Morphological events during the Trypanosoma cruzi cell cycle

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.     

Trypanosoma cruzi: correlations of biological aspects of the life cycle in mice and triatomines.

The infection pattern in Swiss mice and Triatomine bugs (Rhodnius neglectus) of eleven clones and the original stock of a Trypanosoma cruzi isolate, derived from a naturally infected Didelphis marsupialis, were biochemically and biologically characterized. The clones and the original isolate were in the same zymodeme (Z1) except that two clones were found to be in zymodeme 2 when tested with G6PDH. Although infective, neither the original isolate nor the clones were highly virulent for the mice and lesions were only observed in mice infected with the original stock and one of the clones (F8). All clones and the original isolate infected bugs well while only the original isolate and clones E2 and F3 yielded high metacyclogenesis rates. An observed correlation between absence of lesions in the mammal host and high metacyclogenesis rates in the invertebrate host suggest a evolutionary trade off i.e. a fitness increase in one trait which is accompanied by a fitness reduction in a different one. Our results suggest that in a species as heterogeneous as T. cruzi, a cooperation effect among the subpopulations should be considered.     

The gamma-glutamyltranspeptidase of Trypanosoma cruzi

Trypanosoma cruzi epimastigotes show gamma-glutamyltranspeptidase activity which has characteristics significantly different than the mammalian enzyme. The protozoan enzyme is localized in the cytosolic fraction, it has a Km of 1.6 mM and a Vmax of 17.4 nmol/min/mg protein with L-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor, and an optimun pH range from 7.5 to 8.0. The best amino acid acceptors were L-histidine, L-asparagine, L-aspartate, L-glutamate and L-proline, but L-glutamine was a very poor acceptor. The enzyme was very sensitive to inhibition by 6-diazo-5-oxo-L-norleucine (k2 = 4.0 X 10(5)/M per min) and O-diazo-acetyl-L-serine (k2 = 1.1 X 10(4)/M per min). Phenobarbital (k2 = 8.38/M per min) and L-serine borate (Ki = 34 mM) were poor inhibitors. The activity of the enzyme was not correlated with the logarithmic phase of growth of the parasites and steadily decreases with the age of the cultures.     

The carboxylesterases of Trypanosoma cruzi epimastigotes

Carboxylesterase activity in Trypanosoma cruzi was found mainly in the microsomal (40%) and the cytosolic fraction (26%). The Vmax for p-nitrophenyl acetate was 28.50 and 17.60 nmol per min and mg of protein for the microsomal and the cytosolic fractions, respectively. The Km was 0.78 mM for the microsomal activity and 0.55 mM for the cytosolic activity. The inhibition rate constant with N-ethylmaleimide were 38.10 M-1 min-1 and 2.56 M-1 min-1 for the cytosolic and the microsomal enzymes, respectively. The rate constants with Paraoxon were 8,360 M-1 X min-1 and 32,600 M-1 X min-1. Polyacrylamide gel electrophoresis under nondenaturing conditions showed three bands of microsomal activity with M.W. of 63, 136 and 153 kDA. Similarly, three bands of cytosolic activity with M.W. of 126, 294 and 479 kDA were identified.     

The effect of the diterpene 5-epi-icetexone on the cell cycle of Trypanosoma cruzi

Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide. We observed that the diterpene does not affect the growth of the parasites when added within 10 h after removal of hydroxyurea, but the compound was effective on growth when added to the cultures after 12 h. Thymidine incorporation was somewhat inhibited when the diterpene was added at 12 h after removal of hydroxyurea, possibly on the transition S/G2. Using transmission electron microscopy we observed that the diterpene induced a delay in the progression of cell division. We conclude that the compound, at cytostatic dose, affects the cell cycle of T. cruzi, possibly in the transition S/G2 phase and cell division. Further studies will focus to identify the molecular targets for the action of 5-epi-icetexone.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

The development of Trypanosoma cruzi in triatominae

Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. These insects ingest an enormous amount of blood, with ingestion followed very rapidly by a strong diuresis, slow digestion and occasionally long periods of starvation. Resulting changes in the intestinal environment induce the development of dominant stages of T. cruzi--epimastigotes and metacyclic trypomastigotes--and can be correlated with the appearance of specific developmental stages--spheromastigotes and giant cells--which otherwise are only rarely seen. Here, Astrid Kollien and Günter Schaub outline recent research on these developmental steps of T. cruzi in the vector, and the effects of different compounds acting against the parasite in the vector.     

The cAMP receptor protein of Trypanosoma cruzi

The widest used method to determine cAMP-binding activity in cell-free extracts (Gilman, A. G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312) underestimated by about 10-fold the total amount of [3H]cAMP bound by crude extracts of cultured forms of Trypanosoma cruzi (epimastigotes), when compared with the results obtained by the modified Millipore filter technique described by Doskeland and Ueland (Doskeland, S. O., and Ueland, P.M. (1977) Biochem. J. 165, 561-573). After column chromatography on DEAE-Sephacel, the cAMP-binding activity eluted as a single symmetrical peak at about 210 mM NaCl, totally separated from two peaks of cAMP-independent phosphotransferase activities which eluted, respectively, at 90 and 270 mM NaCl. These two protein kinases showed similar specificities for exogenous substrates, preferring in this order: protamine greater than casein greater than histone H2b. Photoaffinity labeling of cell-free extracts with 8-azido[32P]adenosine 3':5'-monophosphate and autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis specifically labeled only one protein of Mr = 62,000. From these results it is concluded that T. cruzi epimastigotes possess one single cAMP-binding protein of monomeric Mr = 62,000, not associated with a phosphotransferase activity and probably very different in nature to known regulatory subunits of protein kinases, given the results obtained with the Millipore filtration technique.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).