Mariana R. Miranda

Characterization of Trypanosoma cruzil-cysteine transport mechanisms and their adaptive regulation

L-Cysteine and methionine are unique amino acids that act as sulfur donors in all organisms. In the specific case of Trypanosomatids, L-cysteine is particularly relevant as a substrate in the synthesis of trypanothione. Although it can be synthesized de novo, L-cysteine is actively transported in Trypanosoma cruzi epimastigote cells. L-Cysteine uptake is highly specific; none of the amino acids assayed yield significant differences in terms of transport rates. L-Cysteine is transported by epimastigote cells with a calculated apparent K(m) of 49.5 microM and a V(max) of about 13 pmol min(-1) per 10(7) cells. This transport is finely regulated by amino acid starvation, extracellular pH, and between the parasite growth phases. In addition, L-cysteine is incorporated post-translationally into proteins, suggesting its role in iron-sulfur core formation. Finally, the metabolic fates of Lcysteine were predicted in silico.

Trypanosoma cruzi: Multiple nucleoside diphosphate kinase isoforms in a single cell

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml(-1). TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.

Trypanosoma cruzi nucleoside diphosphate kinase 1 ( TcNDPK1) has a broad nuclease activity.

Here, we present the characterization of a trypanosomatid nucleoside diphosphate kinase (TcNDPK1) exhibiting nuclease activity. This is the first identification of a NDPK with this property in trypanosomatid organisms. The recombinant TcNDPK1 protein cleaves not only linear DNA, but also supercoiled plasmid DNA. Additionally, TcNDPK1 is capable of degrading Trypanosoma cruzi genomic DNA. ATP or ADP did not affect the nuclease activity, while the absence of Mg2+ completely inhibits this activity. NDPK and nuclease activities were inhibited at the same temperature, suggesting the presence of related catalytic sites. Furthermore, phenogram analysis showed that TcNDPK1 is close to Drosophila melanogaster and human NDPKs. The unspecific nuclease activity could suggest a participation in cellular processes such as programmed cell death.

Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions

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.

Resveratrol inhibits Trypanosoma cruzi arginine kinase and exerts a trypanocidal activity.

Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (<-6.96kcal/mol), resveratrol was chosen for subsequent assays. Resveratrol inhibits 50% of recombinant arginine kinase activity at 325μM. The trypanocidal effect of resveratrol was evaluated on the T. cruzi trypomastigotes bursting from infected CHO K1 cells, with IC50=77μM. Additionally epimastigotes overexpressing arginine kinase were 5 times more resistant to resveratrol compared to controls. Taking into account that: (1) resveratrol is considered as completely nontoxic; (2) is easily accessible due to its low market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease.

Plasmid Vectors and Molecular Building Blocks for the Development of Genetic Manipulation Tools for Trypanosoma cruzi

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.

Singular Features of Trypanosomatids' Phosphotransferases Involved in Cell Energy Management

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.

Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi

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.

Trypanosoma cruzi Proline Transport Presents a Cell Density-dependent Regulation.

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.

The flagellar adenylate kinases of Trypanosoma cruzi.

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.