Physiological aspects of Trypanosoma cruzi gene regulation during heat-shock.

Investigations on the conditions of heat-shock response in Trypanosoma cruzi, the agent of Chagas disease, showed that at 37 degrees C, one of the heat-shock temperatures employed, the parasites from 48 h culture do not display a classical response to the heat treatment, since a general increase in RNA and protein synthesis was detected. The classical heat-shock response was detected only at 40 degrees C. The data also suggest that the heat shock proteins (HSP) mRNA population is sufficient to maintain protein synthesis at a high rate for at least 1 h and, to maintain the same rate of response for a longer period, transcription is necessary. The half life of HSP 70 mRNA is less than 3 h at 37 degrees C. The protein synthesized during the first hour of the heat shock at 37 degrees C is stable for at least 24 h. The parasite seems to be able to reuse the stock of HSP mRNAs stored during the first thermal shock to respond to a second heat treatment. These data are discussed bearing in mind other cell types.

Transcriptional and post-transcriptional control of tubulin gene expression in Trypanosoma cruzi.

We have shown that tubulin mRNA accumulation is regulated at the transcriptional level during metacyclogenesis of Trypanosoma cruzi, although the contribution of post-transcriptional mechanisms is also indicated. mRNA heterogeneity is not restricted to beta-tubulin, and differential regulation of alpha-tubulin mRNAs is observed during this stage of the parasite's life cycle. Treatment of epimastigotes with the microtubule-depolymerizing agent vinblastine resulted in growth inhibition and morphological alterations. Vinblastine also induced a rise in the pool of free tubulin subunits, concomitant with diminished tubulin synthesis and reduced mRNA levels. Tubulin gene transcription remained unaltered during vinblastine treatment, suggesting post-transcriptional control. These observations are in agreement with the autoregulatory model of tubulin gene expression described for a variety of cell types. We conclude that T. cruzi utilizes transcriptional and post-transcriptional control mechanisms for tubulin gene expression.

HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels.

The level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra-optimal temperatures: the total amount of this RNA per cell is increased at 37 degrees C, and slightly decreased at 40 degrees C relative to its level at 29 degrees C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat-shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37 degrees C and 40 degrees C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37 degrees C, in addition, an increase in the total amount of this template is observed in the cells.

Alpha- and beta-tubulin mRNAs of Trypanosoma cruzi originate from a single multicistronic transcript.

The cluster of alternated alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi was shown to be transcribed into a single RNA molecule which upon processing gives rise to the mature alpha- and beta-tubulin mRNAs. This conclusion was based on: (i) nuclear RNA species with the same molecular mass hybridize to both alpha- and beta-tubulin cDNA probes; (ii) S1 nuclease assay of the clustered tubulin genes has shown protected DNA fragments of the same size and of greater molecular mass than that corresponding to the mRNAs, hybridizable to both alpha- and beta-tubulin cDNA probes; (iii) beta-tubulin hybrid selected RNA is still able to hybridize to alpha-tubulin probe.

Trypanosoma cruzi: an in vitro cycle of cell differentiation in axenic culture.

The operation of an in vitro cycle of cell differentiation of Trypanosoma cruzi in axenic culture was obtained. When epimastigote forms, grown in LIT medium, were transferred to a modified LIT medium (E. Chiari, 1981, "Diferenciação do Trypanosoma cruzi em cultura." Ph.D. dissertation, Universidade Federal de Minas Gerais, Brazil), metacyclic trypomastigotes were generated. The latter, upon treatment with fresh human serum, and subsequent incubation in LIT medium gave origin to clusters of spheromastigote cells. The spheromastigotes were resistent to lysis mediated by the complement system and possess a morphology shown by optical and electron microscopy to be very similar to spheromastigotes derived from tissues of infected vertebrates. Blood-like trypomastigotes, or epimastigotes, could be obtained from spheromastigotes depending on the incubation conditions: at high serum concentration (55%) at 37 C, blood-like trypomastigotes were generated; by aging or heating (37 C), at low serum concentration (10%), epimastigotes were formed, closing the whole sequence of cell differentiation of T. cruzi. The molecular characterization of the different cell forms by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolic pulse labeled proteins showed that the in vitro differentiated cells were distinct, not only by morphological criteria, but by differential gene expression as well. All the forms described could be obtained in large amounts (6 x 10(7) to 1 x 10(8)/ml), making it possible to perform preparative biochemical, molecular biological, and immunological experiments.

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature.

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.

Dynamic aspects of cytoplasmic poly(A)+ RNA of Tetrahymena pyriformis.

In the present work a study was made of the compartmentalization of the poly(A)+ RNA populations during the cultural development of cells of T. pyriformis that were pre-starved or derived from stationary cultures. It was found that the poly(A)+ RNA content increases when the cells change from stationary to lag phase. The increase in RNA poly(A)+ is manifested exclusively in the polysome compartment. The level of poly(A)+ RNA in the cytoplasmic non-polysomal compartment does not change. The increase in poly(A)+ RNA is concomitant with an expansion of the polysomes. Pre-starved cells initiate polysome formation soon after being transferred to a growing medium. During this time the poly(A)+ RNA content of the non-polysomal compartment decreases and that of polysomes increases in close proportion. Not only in the starved but also in stationary cells and in those that are beginning to grow, the proportion of poly(A)+ RNA in mRNP is higher than in the polysomes. These data are interpreted as indicating that cells of T. pyriformis, derived from stationary cultures are dependent on RNA synthesis for polysome formation; on the other hand, pre-starved cells use preformed non-polysomal poly(A)+ RNA for the same purpose, in the beginning of the cultural development.

Poly(A)+ RNA metabolism during change of physiological state of Tetrahymena pyriformis cells.

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.

Isolation and properties of flagella of trypanosomatids.

A procedufe is described for the isolation of flagella of Crithidia fasciculata, Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.

Effect of the injection of ribosomes and RNA from Crithidia fasciculata on the experimental infection of mice by Trypanosoma cruzi.

Immunization of mice with Crithidia fasciculata (live suspension, ribosomal fraction and purified RNA) induced a certain degree of protection (decrease of parasitaemia) against infection with Trypanosoma cruzi.

Nalidixic acid and macromolecular metabolism in Tetrahymena pyriformis: effects on protein synthesis.

A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis.