RESUMO
CONTEXT: Currently, Chagas disease represents an important public health problem affecting more than 8 million people worldwide. The vector of this disease is the Trypanosoma cruzi (Tc) parasite. Our research specifically focuses on the structure and aggregation states of the enzyme aldo-keto reductase of Tc (TcAKR) reported in this parasite. TcAKR belongs to the aldo-keto reductase (AKR) superfamily, enzymes that catalyze redox reactions involved in crucial biological processes. While most AKRs are found in monomeric forms, some have been reported to form dimeric and tetrameric structures. This is the case for some TcAKR. To better understand how TcAKR multimers form and remain stable, we conducted a comprehensive computational analysis using molecular dynamics (MD) simulations. Our approach to elucidating the aggregation states of TcAKR involved two strategies. Initially, we explored the dynamic behaviour of pre-assembled TcAKR dimers. Subsequently, we examined the self-aggregation of eight monomers. This investigation led to the identification of crucial residues that contribute to the stabilization of protein-protein interactions. It was also found that TcAKRs can form stable supramolecular assemblies, with each monomer typically surrounded by three first neighbours. These findings align with experimental reports of tetrameric or more complex supramolecular structures. Our computational studies could guide further experimental investigations aiming at drug development and assist in designing strategies to modulate aggregation. METHOD: Atomistic molecular dynamics simulations were carried out. The TcAKR 3D model structure was obtained by homology modelling using the Swiss Model for the TcAKR sequence (GenBank accession no. EU558869). Further, we checked the model with Alphafold2 and found a high degree of similarity between models. Several tools were used to build the dimers including CLUSPRO, GRAMM-Docking, Hdock, and Py-dock. Protein superstructures were built using the PACKMOL package. CHARMM-GUI was used to set up the simulation systems. GROMACS version 2020.5 was used to perform the simulations with the CHARMM36 force field for the protein and ions and the TIP3P model for water. Further analyses were performed using VMD, GROMACS, AMBER tools, MDLovoFit, bio3d, and in-house programs.
Assuntos
Aldo-Ceto Redutases , Simulação de Dinâmica Molecular , Trypanosoma cruzi , Trypanosoma cruzi/enzimologia , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/metabolismo , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Chlorocebus aethiops , Di-Hidroxiacetona/metabolismo , Di-Hidroxiacetona/toxicidade , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Osmorregulação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors, including antigens from the trans-sialidase (TS) superfamily, which modulate the host immune responses. Tc13 antigens belong to group IV of TSs and are characterized by C-terminal EPKSA repeats. Here, we studied the effect of the Tc13 antigen from the Tulahuén strain, Tc13Tul, on primary cultures of splenocytes from naïve BALB/c mice. Recombinant Tc13Tul increased the percentage of viable cells and induced B (CD19+) lymphocyte proliferation. Tc13Tul stimulation also induced secretion of non-specific IgM and interferon-γ (IFN-γ). The same effects were induced by Tc13Tul on splenocytes from naïve C3H/HeJ mice. In vivo administration of Tc13Tul to naïve BALB/c mice increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an enhanced effect on IgM and IFN-γ secretion. Our results indicate that Tc13Tul may participate in the early immunity in T. cruzi infection by favouring immune system evasion through B-cell activation and non-specific Ig secretion. In contrast, as IFN-γ is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favour of the host.
Assuntos
Antígenos de Protozoários/imunologia , Glicoproteínas/imunologia , Neuraminidase/imunologia , Baço/parasitologia , Trypanosoma cruzi/imunologia , Animais , Imunoglobulina G , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Baço/imunologia , Trypanosoma cruzi/enzimologiaRESUMO
Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as ß-lapachone (ß-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by ß-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that ß-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, ß-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only ß-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in ß-Lap activation.
Assuntos
Aldo-Ceto Redutases/metabolismo , Naftoquinonas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Aldo-Ceto Redutases/genética , Animais , Chlorocebus aethiops , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fenótipo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Células VeroRESUMO
Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases.
Assuntos
Nitroimidazóis/metabolismo , Trypanosoma cruzi/enzimologia , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Doença de Chagas/tratamento farmacológico , Doença de Chagas/metabolismo , DNA de Protozoário/genética , Nitroimidazóis/farmacologia , Nitrorredutases/genética , Nitrorredutases/metabolismo , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologiaRESUMO
Tc13Tul antigen is expressed in the mammalian stages of Trypanosoma cruzi, the etiological agent of Chagas' disease. Here, we designed and validated an enzyme-linked immunosorbent assay using the recombinant Tc13Tul (Tc13Tul-ELISA) and found that it had 82.5% sensitivity and 97.05% of specificity. To evaluate whether the decrease in antibodies against Tc13Tul may be used as an early marker of the effect of chemotherapy with benznidazole, sera from 30 T. cruzi-infected children were evaluated by Tc13Tul-ELISA before and after benznidazole treatment. While in Group A (6 months-4 years old, n = 16) the decrease of more than 30% of Tc13Tul-ELISA values showed a sensitivity similar to that of conventional serology (CS); in Group B, (5-12 years old, n = 14) the decrease of Tc13Tul-ELISA values was a better parameter than negativization of CS to monitor the impact of treatment. Therefore, the dosage of anti-Tc13Tul antibodies may be useful as a methodology complementary to CS to evaluate chagasic patients undergoing chemotherapy with benznidazole.
Assuntos
Antígenos de Protozoários/sangue , Doença de Chagas/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática/métodos , Nitroimidazóis/uso terapêutico , Trypanosoma cruzi/isolamento & purificação , Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Criança , Pré-Escolar , Humanos , Lactente , Sensibilidade e Especificidade , Testes Sorológicos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas' disease, have shown side effects and variable efficiency. With the aim to describe parasite enzymes involved in the mechanisms of action of trypanocidal drugs and since it has been reported that reductases are crucial in their metabolism, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi. The percolation of a soluble fraction of epimastigote lysates through a Cibacron Blue-Sepharose column followed by elution by NADPH yielded a predominant protein with an apparent molecular weight of 32 kDa. This protein was identified by MALDI-TOF as an aldo-keto reductase (AKR) and hence denominated TcAKR. TcAKR was mainly localized in the cytosol and was also present in trypomastigote and amastigote lysates. The recombinant TcAKR (recTcAKR) showed NADPH-dependent reductase activity with the AKR substrates 4-nitrobenzaldehyde and 2-dihydroxyacetone. The saturation curves for both substrates were consistent with the Michaelis-Menten model. We also tested whether recTcAKR may reduce naphthoquinones (NQ), since many of these compounds have displayed important trypanocidal activity. recTcAKR reduced o-NQ (1,2-naphthoquinone, 9,10-phenanthrenquinone and beta-lapachone) with concomitant generation of free radicals but did not exhibit affinity for p-NQ (5-hydroxy-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, alpha-lapachone and menadione). The substrate saturation curve with o-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In addition, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. TcAKR is the first member of the AKR family described in T. cruzi. Our results indicate that this enzyme may participate in the mechanisms of action of trypanocidal drugs.