Trypanosoma cruzi: quantification and analysis of the infectivity of cloned stocks

The infection of bovine embryo skin and muscle cells by trypomastigotes of four Trypanosoma cruzi clones (CA-I/71, /72, Miranda/76, /80) was quantified. Stable and reproducible intra-isolate differences were observed; an almost 70-fold difference in infectivity occurred between clones. The CA-I/71 clone was not susceptible to N-acetyl-D-glucosamine at a concentration that inhibits the infection of vertebrate cells by Ernestina and Y-strain parasites. Eight other monosaccharides that are common constituents of vertebrate cell surface glycoproteins also failed to inhibit the infection of vertebrate cells by the CA-I/71 clone.     

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.     

Trypanosoma cruzi: isolation and characterization of membrane and flagellar fractions.

A cell fractionation procedure for obtaining membrane and flagellar fractions was developed using Trypanosoma cruzi epimastigote forms. The cells, swollen in an hypotonic medium, were disrupted in the presence of a nonionic detergent, and fractions were isolated by differential centrifugation. The flagellar fraction, pelleted in 10 min at 10,000g, was further purified on a sucrose gradient. The membrane fraction was obtained by centrifugation of the supernatant at 27,000g for 30 min. Electron microscopy of the isolated fractions demonstrated a high degree of purity of each fraction. The membrane fraction showed homogeneous vesicles with low ribosome content. In frozen-etched preparations, the distribution of intramembranous particles on the vesicles was similar to that of the plasma membrane of intact cells. Enzymatic assays indicated that the membrane and flagellar fractions had low contamination with mitochondria and lysosomes. 5′-Nucleotidase activity was not detected in the membrane fraction; Mg²⁺-dependent ATPase activity was slightly enhanced, although, the enzyme was not sensitive to Na⁺, K⁺, and Ca²⁺ ions. The membrane fraction showed about five times the adenylyl cyclase activity of the whole homogenate. Gel immunodiffusion revealed the whole antigen of T. cruzi extracted by formamide to be identical to the membrane fraction when both were tested against rabbit anti- T. cruzi (epimastigote) immune serum.     

Trypanosoma cruzi: isolation and characterization of two clones derived from neonatal mice

The Tulahuen strain of Trypanosoma cruzi was cloned in 15 C3H/Anf neonatal mice. Ten of these 15 neonates became parasitemic before the 12th day and died before the 19th day after the inoculation of a single bloodstream trypomastigote. Two clones were selected and maintained, while the other isolates which did not grow in a liquid metacyclic stage culture (LMC) medium were eventually discarded. The kinetics of in vitro growth and transformation from epimastigote to metacyclic trypomastigote of these two clones were characterized in LMC medium at 27 degrees C. Infectivities for vertebrate cells in vitro were retained by these two clones during the period of cultivation. The tropism for brain, heart, lungs, esophagus, stomach, large intestine, liver, pancreas, spleen, lymph nodes, kidneys, bladder, and skeletal muscles was also examined in the mice. The communication describes the establishment and characterization of T. cruzi clones. The utilization of these cloned parasites should produce some advantages in generating reproducible data in investigations.     

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.     

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.     

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.     

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.     

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.     

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.     

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.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

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.