On the Fine Structure of the Nucleus in Trypanosoma cruzi in Tissue Culture Forms. Spindle Fibers in the Dividing Nucleus*

The fine structure of the dividing nucleus in the intracellular amastigote forms of Trypanosoma cruzi from tissue cultures has been described. In the first phase of division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metozoan cells then appear, running from one pole to the other. They disappear when the division of the nucleus is complete and the chromatin and the nucleolar material reorganize into their former positions.     

Identification of Trypanosoma cruzi Zymodemes by Kinetoplast DNA Probes

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.     

Complete structure of the glycan of lipopeptidophosphoglycan from Trypanosoma cruzi Epimastigotes.

The lipopeptidophosphoglycan is the major cell surface glycoconjugate of the epimastigote forms of the parasitic protozoan Trypanosoma cruzi. A detailed partial structure for this molecule has been reported (Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M., and Mendonca-Previato, L. (1990) J. Biol. Chem. 265, 2518-2526). In this study, we complete the primary structure assignments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a combination of 1H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes.     

Antigenic Differences Among Epimastigotes,Amastigotes and Trypomastigotes of Trypanosoma cruzi*

Antigenic differences were demonstrated among trypomastigotes, amastigotes, and epimastigotes of Trypanosoma cruzi by the indirect fluorescent antibody method. Tests using cross-absorbed sera were included in the study.     

Incubation in Mice Provides a Signal for the Differentiation of Trypanosoma cruzi Epimastigotes to Trypomastigotes1

The differentiation of Trypanosoma cruzi epimastigotes into trypomastigotes was studied in diffusion chambers sub-cutaneously implanted in mice. Using epimastigotes of the Tulahuén strain, transformation was first evident at 16 h after implantation and reached its maximum (92% trypomastigotes) by 24 h. Shortly before their differentiation into trypomastigotes, epimastigotes were found to develop resistance to lysis by the alternative pathway of complement. Furthermore, implantation of stationary-phase (as opposed to log-phase) parasites resulted in the accumulation of large numbers of complement-resistant epimastigotes in the chambers. These observations suggest that epimastigotes pass through a complement-resistant transitional stage before differentiating into trypomastigotes and that transformation may require cell division. In a further series of experiments, epimastigotes recovered 7 h after implantation in mice were found to differentiate into trypomastigotes when cultured in vitro for an additional 17 h at 37°C. This observation indicates that the events which trigger the morphologic transformation of epimastigotes into trypomastigotes can be dissociated operationally from the differentiation process itself.     

Evidence for NADH- and NADPH-linked Glutamate Dehydrogenases in Trypanosoma cruzi Epimastigotes*

SYNOPSIS Two glutamate dehydrogenases, NADH-linked (EC 1.2.1.2) and NADPH-linked (EC 1.2.1.4.) were isolated from the epimastigote forms of Trypanosoma cruzi and purified. Both enzymes exist as hexamers. The molecular weights of the native NADH-and NADPH-linked glutamate dehydrogenases were estimated to be 360,000 and 265,000, respectively, and those of the subunits to be 58,000 and 43,000, respectively. The isoelectric point of the NADH-linked dehydrogenase is at pH 5.25 and that of the NADPH-linked enzyme at pH 5.1. The activities of both enzymes are regulated by product inhibition. In addition, purine nucleotides were shown to be potent inhibitors of the NADH-linked glutamate dehydrogenase.     

Fine Structure and Cytochemistry of the Hydrogenosome of Tritrichomonas foetus1

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca⁺⁺-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl₂ and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.     

Kinetoplast DNA in Trypanosoma equinum1

Identification and characterization of kDNA is described in the naturally occurring totally dyskinetoplastic species Trypanosoma equinum. Fluorescence microscopy of live cells, using the highly sensitive and specific probe DAPI (4,6,-diamidino-2-phenyl-indole), showed the presence of a diversity of extranuclear fluorescent bodies scattered along the length of the organism. Transmission electron microscopic studies revealed a close similarity between the distribution of these DAPI-fluorescing particles and of dense aggregates of nonfibrillar material resembling the kDNA of dyskinetoplastic strains of other species. Variable sized remnants of kDNA, occurring singly or in clusters, were found scattered throughout the mitochondrion. Analytical cesium chloride ultracentrifugation of total cellular DNA extracts showed a kDNA banding profile at a buoyant density equal to 1.691 gm/cm³, representing approximately 11% of the total cellular DNA content. Molecular spreads of isolated kDNA revealed a population of open circular molecules ranging in contour length from 0.11–9.69 μm.     

