A New Liquid Medium for Trypanosoma (Schizotrypanum) cruzi*

A new liquid medium, with fetal calf serum as the sole undefined component, was devised for the cultivation of Trypanosoma cruzi. The need for the serum is ascribed to its mitogenic proteins, which stimulate division of, and the uptake of [³H]thymidine by the parasites. In the new medium, T. cruzi has a cycle culminating in the appearance of up to 90% metacyclic forms in the stationary phase. This cycle is repeated on each serial transfer.     

Genomic DNA sequence polymorphism of Trypanosoma (Schizotrypanum) cruzi isolates with phenotypic variation

1. Single-copy DNA sequences were analyzed by molecular hybridization to determine both the extent of complementarity and time of evolutionary divergence between three isolates obtained from Argentina, Mexico or Venezuela. 2. The Mexico-Venezuela pair appeared much closer than the Mexico-Argentina pair. 3. The tentative values for time of evolutionary divergence were higher than those estimated previously by allozymic variation.     

Trypanosoma (Schizotrypanum) cruzi: repetitive DNA sequence evolution in three geographically distinct isolates

1. Middle-repetitive DNA sequences were analyzed by molecular hybridization to determine both the extent of complementarity and time of evolutionary divergence between isolates of Trypanosoma cruzi from Argentina, Mexico, or Venezuela. 2. Although molecular hybridizations showed no significant difference between the middle-repetitive DNAs of the Mexican and Venezuelan isolates, there was a 2.7% base pair mismatch in hybrid molecules formed by association of strands from both the Mexican and Argentine isolates. 3. Using the rates for divergence of middle-repetitive DNA in sea urchins, the Mexican and Argentine isolates were estimated to have diverged approximately 20-25 million years ago. 4. Analysis of the kinetics for the DNA hybridizations indicates that only minor amplifications of specific gene sequences or changes in the complexity of the genomes could have occurred during the divergence of the three isolates studied.     

The Fine Structure of the Flagellum and Kinetoplast-Chondriome of Trypanosoma (Schizotrypanum) cruzi in Tissue Culture*

SYNOPSIS. Trypanosoma cruzi in tissue cultures was studied with the electron microscope after double fixation in glutaraldehyde and osmium tetroxide, and embedding in Epon. Previous findings on its fine structure were confirmed, and some new structures were found in the flagellum and kinetoplast-chondriome. In the flagellum, an intraflagellar body was found, similar to that observed in other trypanosomes, beginning at the base of the flagellum and running along the axial fibre bundle thruout its length. The axial fibre bundle is formed by interconnecting tubules, the outer ones apparently smooth, the inner ones with a helical substructure. Lateral extensions from the outer tubules in the flagellar bundle seem to enter the intraflagellar body. The kinetopiast in the leishmania bodies has the same electrondense structure described before. In the trypanosome form it has assumed a large spherical shape, in which the formerly short, compressed fibres have grown in length, are more dispersed and have an irregular shape. They are oriented in the direction of the body's length in parallel array. The whole formation is continuous with a long mitochondrion which begins in the region of the nucleus and extends up almost to the tip of the trypanosome. The matrix of the kinetoplast in these forms is electron-transparent; the matrix of the mitochondria is rather dense. In a few extracellular trypanosomes, a special structure was found in which the kinetoplast is composed of electron-transparent formations, arranged in orderly horizontal lines quite similar to the mitochondrial cristae of the parasite. The significance of this structure is uncertain.     

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp. nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World.     

Enzyme variation among clones of Trypanosoma cruzi

The genetic relationships within and between stocks of Trypanosoma cruzi were studied by the in vitro isolation of clones and sub-clones and by the comparison of their isoenzyme patterns in thin-layer starch-gel electrophoresis. Altogether 13 clones and 36 sub-clones were isolated from stocks CL89 and Y207 of T. cruzi. Employing the enzymes L-alanine aminotransferase (ALAT), L-aspartate aminotransferase (ASAT), glucose phosphate isomerase (GPI), malic enzyme (ME), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoglucomutase (PGM), two zymodemes, B and C, emerged among the clones with distinct banding patterns. These zymodemes were consistently distinguished by ALAT, ASAT, GPI, G6PD, 6PGD, and PGM and the differences in enzyme profiles were simultaneously reflected in all six enzyme systems. That the enzymic characters as genetic determinants are stable was demonstrated after recloning and successive replica-platings, i.e., the distinct enzyme patterns remained consistent and homogeneous, the siblings retained the enzyme profiles of their parental clones, and no segregation of the enzyme patterns was observed. Our data from clone and sub-clone examinations show that the isoenzymes act as labels to characterize T. cruzi stocks. Furthermore, enzyme variation was demonstrated among clones isolated from stock CL89.     

Purification of tissue forms (Amastigotes) of Trypanosoma cruzi by immunoaffinity chromatography

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Sepharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Effect of Fetal Calf Serum Fractions and Proteins on Division and Transformation of Trypanosoma (Schizotrypanum) cruzi in Vitro*

SYNOPSIS. Division and epimastigote-to-trypomastigote transformation of Trypanosoma cruzi were observed in O'Daly's SM medium supplemented, in place of whole fetal calf serum, with fractions of this serum, its partially purified proteins, or with mixtures of these fractions and proteins. In addition to their division-promoting effects, most but not all serum fractions stimulated [³H]thymidine uptake by the flagellates. As revealed by TEAE-cellulose column chromatography and immunoelectrophoresis, the serum fractions were altered during the logarithmic growth phase of the trypanosomes.     

Crossed-immunoelectrophoretic analyses of Trypanosoma cruzi epimastigote, metacyclic, and bloodstream forms

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.     

Glycolipid components of epimastigote forms of Trypanosoma cruzi

Glycosphingolipids were isolated from a lipid extract of epimastigote forms of Trypanosoma cruzi via Florisil and silicic acid column chromatography. The carbohydrate components of neutral glycolipid consisted of mannose and galactose in a ratio of 1:2. The fatty acids of the glycolipid were analyzed by gas liquid chromatography-mass spectrometry (g.l.c.-m.s.). Normal and 2-hydroxy fatty acids were found. The sphingosine bases were C18 dihydrosphingosine and 17-methyl sphingosine.     

Lipid composition of three morphological stages of Trypanosoma cruzi

The neutral and phospholipid content at each of the three morphological stages of the parasite, Trypanosoma cruzi, was analyzed by thin-layer chromatography. Total lipid fatty acid composition at each stage was analyzed by gas-liquid chromatography and the results were compared. Change in lipid composition at each stage was observed.     

Acetate oxidation by bloodstream forms of Trypanosoma cruzi.

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was a sensitive to antimycin A inhibition as was CO2 production. There was a 6--7% residual O2 uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.     

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.     

Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi

Acid phosphatase activity in Trypanosoma cruzi was found to be located on the external surface of the plasma membranes. Both specific activity and activity per cell of this bound enzyme were significantly higher in the cells of amastigote (an intracellular form) than that of trypomastigote (a bloodstream form) and epimastigote (culture form). During the transformation of epimastigotes to amastigotes in vitro the activity of surface acid phosphatase was elevated concomitant with the increase in population of amastigotes. These results were interpreted as that the elevated enzyme activity is required for the intracellular parasitization of this organism or is a consequence of the morphological transformation.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.