Immunization of mice against infection with Trypanosoma cruzi. Cross-immunization between five strains of the parasite using freeze-thawed vaccines containing epimastigotes of up to five strains.

McHardy N. and Elphick J. P. 1978. Immunization of mice against infection with Trypanosoma cruzi. Cross-immunization between five strains of the parasite using freeze-thawed vaccines containing epimastigotes of up to five strains. International Journal for Parasitology8: 25–31. Groups of male CD-1 mice were immunized with two doses of vaccines containing 10⁸ freeze-thawed cultured epimastigotes of Trypanosoma cruzi of five strains—Y, M, BG, Peru and Tulahuen, with saponin as adjuvant. Each vaccine contained 1, 2, 3 or 5 strains of the parasite. The mice were challenged with each of the five strains. All the single-strain vaccines gave good protection against both homologous and heterologous challenges, with the exception of the strain Y vaccine, which gave poor protection against homologous challenge, but good protection against all four heterologous challenges. The inclusion of more than one strain of epimastigote in the vaccine failed to increase protection, and in some instances appeared to reduce it, in comparison with vaccines containing only one of the component strains. It is suggested that this is due to antigenic competition.     

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC₅₀ = 5 µM and 7.5 µM, respectively) and less than nifurtimox (IC₅₀ = 3.6 µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC₅₀ values of 160.64 and 160.66 µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.     

Humoral antibody response and Ig characterization of the specific agglutinins in rabbits during experimental American trypanosomiasis

A comparative follow up study of the specific agglutinins detected by direct agglutination (DA) test and the immune response detected by specific lysis (SL), indirect immunofluorescence (IFA), indirect hemagglutination (IHA) and complement fixation (CF) tests in rabbits inoculated with trypomastigotes of T. cruzi is reported here.The specific antibody response was detected first by DA test. Reductive cleavage of sera with 2-mercaptoethanol produced a drop in the agglutinin titer of the sera during the first 30 days of infection.The next test to become positive was SL and later on the IFA, IHA and CF tests became positive simultaneously.When fractions obtained by column chromatography in Sephadex G-200 were tested serologically it was demonstrated that specific antibodies were detected mainly in fraction I (IgM) of the pooled rabbit sera obtained 15 days after inoculation (acute stage), and in fraction II (IgG) of the pooled sera obtained from rabbits 90 days after inoculation (chronic stage).Antigens prepared with trypsinized and formolized epimastigotes of three T. cruzi strains, belonging to each one of the different immunological groups described, worked similarly in the detection of specific agglutinin antibodies.Trypanosoma cruzi agglutinins were highly specific in their reaction with their homologous T. cruzi antigens as was proved by the low agglutinin titer obtained in sera from infected rabbits when, instead of T. cruzi epimastigotes, promastigotes of L. donovani were used as antigen, and by the incapacity of this parasite to absorb the T. cruzi agglutinins.     

Proliferation and Differentiation of Trypanosoma cruzi inside Its Vector Have a New Trigger: Redox Status

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H₂O₂ and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.     

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.     

Cruzipain Activates Latent TGF-β from Host Cells during T. cruzi Invasion

Several studies indicate that the activity of cruzipain, the main lysosomal cysteine peptidase of Trypanosoma cruzi, contributes to parasite infectivity. In addition, the parasitic invasion process of mammalian host cells is described to be dependent on the activation of the host TGF-β signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-β and thereby trigger TGF-β-mediated events crucial for the development of Chagas disease. We found that live epimastigotes of T. cruzi, parasite lysates and purified cruzipain were able to activate latent TGF-β in vitro. This activation could be inhibited by the cysteine peptidase inhibitor Z-Phe-Ala-FMK. Moreover, transfected parasites overexpressing chagasin, a potent endogenous cruzipain inhibitor, prevented latent TGF-β activation. We also observed that T. cruzi invasion, as well as parasite intracellular growth, were inhibited by the administration of Z-Phe-Ala-FMK or anti-TGF-β neutralizing antibody to Vero cell cultures. We further demonstrated that addition of purified cruzipain enhanced the invasive activity of trypomastigotes and that this effect could be completely inhibited by addition of a neutralizing anti-TGF-β antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-β in the process of cell invasion are functionally linked. Our data suggest that cruzipain inhibition is an interesting chemotherapeutic approach for Chagas disease not only because of its trypanocidal activity, but also due to the inhibitory effect on TGF-β activation.     

