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.     

Schizodeme analyses of Trypanosoma cruzi zymodemes from Chile

Kinetoplast DNA was isolated from Chilean Trypanosoma cruzi populations and digested with the restriction endonucleases EcoRI, HinfI, HpaII, MspI, and HaeIII. Three major schizodeme groups were discriminated. There was a correlation between the Chilean schizodeme groups (S1, S2, or S3) and the zymodemes known to occur in Chile (Z1, Brazilian Z2 and Bolivian Z2, respectively), although heterogeneity was seen within the schizodeme groups S2 and S3. Standard Brazilian and Bolivian T. cruzi clones (X10 clone 1, Esmeraldo clone 3, SC43 clone 1, and CAN III clone 1) and laboratory strains (Tulahuen and Y) were included in the schizodeme comparisons. SC43 clone 1 had obvious affinities with S3 and X10 clone 1 shared some features with S1 but the other reference stocks could not be definitely assigned to S1, S2, or S3. Fragment patterns and densitometric traces following digestion with HpaII or MspI suggested that kDNA sequences were not methylated.     

Tissue tropism of different Trypanosoma cruzi strains.

A systematic study of the distribution of intracellular parasites in the organs and tissues was performed in groups of mice inoculated with 4 different Trypanosoma cruzi strains. An extremely high parasitism of spleen, liver, and bone marrow was observed in mice inoculated with Y and Berenice strains; with CL strain, however, parasites were almost absent in those organs. Bloodstream forms apparently present differences which facilitate or prevent their uptake by macrophages from the mononuclear phagocytic system. Parasitism of the smooth muscle from hollow organs was significantly higher with ABC and Berenice strains than with Y and CL. The importance of the distribution of intracellular stages in the pathogenesis of the disease is discussed.     

Delimitation of Trypanosoma cruzi zymodemes by numerical taxonomy

Summary Based on the absence or presence of 24 isoenzyme characters, Brazilian Trypanosoma cruzi stocks can be separated by methods of numerical taxonomy into three principal, distinct groups; these groups correspond to the three T. cruzi zymodemes previously reported from north Brazil. A distinction is drawn between taxonomy, which may be of zoological interest only, and identification of medically important groups according to one or a few distinguishing characters.This paper is reprinted from Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 238–242.This paper is reprinted from Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 238–242.     

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.     

Trypanosoma cruzi: studies on the interactions of lectins with glycoconjugates of different zymodemes

Detergent extracts were made of eight strains of Trypanosoma cruzi which were representative of the principal zymodemes. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the glycoproteins were reacted with 21 different 125I-labeled lectins and autoradiographed. The staining patterns with particular lectins varied considerably between strains. Concanavalin A stained up to 17 distinct bands in some strains. Other lectins such as peanut lectin only stained two bands in zymodeme 1 strains and none in the other zymodemes. The reaction of N-acetylgalactosamine-specific lectins with some bands indicated the presence of this sugar and this was confirmed by analysis of the extracts. The lectin staining patterns provided an insight into the glycoprotein composition of the bands and should indicate whether combinations of lectins can be used in affinity chromatography systems to purify the glycoproteins.     

Trypanosoma cruzi: characterization of in vitro and in vivo synthesized polypeptides from epimastigote forms of different strains

Intact RNAs were isolated from epimastigote forms of different Trypanosoma cruzi strains. Translation of the mRNAs using rabbit reticulocyte lysate and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed protein profiles comparable to those observed by labeling cells in vivo. No major interstrain differences were observed in the patterns of the polypeptides synthesized in vitro and in vivo, indicating that metabolic proteins are similar among distinct strains. Several T. cruzi polypeptides produced in the rabbit reticulocyte lysate system were immunoprecipitated by specific antisera. The patterns of antigenic polypeptides recognized by antisera raised against epimastigotes from different strains were also very similar.     

Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from South Carolina

Biological and molecular characteristics of a raccoon isolate of Trypanosoma cruzi (R36) were compared with those of a known virulent strain (Brazil). Included in the characterization were growth rate in liver infusion tryptose medium, infectivity for murine fibroblasts, intracellular amastigote replication and trypomastigote release rates, polymerase chain reaction (PCR) profiling of the mini-exon gene, isoenzyme and random amplified polymorphic DNA (RAPD) profiles, and in vivo virulence for C3H/HeJ mice. Similar growth curves were noted for both strains; however, infectivity and rates of intracellular amastigote replication and trypomastigote release were significantly lower for the R36 isolate than for the Brazil strain. To determine virulence, C3H/ HeJ mice were exposed intraperitoneally to the R36 isolate. No parasite was observed in blood by direct examination or in tissues by histology; however, T. cruzi was detected by PCR in tissues (quadriceps and spleen) at 21 days postinfection. Analyses of the mini-exon gene, isoenzyme, and RAPD profiles indicate that R36 is in the T. cruzi II group and the Brazil strain is in the T. cruzi I group. Although infectivity and virulence of the raccoon isolate were lower than those for the Brazil strain, autochthonous infections in the United States have been reported, which suggests the need for further study of local T. cruzi isolates.     

Comparative biological characterization of Berenice and Berenice-78 strains of Trypanosoma cruzi isolated from the same patient at different times

The Berenice-78 strain of T. cruzi is very different from the Berenice strain isolated 16 years earlier from the same patient. The authors verified its high infectivity and low virulence for C3H inbred mice that survived the acute phase of infection. In these animals, it was verified that the tropism of parasites was more accentuated for cardiac and skeletal musculature and the parasitaemic level progressively increased with successive blood passages with posterior stability. In relation to Berenice strain the same characteristics were observed as described by Brener, Chiari & Alvarenga (1974). The increase in its virulence for albino mice was again demonstrated. The authors discussed the possibility of reinfection of the patient called Berenice and the importance of knowledge about T. cruzi strains of low virulence for laboratory animals.     

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.     

Trypanosoma cruzi: protection of mice with epimastigote antigens from immunologically different parasite strains

Antigens, prepared by the aid of pressure, from epimastigotes of strains of T. cruzi belonging to the different immunological groups described, conferred equal protection in mice against lethal infections of T. cruzi trypomastigotes of the T strain, which belongs to one of those immunological groups. Humoral antibodies were detected by the direct agglutination and immune fluorescent tests in all the immunized groups. The B and T strains produced earlier antibody responses than G and L strains. The weakest antibody response was induced by antigens obtained from the L strain. All the immunized mice sacrificed 21 days after challenge infection showed prominent inflammatory reactions at the tissue level, as well as free amastigote-like bodies. Four months after challenge injection, myocardium, liver, and spleen appeared histologically normal when compared to uninfected control mice. However, histological alterations were detected usually in striated muscle. The latter tissue seemed to be the best to check residual infections.     

Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi

Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.     

Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.     

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.     

Biological characterization of a beta-galactosidase expressing clone of Trypanosoma cruzi CL strain

Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.     

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.