Trypanosoma cruzi: use of herpes simplex virus-thymidine kinase as a negative selectable marker

Trypanosoma cruzi, the protozoan that causes Chagas' disease, was transfected with a fusion gene of hygromycin phosphotransferase and herpes simplex virus-thymidine kinase, HyTK. Transfectants selected in hygromycin had thymidine kinase activity, whereas controls did not. In vitro growth of the mammalian life-stage forms, amastigotes and trypomastigotes, was inhibited 98% by the nucleoside analogue ganciclovir (5 micrograms/ml). Growth of the insect-stage form, epimastigotes, was not inhibited by ganciclovir (up to 250 micrograms/ml) or other nucleoside analogues. Intracellular uptake of ganciclovir by epimastigotes was found to be 10-fold less than that by amastigotes. Mice infected with the HyTK-expressing parasites and treated with ganciclovir had a statistically significant reduction of parasitemia by 57%; however, complete eradication of parasites was not achieved. The parasites recovered from the treated mice continued to be susceptible to ganciclovir in vitro. Parasite clones with higher expression of thymidine kinase were more sensitive to ganciclovir, suggesting that greater expression of the thymidine kinase gene may lead to parasites that can be fully eradicated from infected experimental animals.     

Trypanosoma cruzi: maintenance in culture modify gene and antigenic expression of metacyclic trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi.     

Superantigens but not mitogens are capable of inducing upregulation of E-selectin ligands on human T lymphocytes

A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79% and an 85.36% identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi.     

Neolignans inhibit Trypanosoma cruzi infection of its triatomine insect vector, Rhodnius prolixus

Two neolignans, burchellin and nordihydroguaiaretic acid (NDGA), were toxic only to Trypanosoma cruzi clone Dm28c maintained in brain heart infusion (BHI) medium at a concentration of 100 microg/ml, not 10 microg/ml. When Rhodnius prolixus was fed with epimastigotes of T. cruzi and treated simultaneously with a single dose of burchellin or NDGA at 10 pg/ml of blood meal the number of parasites in the gut decreased. Whereas burchellin was only partially active, NDGA drastically reduced the number of epimastigotes and metacyclic trypomastigotes of T. cruzi in the excreta (urine plus feces). When the insect larvae were pretreated with burchellin or NDGA at 20 days before the infection with T. cruzi a significant reduction in the number of parasites in the gut occurred. However, when both compounds were applied at 20 days after the establishment of T. cruzi infection, although burchellin significantly reduced the gut infection, neither compound could abolish the infection entirely within the subsequent 15 days.     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiopen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reducatase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 microM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 degrees C against trypomastigotes in murine blood as well against the enzyme trypanothione reducatase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.     

Determination of DNA damage in F344 rats induced by geometric isomers of tamoxifen and analogues

The transformation of Trypanosoma cruzi epimastigotes to mammal-infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions (TAU 3AAG medium). During this process, changes in the nature of cell surface sugar composition and sugar distribution was evaluated using FITC and gold-labeled lectins and observed by flow cytometry and transmission electron microscopy. The pattern of labeling with the lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Limax flavus (LFA), Canavalia ensiformis (Con-A), and Ricinus communis (RCA-I) significantly changed during the metacyclogenic process. The results obtained are discussed in relation to the role played by T. cruzi cell surface carbohydrate residues on the process of parasite-host cell interaction.     

Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristics J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated resides characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal outbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B-lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., 3H-thymidine incorporation) and cell membrane level of interleukin-2 receptors (IL-2R; determined by flow cytometry) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

Biological characterization of Trypanosoma cruzi stocks from Chilean insect vectors

Fifty-seven Trypanosoma cruzi stocks isolated from Triatoma infestans and Triatoma spinolai of the five different geographic endemic areas of Chile were studied by schizodeme and molecular karyotype analysis. Four different genotypes are found in the sylvatic T. spinolai vector and five in the T. infestans domiciliary vector. Of these genotypes, two common genotypes overlap on both transmission cycles exclusively in the extreme northern endemic areas of Chile. Metacyclic trypomastigotes obtained in vitro or cell-derived trypomastigotes proved to be infective in gamma-irradiated Balb/c mice for the study of the immune response and biological behavior. Of a total of 57 T. cruzi stocks obtained, 19 of them, representing all the different genotypes found in Chile, were tested on a murine experimental model and then fully studied. Female compared with male animals demonstrated greater resistance to Chagas disease with all the T. cruzi stocks tested. The immune response was assessed by lytic antibodies that were studied by the in vitro antibody-dependent complement-mediated lytic assay with the use of bloodstream trypomastigotes as target cells. In one unique parasite genotype the elicited lytic antibodies reacted in a genotype-specific manner, in contrast with lytic antibodies generated by other T. cruzi genotypes. Parasitemias were high, moderate, and low, with mortality ranges of 6-50%, 0-45%, and 0-10%, respectively. No association was found between specific infective genotypes and virulence or mortality. Independently of the T. cruzi strain studied, each population displayed a characteristic parasitemia curve and prepatent period. A considerable number of the parasite stocks proved to be mixed populations, according to molecular karyotype patterns obtained before and after differentiation and amplification of the parasites. This fact created difficulty in assessing the identity of the genotype really infective to mice.     