The effect of the biflavonoid 2″,3″-dihydroochnaflavone on Trypanosoma cruzi Y strain

Trypanosoma cruzi is the causative agent of Chagas disease which affects 8 million people in Latin America. The parasite possesses high capacity to evade host immune system and the available drugs to treat Chagas disease present low efficacy combined to serious side effects to patients. Therefore, the identification of alternative therapeutics is essential. Brazilian flora exhibits an immense diversity of metabolites with great potential to be developed into new drugs. We investigated the action of 2″,3″-dihydroochnaflavone a biflavonoid extracted from Luxemburgia nobilis Eichler ex Engl. (Ochnaceae) against T. cruzi (Y strain). Our experiments showed that this compound is effective against parasite epimastigote forms, presenting IC₅₀ value of (2.5 ± 0.1) μM after 96 h of treatment. Ultrastructure alterations were also detected in treated epimastigotes especially mitochondrial enlargement at the kinetoplast region. At the concentration of 30 μM, the compound killed (61.6 ± 3.37)% of the parasite in its amastigote form. In addition, at the same concentration, the compound killed all trypamastigotes growing within murine macrophages after 7–9 days of infection. Nonetheless, the biflavonoid concentrations were harmless to murine enriched population of lymphocytes and peritoneal macrophages. These results indicate that 2″,3″- dihydroochnaflavone presents activity against T. cruzi.     

MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host.

Methodology/Principal Findings

In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption.

Conclusions/Significance

The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi.     

Synthesis and biological evaluation of isoxazolyl-sulfonamides: A non-cytotoxic scaffold active against Trypanosoma cruzi, Leishmania amazonensis and Herpes Simplex Virus

In this study we report the synthesis, characterization, biological evaluation, and druglikeness assessment of a series of 20 novel isoxazolyl-sulfonamides, obtained by a four-step synthetic route. The compounds had their activity against Trypanosoma cruzi, Leishmania amazonensis, Herpes Simplex Virus type 1 and cytotoxicity evaluated in phenotypic assays. All compounds have drug-like properties, showed low cytotoxicity and were promising regarding all other biological activities reported herein, especially the inhibitory activity against T. cruzi. The compounds 8 and 16 showed significant potency and selectivity against T. cruzi (GI₅₀?=?14.3?µM, SI?>?34.8 and GI₅₀?=?11.6?µM, SI?=?29.1, respectively). These values, close to the values of the reference drug benznidazole (GI₅₀?=?10.2?µM), suggest that compounds 8 and 16 represent promising candidates for further pre-clinical development targeting Chagas disease.     

Memantine,an Antagonist of the NMDA Glutamate Receptor,Affects Cell Proliferation,Differentiation and the Intracellular Cycle and Induces Apoptosis in Trypanosoma cruzi

Chagas'' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed) remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC₅₀ 172.6 µM). Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction) and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca²⁺ and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC₅₀ 31 µM). Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote) were more affected as were the processes of differentiation and cell invasion.     

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.     

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.     

3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile): A new highly active compound against epimastigote and trypomastigote form of Trypanosoma cruzi

We have synthesized the Morita-Baylis-Hillman adduct (MBHA) 3-hydroxy-2-methylene-3-(4-nitrophenyl)-propanenitrile (3) in quantitative yield and evaluated on Trypanosoma cruzi epimastigote and bloodstream trypomastigote forms. Compound 3 strongly inhibited epimastigote growth, with IC₅₀/72 h of 28.5 μM and also caused intense trypomastigotes lysis, with an IC₅₀/24 h of 25.5 μM. Ultrastructural analysis showed significant morphological changes on both parasite forms treated with 3, including increase of cell volume and rounding of cell body as well as intense intracellular disorganization. Morphological changes indicative of apoptosis, autophagy or necrosis were observed in most affected cells. Docking calculations of 1, 2 and 3 pointed out the possibility of T. cruzi Farnesyl Pyrophosphate Synthase (TcFPPS) enzyme inhibition in 3 mechanism of action.     

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.     

Antibody-dependent cytotoxicity of human and mouse mononuclear cells against Trypanosoma cruzi epimastigotes

Human peripheral mononuclear cells were cytotoxic to antibody-sensitized Trypanosoma cruzi epimastigotes. The cytotoxic effect depended on the concentration of effector cells and antiserum, and was progressive until 17 hr of incubation at 28 °C. After 3 hr of incubation the highest specific activity was achieved at a 50:1 effector to target cell ratio. A nonspecific cytotoxic effect in the absence of antiserum was observed at a 100:1 parasite to cell ratio or after 17 hr of incubation. When the human mononuclear cell population was depleted of adherent cells by Sephadex G-10 filtration or adsorption to glass, the cytotoxic effect was greatly reduced. Similar results were obtained using mouse spleen cells, indicating that only the adherent cells were cytotoxic to sensitized T. cruzi in both systems. When human mononuclear cells were incubated with amobarbital, cyanide, azide, or aminotriazole, an inhibition of cytotoxicity against sensitized T. cruzi was observed, suggesting that oxygen reduction products and myeloperoxidase were involved in the destruction of sensitized T. cruzi epimastigotes by normal human mononuclear cells.     

Involvement of STI1 protein in the differentiation process of Trypanosoma cruzi

The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.     

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell‐derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long‐term cultured epimastigotes: resistance to complement‐mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up‐regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host‐parasite interactions, showing that rdEpi can be infective to the mammalian host.     

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure–activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a–h and 6a–h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC₅₀ of 9.5 ± 2.8 and 3.5 ± 1.8 μM for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC₅₀ of 11.3 ± 2.8 μM. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50 mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection.     

Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

Background

Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.

Methodology/Principal Findings

In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.

Conclusions/Significance

Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.     

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.