over an ∼ 12,000 compound structures database to find drugs targeted to this enzyme with trypanocidal activity. Four medicines were selected and evaluated in vitro , ketorolac (KET, an anti-inflamatory), dutasteride (DUT, used to treat benign prostatic hyperplasia), nebivolol and telmisartan (NEB and TEL, used to treat high blood pressure). The four compounds were weak inhibitors and presented different trypanocidal effect on epimastigotes, trypomastigotes and intracellular stages. NEB and TEL were the most active drugs with increased effect on intracellular stages, (IC 50 = 2.25 µM and 13.21 µM respectively), and selectivity indexes of 13.01 and 8.59 respectively, showing comparable effect to BZL, the first line drug for Chagas' disease treatment. In addition, both presented positive interactions when combined with BZL. Finally, transgenic epimastigotes with increased expression of TcNDPK1 were more resistant to TEL and NEB, suggesting that TcNDPK1 is at least one of the molecular targets. In view of the results, NEB and TEL could be repurposed medicines for Chagas' disease therapy. [ABSTRACT FROM AUTHOR]

Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6–10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease. [ABSTRACT FROM AUTHOR]

Benznidazole is the first-line drug used in treating Chagas disease, which is caused by the parasite Trypanosoma cruzi (T. cruzi) . However, benznidazole has limited efficacy and several adverse reactions. Pentamidine is an antiprotozoal drug used in the treatment of leishmaniasis and African trypanosomiasis. In T. cruzi , pentamidine blocks the transport of putrescine, a precursor of trypanothione, which constitutes an essential molecule in the resistance of T. cruzi to benznidazole. In the present study, we describe the effect of the combination of benznidazole and pentamidine on isolated parasites, mammalian cells and in mice infected with T. cruzi . In isolated trypomastigotes, we performed a dose-matrix scheme of combinations, where pentamidine antagonized the effect of benznidazole, mainly at concentrations below the EC 50 of pentamidine. In T. cruzi- infected mammalian cells, pentamidine reversed the effect of benznidazole (measured by qPCR). In comparison, in infected BALB/c mice, pentamidine failed to get synergy with benznidazole, measured on mice survival, parasitemia and amastigote nest quantification. To further explain the in vitr o antagonism, we explored whether pentamidine affects intracellular trypanothione levels, however, pentamidine produced no change in trypanothione concentrations. Finally, the T. cruzi polyamine permease (TcPAT12) was overexpressed in epimastigotes, showing that pentamidine has the same trypanocidal effect, independently of transporter expression levels. These results suggest that, in spite of the high potency in the putrescine transport blockade, TcPAT12 permease is not the main target of pentamidine, and could explain the lack of synergism between pentamidine and benznidazole. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. Author Summary: Infection with Trypanosoma cruzi produces a condition known as Chagas disease which affects at least 17 million people. Adenylate kinases, so called myokinases, are involved in a wide variety of processes, mainly related to their role in nucleotide interconversion and energy management. Recently, nuclear isoforms have been described in several organisms. This "atypical" isoform in terms of primary structure was associated to ribosomes biogenesis in yeast and to Cajal body organization in humans. Moreover nuclear adenylate kinases are essential for maintaining cellular homeostasis. In this manuscript we characterized T. cruzi nuclear adenylate kinase (TcADKn). TcADKn localizes in the nucleolus or cell cytoplasm. Nuclear shuttling mechanisms were also studied for the first time, being dependent on nutrient availability, oxidative stress and by the equivalent of the mammalian TOR pathway in T. cruzi. Furthermore we characterized the signals involved in nuclear importation and exportation processes. In addition, TcADKn expression levels are regulated at an mRNA level, being its 3′UTR involved in this process. These findings are the first steps in the understanding of ribosome processing in trypanosomatids. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N- terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 39 UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. [ABSTRACT FROM AUTHOR]

Trypanosomatids are responsible for economically important veterinary affections and severe human diseases. In Africa, Trypanosoma brucei causes sleeping sickness or African trypanosomiasis, while in America, Trypanosoma cruzi is the etiological agent of Chagas disease. These parasites have complex life cycles which involve a wide variety of environments with very different compositions, physicochemical properties, and availability of metabolites. As the environment changes there is a need to maintain the nucleoside homeostasis, requiring a quick and regulated response. Most of the enzymes required for energy management are phosphotransferases. These enzymes present a nitrogenous group or a phosphate as acceptors, and the most clear examples are arginine kinase, nucleoside diphosphate kinase, and adenylate kinase. Trypanosoma and Leishmania have the largest number of phosphotransferase isoforms ever found in a single cell; some of them are absent in mammals, suggesting that these enzymes are required in many cellular compartments associated to different biological processes. The presence of such number of phosphotransferases support the hypothesis of the existence of an intracellular enzymatic phosphotransfer network that communicates the spatially separated intracellular ATP consumption and production processes. All these unique features make phosphotransferases a promising start point for rational drug design for the treatment of human trypanosomiasis. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is exposed to extremely different environment conditions during its life cycle, and transporters are key molecules for its adaptive regulation. Amino acids, and particularly arginine, are essential components in T. cruzi metabolism. In this work, a novel T. cruzi arginine permease was identified by screening different members of the AAAP family (amino acid/auxin permeases) in yeast complementation assays using a toxic arginine analogue. One gene candidate, TcAAAP411, was characterized as a very specific, high-affinity,l-arginine permease. This work is the first identification of the molecular components involved specifically in amino acid transport in T. cruzi and provides new insights for further validation of the TcAAAP family as functional permeases. [ABSTRACT FROM AUTHOR]

Enolase is a glycolytic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In trypanosomatids, enolase was proposed as a key enzyme after in silico and in vivo analysis and it was validated as a protein essential for the survival of the parasite. Therefore, enolase constitutes an interesting enzyme target for the identification of drugs against Chagas disease. In this work, a combined virtual screening strategy was implemented, employing similarity virtual screening, molecular docking, and molecular dynamics. First, two known enolase inhibitors and the enzyme substrates were used as queries for the similarity screening on the Sweetlead database using five different algorithms. Compounds retrieved in the top 10 of at least three search algorithms were selected for further analysis, resulting in six compounds of medical use (etidronate, pamidronate, fosfomycin, acetohydroxamate, triclofos, and aminohydroxybutyrate). Molecular docking simulations and pose re-scoring predicted that binding with acetohydroxamate and triclofos would be weak, while fosfomycin and aminohydroxybutyrate predicted binding is experimentally implausible. Docking poses obtained for etidronate, pamidronate, and PEP were used for molecular dynamics calculations to describe their mode of binding. From the obtained results, we propose etidronate as a potential Tc ENO inhibitor and describe molecular motifs to be taken into account in the repurposing or design of drugs targeting this enzyme active site. Image 1 [ABSTRACT FROM AUTHOR]

The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel designs. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted. [ABSTRACT FROM AUTHOR]