Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

The identification of new targets for vaccine and drug development for the treatment of Chagas’ disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite’s genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6–8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.     

The Combination of Vitamin K3 and Vitamin C Has Synergic Activity against Forms of Trypanosoma cruzi through a Redox Imbalance Process

Chagas’ disease is an infection that is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas’ disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K₃. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K₃ and vitamin C, alone and in combination, against T. cruzi. The vitamin K₃ + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K₃ + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death.     

Trypanosoma cruzi: Role of macrophage membrane components in the phagocytosis of bloodstream forms

The phagocytosis of Trypanosoma cruzi bloodstream forms is mediated by macrophage Pronase-sensitive membrane components. Trypsin and chymotrypsin treatment of macrophages, which prevents the uptake of T. cruzi culture forms, does not inhibit the phagocytosis of bloodstream parasites. The phagocytosis activity of the macrophages was recovered within 6–8 hr after the removal of Pronase. Inhibition of protein synthesis after Pronase treatment prevents the recovery of the endocytic activity of the macrophages. Fc and C3b receptors are not apparently essential for the phagocytosis of T. cruzi bloodstream forms. The described membrane components may help to explain the tropism of some T. cruzi strains for cells of the mononuclear phagocytic system in the living host.     

Determination of chemical structure and anti-Trypanosoma cruzi activity of extracts from the roots of Lonchocarpus cultratus (Vell.) A.M.G. Azevedo & H.C. Lima

Trypanosoma cruzi is the agent of Chagas disease, an infection that affects around 8 million people worldwide. The search for new anti-T. cruzi drugs are relevant, mainly because the treatment of this disease is limited to two drugs. The objective of this study was to investigate the trypanocidal and cytotoxic activity and elucidate the chemical profile of extracts from the roots of the Lonchocarpus cultratus. Roots from L. cultratus were submitted to successive extractions with hexane, dichloromethane, and methanol, resulting in LCH, LCD, and LCM extracts, respectively. Characterization of extracts was done using ¹H-RMN, ¹³C-RMN, CC and TLC. Treatment of T. cruzi forms (epimastigotes, trypomastigotes, and amastigotes) with crescent concentrations of LCH, LCD, and LCM was done for 72, 48, and 48 h, respectively. After this, the percentage of inhibition and IC₅₀/LC₅₀ were calculated. Benznidazole was used as a positive control. Murine macrophages were treated with different concentrations of both extracts for 48 h, and after, the cellular viability was determined by the MTT method and CC₅₀ was calculated. The chalcones derricin and lonchocarpine were identified in the hexane extract, and for the first time in the genus Lonchocarpus, the presence of a dihydrolonchocarpine derivative was observed. Other chalcones such as isocordoin and erioschalcone B were detected in the dichloromethane extract. The dichloromethane extract showed higher activity against all tested forms of T. cruzi than the other two extracts, with IC₅₀ values of 10.98, 2.42, and 0.83 µg/mL, respectively; these values are very close to those of benznidazole. Although the dichloromethane extract presented a cytotoxic effect against mammalian cells, it showed selectivity against amastigotes. The methanolic extract showed the lowest anti-T. cruzi activity but was non-toxic to peritoneal murine macrophages. Thus, the genus Lonchocarpus had demonstrated in the past action against epimastigotes forms of T. cruzi but is the first time that the activity against infective forms is showed, which leading to further studies with in vivo tests.     

Beneficial effects of acetylsalicylic acid (aspirin) on the actions of extracellular vesicles shed by Trypanosoma cruzi in macrophages

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease, shed extracellular vesicles (EVs) that promote the susceptibility of host cells to infection. During T. cruzi infection, the immune response of the host is important for controlling parasitism, which is necessary for survival. Macrophages produce inflammatory mediators, such as eicosanoids and nitric oxide (NO), with trypanocidal effects that control the parasite load in the early stages of the disease. In this study, we evaluated the contribution of host cyclooxygenase (COX) to the actions of EVs shed by T. cruzi strain Y (EVs-Y) in infected macrophages. RAW 264.7 macrophages exposed to EVs-Y and then infected with trypomastigote forms of T. cruzi produced less NO, and an increased number of trypomastigote forms were internalized in the cell compared to the controls, indicating that the effects exerted by EVs-Y favor the parasite. Interestingly, when macrophages were pretreated with acetylsalicylic acid, a dual COX inhibitor, before exposure to EVs-Y and subsequent infection with trypomastigote forms, there was an increase in NO production and a decrease in trypomastigote uptake compared to the controls. These results suggest that EVs-Y modulates the macrophage response in favor of T. cruzi and indicate a role for COX in the effects of EVs.     

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 Δ¹-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ¹-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 (K_m, k_cat, and k_cat/K_m) 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.     

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

Background

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ Principal Findings

Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/Significance

We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.     

The characterization of anti-<Emphasis Type="Italic">T. cruzi</Emphasis> activity relationships between ferrocenyl,cyrhetrenyl complexes and ROS release

Trypanosoma cruzi (T. cruzi) is the parasite that causes Chagas disease. Nifurtimox is the most used drug against the T. cruzi, this drug increases intermediaries nitro group, being mainly responsible for the high toxicity component, for this reason it is important to study new organic compounds and thus improve therapeutic strategies against Chagas disease. The electronic effects of ferrocenyl and cyrhetrenyl fragments were investigated by DFT calculation. A close correlation was found between HOMO–LUMO gap of nitro radical NO ₂ ^? with the experimental reduction potential found for nitro group and IC₅₀ of two forms the T. cruzi (epimastigote and trypomastigote). The IC₅₀ on human hepatoma cells is higher for both compounds compared to IC₅₀ demonstrated in the two forms the T. cruzi, and additionally show reactive oxygen species release. The information obtained in this paper could generate two new drugs with anti-T. cruzi activity, but additional studies are needed.     

