Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.     

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.     

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A ₆₀₀ 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k _cat of 1.7 s⁻¹ and limiting K _m values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively. Received: 3 September 1999 / Accepted: 1 November 1999     

Antigenic analysis of Trypanosoma cruzi strains by crossed immunoelectrophoresis: demonstration and isolation of antigens particular to some strains

Antigenic differences among soluble extracts of Y, CL, SF, and Colombian strain epimastigotes of Trypanosoma cruzi have been demonstrated by crossed immunoelectrophoresis with an intermediate gel containing the heterologous antiserum. Using crossed immunoelectrophoresis with the homologous antiserum, over 30 precipitin lines could be demonstrated for each strain and, even though marked differences were observed in experimental infections, the strains shared a significant number of antigens. In addition, some strain-particular antigens were isolated using affinity chromatography. These antigens could be valuable in the study of biological, immunological, and pathological characteristics of experimental and natural T. cruzi infections.     

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

Chimeric synthetic peptides as antigens for detection of antibodies to Trypanosoma cruzi

Six chimeric synthetic peptides (QCha-1, QCha-2, QCha-3, QCha-4, QCha-5, and QCha-6) incorporating antigenic sequences of two immunodominant repeat B-cell epitopes of Trypanosoma cruzi were synthesized by conventional solid-phase peptide synthesis. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of positive Chagasic sera (n=82), while specificity was evaluated with samples from healthy blood donors (n=44) and patients with other infectious diseases (n=86). The antigenicity of the chimeric peptides in solid-phase immunoassays was compared with that of the monomeric peptides. Data demonstrated that the chimeric peptide QCha-5 was the most reactive because it detected antibodies to parasite efficiently. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of Chagas' disease.     

Trypanosoma cruzi: shedding of surface antigens as membrane vesicles

Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CA1, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter.     

Guerreiro Machado reaction. Chromatographic analysis of Trypanosoma cruzi antigens

The aqueous extract of Trypanosoma cruzi, previously treated by benzene, was filtered through a column of Sephadex G-200. The eluted fractions were tested by complement-fixation with the chagasic reference serum. It was found that the CF antigens were eluted in the first fractions and the contaminants in the last ones. The aqueous antigen, when dried and extracted with methanol, showed a very high titer in the complement-fixation quantitative test. This antigen is free of contaminants and can be recommended for use in the serological test for the diagnosis of Chagas' disease.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop.

A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote

Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [³H]palmitic acid or [³H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using ³H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A₁, phospholipase A₂, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.     

Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin 1

Trypanosoma cruzi tryparedoxin 1 (TcTXN1) is an oxidoreductase belonging to the thioredoxin superfamily, which mediates electron transfer between trypanothione and peroxiredoxins. In trypanosomes TXNs, and not thioredoxins, constitute the oxido-reductases of peroxiredoxins. Since, to date, there is no information concerning TcTXN1 substrates in T. cruzi, the aim of this work was to characterize TcTXN1 in two aspects: expression throughout T. cruzi life cycle and subcellular localization; and the study of TcTXN1 interacting-proteins. We demonstrate that TcTXN1 is a cytosolic and constitutively expressed protein in T. cruzi. In order to start to unravel the redox interactome of T. cruzi we designed an active site mutant protein lacking the resolving cysteine, and validated the complex formation in vitro between the mutated TcTXN1 and a known partner, the cytosolic peroxiredoxin. Through the expression of this mutant protein in parasites with an additional 6xHis-tag, heterodisulfide complexes were isolated by affinity chromatography and identified by 2-DE/MS. This allowed us to identify fifteen TcTXN1 proteins which are involved in two main processes: oxidative metabolism and protein synthesis and degradation. Our approach led us to the discovery of several putatively TcTXN1-interacting proteins thereby contributing to our understanding of the redox interactome of T. cruzi.