Changes in Trypanosoma cruzi Kinetoplast DNA Minicircles Induced by Environmental Conditions and Subcloning

ABSTRACT. Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood-free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.     

The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

Background

The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients.

Methodology/Principal Findings

We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test.

Conclusions/Significance

Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.     

Comparative Kinetics of Arginine and Lysine Transport by Epimastigotes and Trypomastigotes from Two Strains of Trypanosoma cruzi*

SYNOPSIS. Three-day-old cultures of Y and MR strains of Trypanosoma cruzi had a higher rate of lysine and arginine uptake than 10-day cultures. Amino acid uptake by cells of the MR strain was consistently higher than that of the Y strain. Flagellates separated on DEAE-cellulose columns have normal structure, motility, and infectivity; they have higher rates of lysine and arginine uptake than the original 3- and 10-day cultures. In addition, passage through DEAE-cellulose columns modified the kinetic behavior of amino acid transport systems in the flagellate membranes. Methionine inhibited uncompetitively uptake of lysine and arginine by MR and Y strains. Lysine inhibited arginine uptake by both strains by an uncompetitive mechanism. Lysine, however, inhibited the uptake of arginine by 10-day culture cells of the Y strain by a mixed-type of inhibition. Arginine also inhibited the lysine uptake of both strains by an uncompetitive mechanism. In all experiments, beyond a certain level, a further increase in inhibitor concentration resulted in a decreased inhibition, which eventually disappeared altogether. Inhibition of amino-acid uptake by any of the substances tested was not observed after passage of flagellates through a DEAE-cellulose column. A model for amino acid transport was formulated which includes a recognition site amenable to modulation by effectors.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.     

Fine Structure of Sarcocystis cruzi Schizonts

SYNOPSIS Schizogony of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) takes place in vascular endothelial cells 26 to 33 days after cattle ingest sporocysts from dogs. Kidney cortex from a heavily infected, dexamethasone-treated bovine was fixed for electron microscopy to determine the method of schizogonie development. Schizogony takes place by endopolygeny characterized by marked enlargement of the parasite nucleus, formation of nuclear lobes, presence of numerous spindles with adjacent pairs of centrioles along the nucleus, and simultaneous formation of daughter merozoites in the cytoplasm adjacent to the spindle poles. Endopolygeny in S. cruzi differs from that in other Sporozoa in that merozoite anlagen are seen in the cytoplasm before any nuclei divide. The resultant merozoites continue development and, when mature, resemble other sporozoan zoites. Upon release from the host cell into capillaries, they travel to muscle tissue to continue the life cycle by forming sarcocysts.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. I. Fine Structure and Cytochemistry*†

SYNOPSIS. An extracellular surface coat was observed at the fine-structural level on the outer lamina of the pellicular and flagellar membranes of intact Trypanosoma musculi bloodstream forms. The surface coat had a mean width of 9.2 nm, and was composed of a somewhat electron dense, uneven, fibrillar-like matrix. Brief trypsin treatment of living blood forms completely removed the cell surface coat. Several cytochemical methods applicable to electron microscopy were used to detect the presence and distribution of carbohydrates in the trypanosome's surface coat and pellicular membrane. The polycationic dye compounds employed were: ruthenium red, ruthenium violet, Alcian blue chloride, and lanthanum nitrate. Electron-dense stain reaction products, indicative of polysaccharides, were evident in the surface coat of cells treated with these dyes, which also agglutinated both living and glutaraldehyde fixed cells. Like the surface coat, the pellicular membrane of trypsinized cells gave strong positive staining reactions with the several dyes, indicating the presence of membrane bounded carbohydrates, and living and glutaraldehyde-fixed trypsinized cells were agglutinated with the polycationic stains. Bloodstream forms were treated with the enzymes, α-amylase, dextranase, and neuraminidase. No obvious morphologic difference, however, was apparent between the surface coat of untreated cells and those subjected to treatment with any of the various glycoside hydrolase enzymes. Further, these enzymes had no apparent gross effect on the staining affinity of the surface coat for the several polycationic dyes. Cationized ferritin was used to visualize the negative cell surface charge of T. musculi bloodstream forms. Large quantities of cationized ferritin were bound in the surface coat matrix. Glycoside hydrolase enzyme treatments had no apparent effect on the amount of ferritin bound in the surface coat. Cationized ferritin was bound also to the outer lamina of the pellicular membrane in trypsinized cells, which had quantitatively less ferritin bound per surface unit area than bloodstream forms untreated by the enzyme. Living and glutaraldehyde-fixed cells were agglutinated with cationized ferritin. The results obtained in the various experiments indicated that polyanionic polysaccharides were constituent terminal ligands of the surface coat matrix and pellicular membrane in T. musculi bloodstream forms.     