Stage-Regulated GFP Expression in Trypanosoma cruzi: Applications from Host-Parasite Interactions to Drug Screening

Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.     

Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages

Kierszenbaum F., Lima M. F. and Wirth J. J. 1985. Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages. International Journal for Parasitology15: 409–413. The contribution of phagocytic function to host defense against infection with metacyclic forms of Trypanosoma cruzi isolated from insect vectors was investigated in mice passively transferred with anti-T. cruzi serum. The protective effect resulting from the passive transfers was significantly reduced by administration of either silica or cobra venom factor (CVF). A more pronounced curtailment of the protective effect was seen when both silica and CVF were administered to the mice. This effect was greater than that calculated by adding the effects produced by silica and CVF alone. In in vitro experiments, presence of anti-T. cruzi antibodies enhanced the capacity of mouse macrophages to take up the metacyclic organisms and increased the proportion of macrophages associating with the parasites. Increased macrophage-parasite association was also seen when either the flagellates or the macrophages were preincubated with the antiserum. Antibody-treated metacyclic forms of T. cruzi were more rapidly cleared by untreated macrophages than parasites pretreated with normal mouse serum. These results support a role for macrophages in host defense against the form of T. cruzi responsible for natural infections and emphasize the role played by anti-T. cruzi antibodies. The combined effect of the silica and CVF treatments suggests that C activity may contribute to the protective action of antibodies through its opsonic properties, though a concomitant role for C-dependent immune lysis cannot be ruled out. These results highlight the protective role of antibodymediated mechanisms against infection with the form of T. cruzi responsible for natural infections.     

Genome-Wide Screening and Identification of New Trypanosoma cruzi Antigens with Potential Application for Chronic Chagas Disease Diagnosis

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.     

Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

Background

Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels.

Results

Different IEF conditions using 6–11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages.

Conclusion

High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.     

Lack of protection against Trypanosoma cruzi by multiple doses of T. lewisi culture forms. A discussion on some strains of "lewisi"

No protection against Trypanosoma cruzi was afforded to mice by previously inoculating multiple doses of T. lewisi blood stream forms, strain RU or culture forms of the IMT strain.Since these results conflicted with those obtained by other authors, we secured a sample of the strain used by them. This strain, labeled “T. lewisi” and which we called “F”, was verified to be actually T. cruzi. It was characterized by its morphologic features in axenic cultures, its capability to infect HeLa cells where the intracellular cycle was completed, by its infectivity for white mice which had amastigotes in their heart muscle, and in which the strain has been maintained by serial passages, and by the completion of its biologic cycle in triatoma bugs.The necessity for carefully testing doubtful strains, and the criteria to be adopted in these cases are discussed.     

Post-translational processing, metabolic stability and catalytic efficiency of oat arginine decarboxylase expressed in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)₆-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)₆-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.     

Trypanosoma cruzi: Role of δ-Amastin on Extracellular Amastigote Cell Invasion and Differentiation

Trypanosoma cruzi is a protozoan parasite that comprises different phylogenetic groups and is the causative agent of Chagas’ disease. Different T. cruzi strains present differences in infectivity in in vitro and in vivo experimental models, which are likely related to the expression of different virulence factors. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the parasite. In this study, we showed that a highly infective strain (G strain) of extracellular amastigote (EA) invasive forms expressed reduced RNA levels of amastin compared to a less infective strain (CL). The treatment of HeLa cells with recombinant δ-amastin reduced infectivity of EA forms. However, the ectopic expression of δ-amastin accelerated amastigote differentiation into trypomastigotes. Corroborating the virulence behavior in association with amastin expression, the EAs overexpressing amastin were precociously and robustly observed in the liver of susceptible mouse strains (A/JUnib), whereas parasitemia was never detected in in vivo assays. This is the first report on the regulatory role of amastin in the course of both in vitro and in vivo T. cruzi infection.     

Trypanosoma cruzi: Antigenic invariance of the cell surface glycoprotein

The cell surface antigens of Trypanosoma cruzi have been studied for evidence of antigenic variation. The majority of the cell surface antigens found on epimastigotes were also present on trypomastigote and amastigote forms. Serum absorption studies and peptide mapping of the major cell surface glycoprotein from a series of clones and strains of Trypanosoma cruzi failed to find evidence of antigenic variation. Differences found between geographically distinct strains of Trypanosoma cruzi were minor and not associated with the major glycoprotein. Components present in normal mouse serum were capable of binding to the surface of Trypanosoma cruzi and these components could interfere in subsequent radioimmune assays, particularly with bloodstream derived trypomastigotes.     

Differentiation and Multiplication of Dyskinetoplastic Trypanosoma cruzi in Tissue Culture and in the Mammalian Host*

SYNOPSIS. In cultures of the Y strain of Trypanosoma cruzi treated with acriflavine, a very high proportion of the crithidiae may be dyskinetoplastic. These crithidiae cannot be maintained in subcultures but are able to differentiate into (dyskinetoplastic) metatrypanosomes. In tissue cultures infected with these metatrypanosomes, or with blood trypanosomes and treated with acriflavine, dyskinetoplastic T. cruzi is able to go thru the whole sequence of stages that characterizes its cycle in the vertebrate host: penetration by trypanosomes into cells, differentiation into leishmaniae, multiplication in this phase and differentiation again into trypanosomes. The same may occur in the mammalian host itself. The physiological implications of these findings are discussed and special attention is called to polymorphism of blood forms of T. cruzi, which probably has the same significance it has in the brucei-evansi group of trypanosomes.