Proteasome activity is required for the stage-specific transformation of a protozoan parasite

A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan.     

One fate of epimastigote forms of Trypanosoma cruzi injected in mice. An ultrastructural study

Recently, we suggested that epimastigote forms of Trypanosoma cruzi are cleared from circulation of mice by a mechanism independent of lysis and that platelets play an important role in this process. These observations prompted us to look at the fate of epimastigotes in the lung, liver, and spleen of mice injected intravenously with these parasite forms. Using transmission electron microscopy, we observed clumps of epimastigotes and platelets in direct contact with phagocytes in the lumen of capillaries. However, the platelets and parasites were probably separated before phagocytosis because only parasites were found inside the phagocytes. Indeed, most of the phagocytes, although containing epimastigotes in different stages of disintegration, contained no platelets. The removal of parasites from platelets was probably mediated by phagocytes through a mechanism similar to the removal of bacteria from the surface of erythrocytes in humans. These observations suggest that the nonvirulence of T. cruzi epimastigotes in mice is not due to lysis but probably to the inability of these parasite forms to escape destruction by the phagocytes.     

Echocardographic features of papillary fibroelastoma and their consequences and management

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28 degrees C is 58 +/- 13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace's medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37 degrees C; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24S alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.     

Trans-sialidase and sialidase activities discriminate between morphologically indistinguishable trypanosomatids

The expression of trans-sialidase and sialidase activities in the kinetoplastid protozoa was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from human, insects and vertebrate reservoir hosts. By virtue of the differences observed in the ratios of these enzyme activities, a collection of 52 species and strains comprising the major taxa of these parasites could be separated into four expression types. Type-I parasites express comparable levels of both trans-sialidase and sialidase activities (Endotrypanum species and Trypanosoma lewisi). Type-II parasites express predominantly trans-sialidase activity (Trypanosoma cruzi and Trypanosoma conorhini). Type-III parasites express sialidase activity exclusively (Trypanosoma rangeli and Trypanosoma leeuwenhoeki). Type-IV parasites do not express either activity (Leishmania species and Trypanoplasma borreli). The measurement of trans-sialidase and sialidase activities thus permits the differentiation of parasites frequently found in the same insect vectors that are difficult to distinguish, such as T. cruzi and T. rangeli, or in the same sylvatic vertebrate and invertebrate hosts, such as Leishmania and Endotrypanum.     

Trypanosoma cruzi: effect of immunization on the risk of vector-delivered infection in guinea pigs

The protective effect of experimental immunization was studied in guinea pigs exposed to vectorial infection by Trypanosoma cruzi. Immunized animals received an inoculum of live-attenuated T. cruzi epimastigotes into a granuloma previously induced by Freund's complete adjuvant in the hind footpad. Seven days later, a delayed-type hypersensitivity reaction was triggered by reinjection of the parasites in the front footpad. The animals were then placed in Triatoma infestans-colonized corrals and exposed to vectorial T. cruzi transmission of the parasite for up to 200 days. The effectiveness of this immunizing protocol was controlled in terms of the number of bites necessary for infection (NBNI) in immunized as compared with control animals. Periodic entomological census allowed for the determination of vector biting and infection rates and the calculation of NBNI. Although this measurement was quite variable between yards, an overall average of 4,973 bites was enough to infect a control guinea pig in 4 separate experiments. The corresponding figure for the experimental group was 21,307 bites, implying that immunized animals could resist a 4.28-fold increase (range: 1.99-8.32) in the number of vector bites before becoming infected.     

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.     

Induction of resistance to azole drugs in Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133-138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.     

Overexpression of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, is associated with enhanced metacyclogenesis

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected T. cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandemly repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteine proteinase inhibitor Cbz-Phe-Phe-CHN2 (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.     

In vitro cultivation of Trypanosoma acomys: production of insect stages and bloodstream forms

When Trypanosoma acomys bloodstream forms were cultivated at 37 degrees C in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum (FCS), with Microtus agrestis embryonic fibroblasts in RPMI 1640 medium supplemented with 20% FCS or in Baltz's medium supplemented with 10% FCS, the parasites transformed and largely remained as epimastigotes. Epimastigotes were also usually the commonest stage observed when the parasites were co-cultivated with a mosquito cell line at 27 degrees C. However, if these cultures were initiated with the supernatant suspensions from fibroblast cultures that had been cryopreserved, trypomastigotes, including bloodstream-like forms, were the predominant stage for the first 4 days of culture. It is suggested that the glycerol supplement or the temperature changes stimulated this unusual morphogenesis. At 27 degrees C, T. acomys was incapable of multiplying and died when cultured in fresh Schneider's Drosophila medium supplemented with 20% FCS, but co-cultivation with the mosquito cell lines or cultivation in cell-free supernatants from 1-week-old mosquito cell cultures was successful at this temperature; most of the parasites multiplied as epimastigotes.     

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.