Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.     

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.     

Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains

Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.     

Molecular mechanisms of host cell invasion by Trypanosoma cruzi

The protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular protozoan pathogen. Overlapping mechanisms ensure successful infection, yet the relationship between these cellular events and clinical disease remains obscure. This review explores the process of cell invasion from the perspective of cell surface interactions, intracellular signaling, modulation of the host cytoskeleton and endosomal compartment, and the intracellular innate immune response to infection.     

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.     

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites’ antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson’s coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.     

The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway

Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP⁺/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi

Trypanosoma cruzi is the ethiological agent for Chagas disease in Latin America. This study aimed to test the trypanocidal effect of licarin A and burchellin isolated from plants in northeastern Brazil. These neolignans were tested on T. cruzi and on peritoneal macrophages, to evaluate drug toxicity. Epimastigote growth was inhibited in 45% with licarin A and 20% with burchellin with an IC₅₀/96 h of 462.7 μM and 756 μM, respectively. Epimastigotes treated with licarin A presented swollen mitochondria and disorganized mitochondrial cristae, kDNA and Golgi complex. When treated with burchellin, they presented enormous autophagosomes and chromatin disorganization. Licarin A and burchellin were able to induce trypomastigote death with IC₅₀/24 h of 960 μM and 520 μM, respectively. Although licarin A presented an IC₅₀ for trypomastigotes higher than for epimastigotes, both substances acted as therapeutic trypanocidal agents, because they were able to kill parasites without affecting macrophages. Due to our results, burchellin and licarin A need to be further analysed to observe if they may be used as alternative blood additive prophylaxis against Chagas disease, since it has been established that blood transfusion is an important mechanism in the transmission process.     

Canova medication modifies parasitological parameters in mice infected with Trypanosoma cruzi

The goals of this study were to evaluate the effect of the Canova® medication, a homeopathic immune-system modulator, on the evolution of infection induced by the Trypanosoma cruzi Y strain in mice. The animals were divided into five groups: (i) untreated infected controls (I), (ii) infected animals treated with benznidazole (Bz), (iii) infected animals treated with the Canova medication (CM), (iv) infected animals treated with benznidazole and the Canova medication (Bz + CM), and (v) uninfected controls that received only the vehicle (grain alcohol) (C). The parameters evaluated were: parasitemia, mortality, control of cure, and tissue parasitism analysis. Our results showed that the evolution of the experimental infection was modified by treatment with CM, and that daily and consecutive doses were harmful to the animals, causing death in 100% of the infected animals in a brief period. The analysis of parasitism performed on the organs on the 12th day postinfection showed that in infected animals treated with CM, the number of amastigote/nests in the spleen was significantly reduced, while in cardiac tissue, intestine, and liver the number was significantly increased compared with infected control animals. These results indicate that CM has a negative influence on the host-parasite relationship, modifying the tropism of the parasite for tissues, and increasing the parasitemia peak in this experimental model.     

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner [1]. To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Trypanosoma cruzi: Biodistribution of technetium-99m pertechnetate in infected rats

With the aim of investigating the biodistribution of technetium-99 m pertechnetate () in rats infected with Y strain of Tripanosoma Cruzi, at the peak of parasitemia, (14th day of infection), we injected Wistar rats with 0.1 ml of (3.7 MBq). After 60 min, the percentage of radioactivity per gram was counted in several isolated organs and blood, using a gamma counter (1470 Wizard, PerkinElmer Finland). The uptake of increased significantly in blood and decreased in the colon of infected animals (p < 0.05). A significant reduction in serum iron and red blood cells and a significant increase in total proteins, leukocytes and lymphocytes in the infected rats were observed, compared with controls (p < 0.05). A reduction in muscle layer thickness of the colon and mononuclear inflammation were observed. These results conclusively demonstrate that T. cruzi infection would be associated with changes in the biodistribution of and in colon morphology, with potential clinical implications.     

Synthesis of trisaccharides containing internal galactofuranose O-linked in Trypanosoma cruzi mucins

The trisaccharides β-d-Galf-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (5) and β-d-Galp-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (6) constitute novel structures isolated as alditols when released by reductive β-elimination from mucins of Trypanosoma cruzi (Tulahuen strain). Trisaccharides 5 and 6 were synthesized employing the aldonolactone approach. Thus, a convenient d-galactono-1,4-lactone derivative was used for the introduction of the internal galactofuranose and the trichloroacetimidate method was employed for glycosylation reactions. Due to the lack of anchimeric assistance on O-2 of the galactofuranosyl precursor, glycosylation studies were performed under different conditions. The nature of the solvent strongly determined the stereochemical course of the glycosylation reactions when the galactofuranosyl donor was substituted either by 2-O-Galp or 2-O-Galf.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Trypanosoma cruzi calmodulin: Cloning, expression and characterization

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the α-helix content upon binding with Ca²⁺, when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca²⁺ pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.     

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8 T cells against Trypanosoma cruzi infection

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8⁺ T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8⁺ T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8⁺ T-cell immunity in terms of the number of antigen-specific CD8⁺ T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T_EM) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8⁺ T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8⁺ T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8⁺ T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8⁺ T cells of high quality.     

Experimental Chagas’ disease in orchiectomized Calomys callosus infected with the CM strain of Trypanosoma cruzi

The incidence and progression of disorders associated with an unbalanced immune response has among many factors the gender as a contributory factor. The aims of this work were to evaluate the effects of orchiectomy and the immune response during the experimental Trypanosoma cruzi infection. Young adult, male Calomys callous were i.p. inoculated with 1 × 10⁵ blood trypomastigotes of the CM strain of T. cruzi and divided in groups: Control, Sham and Castrated. Castrated group displayed significantly lower values for prostate and seminal vesicle weights indicating a drastic drop of testosterone plasmatic levels. Orchiectomized animals also displayed lesser number of blood parasites, enhanced lytic antibody percentage, splenocyte proliferation and NO concentration when compared to its sham and control counterparts, indicating that steroid gonadal ablation actually influences immune response triggering a more efficient cellular and humoral response which led animals to become more resistant against T. cruzi infection.