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.     

Attachment of Trypanosoma cruzi Epimastigotes to Hydrophobic Substrates and Use of this Property to Separate Stages and Promote Metacyclogenesis

In vivo, epimastigotes of Trypanosoma cruzi colonize a lipidic superficial layer of the rectal cuticle of the vector Triatoma infestans. In vitro, epimastigotes of four cultured strains and one strain from reduviids use a terminal area of the flagellum to attach to a variety of artificial hydrophobic substances, such as hydrocarbons and a range of synthetic plastics. Trypomastigotes did not attach to these substrates. Hydrophilic molecules, such as neutral or negatively charged polysaccharides. did not facilitate binding. Epimastigotes and trypomastigotes were artificially bound by electrostatic forces to positively charged chitosan or DEAE-Sephacel over their entire surface. Tween 20 and lipid-binding serum albumin effectively inhibited the hydrophobic attachment. Based on this hydrophobic interaction of epimastigotes. a new chromatography technique has been devised to gently separate trypomastigotes from epimastigotes using octacosane-coated beads. Furthermore, the in vitro transformation of epimastigotes to trypomastigotes was enhanced if epimastigotes were permitted to attach to hydrophobic, wax-coated culture vessels.     

Fine Structure and Cytochemistry of Tritrichomonas foetus and Rat Neutrophil Interaction

ABSTRACT. The fine structure of normal and antibody-coated Tritrichomonas foetus cells and their interaction with rat peritoneal neutrophils was studied. Peritoneal neutrophils were obtained by glycogen stimulation. The neutrophils readily associated with and killed the parasites, which were subsequently ingested. The process involved activation of the respiratory burst, as demonstrated by the use of cytochemical methods. Images were obtained indicating that binding of parasites to the neutrophil surface triggers an exocytic response with release of oxygen-derived products. Cytochemical localization of acid phosphatase and peroxidase activities showed that leukocyte granules fused with the parasite-containing phagocytic vacuoles. We also showed the cytochemical localization of alkaline phosphatase in the parasite-neutrophil interaction.     

Monocercomonas sp.: Cytochemistry and Fine Structure of Freeze-Fractured Membranes

Monocercomonas sp. from the wood-snake Tropidophis melanurus was studied using fast freezing of alive and fixed cells followed by freeze-fracture and deep-etching. Cytochemistry for enzymes (acid phosphatase, neutral phosphatase, and thiaminopyrophosphatase) and for carbohydrates and endocytosis of gold-labeled albumin were also performed. The Golgi complex is formed by 12-14 cisternae with typical cis and trans faces connected to a network of tubular and cisternal structures, and is positive for thiaminopyrophosphatase at the trans face. Intraluminal filamentous structures are seen connecting the two faces of the cisternae of the Golgi complex. Lysosomes appeared to contain acid and neutral phosphatases. Cytochemistry showed that lysosomes predominate among the unidentified vacuoles in the cytoplasm. Some vesicles are involved in the endocytic pathway, while others are derived from the Golgi complex. Hydrogenosomes have a rod-like or dumb-bell shape. Two of the anterior flagella present rosettes, when observed in replicas of freeze-fractured material, formed by circular arrangement of intramembranous particles on both P and E faces. The other anterior and the recurrent flagella do not show such rosettes but showed ribbon-like arrays of particles at the point where they emerge from the cell body.