Gene expression and molecular modeling of the HSP104 chaperone of Trypanosoma cruzi.

Heat shock protein (HSP) 104 is a highly conserved molecular chaperone that catalyzes protein unfolding, disaggregation and degradation under stress conditions. We characterized HSP104 gene structure and expression in Trypanosoma cruzi, a protozoan parasite that causes Chagas' disease. The T. cruzi HSP104 is an 869 amino-acid protein encoded by a single-copy gene that has the highest sequence similarity (76%) with that of T. brucei and the lowest (23%) with that of the human protein. HSP104 transcripts were detected at room temperature, and levels increased after incubation at 37° or 40°C. The HSP104 protein was found at low levels in non-heat-shocked cells, and accumulated continuously up to 24 h at elevated temperatures. We developed a predicted structural model of hexameric T. cruzi HSP104, which showed some conserved features.

Control of tubulin gene expression during metacyclogenesis of Trypanosoma cruzi.

During differentiation of the dividing epimastigote to the non-dividing metacyclic trypomastigote form of the parasitic protozoan Trypanosoma cruzi there is a marked reduction in the rate of synthesis of the major proteins alpha- and beta-tubulin. Our results indicate that the control of synthesis of these proteins during the differentiation event is exerted at the level of alpha- and beta-tubulin mRNA accumulation.

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.

Predicted amino acid sequence and genomic organization of Trypanosoma cruzi hsp 60 genes.

We describe the isolation of a Trypanosoma cruzi protein-coding gene that exhibited about 50% identity with members of the family of heat shock protein (hsp) 60 genes. Since this homology extended for most of the predicted 562 amino acid open reading frame and was comparable to the level of sequence identity between the individual hsp 60 genes from diverged species, we conclude that we have isolated and characterized a T. cruzi hsp 60 gene. The T. cruzi hsp 60 genes are arranged in tandem arrays, and the presence of restriction fragment length polymorphisms (RFLPs) among the different hsp 60 genes suggests the presence of several separate gene arrays encoding hsp 60 members.

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.

The association of ACE gene D/I polymorphism with cardiovascular risk factors in a population from Rio de Janeiro.

Our aim was to determine the frequencies of the angiotensin-converting enzyme (ACE) gene alleles D and I and any associations to cardiovascular risk factors in a population sample from Rio de Janeiro, Brazil. Eighty-four adults were selected consecutively during a 6-month period from a cohort subgroup of a previous large cross-sectional survey in Rio de Janeiro. Anthropometric data and blood pressure measurements, echocardiogram, albuminuria, glycemia, lipid profile, and ACE genotype and serum enzyme activity were determined. The frequency of the ACE*D and I alleles in the population under study, determined by PCR, was 0.59 and 0.41, respectively, and the frequencies of the DD, DI, and II genotypes were 0.33, 0.51, and 0.16, respectively. No association between hypertension and genotype was detected using the Kruskal-Wallis method. Mean plasma ACE activity (U/mL) in the DD (N = 28), DI (N = 45) and II (N = 13) groups was 43 (in males) and 52 (in females), 37 and 39, and 22 and 27, respectively; mean microalbuminuria (mg/dL) was 1.41 and 1.6, 0.85 and 0.9, and 0.6 and 0.63, respectively; mean HDL cholesterol (mg/dL) was 40 and 43, 37 and 45, and 41 and 49, respectively, and mean glucose (mg/dL) was 93 and 108, 107 and 98, and 85 and 124, respectively. A high level of ACE activity and albuminuria, and a low level of HDL cholesterol and glucose, were found to be associated with the DD genotype. Finally, the II genotype was found to be associated with variables related to glucose intolerance.

Conservation of heat-shock proteins in Trypanosoma cruzi.

Heat shock is an integral part of the life cycle of Trypanosoma cruzi. Here, Edson Rondinelli reviews the parasite's response to stress.

Characterization and cellular distribution of heat-shock proteins HSP70 and HSP60 in Trypanosoma cruzi.

At least eight protein members of HSP70 and three of the HSP60 families have been identified in Trypanosoma cruzi; of these, five HSP70 isoforms and one HSP60 isoform were respectively induced by a 2-hr heat-shock treatment at 37 degrees C. Immunoelectronmicroscopy of epimastigote, spheromastigote, and metacyclic cells obtained in vitro showed anti-HSP60 reactive proteins in the mitochondria and near the kinetoplast. Anti-HSP70 reactive proteins presented a more complex pattern. They were observed in different cellular compartments, and after heat shock all the three cell forms analyzed presented gold particles associated with the cellular membrane.

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos.

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.

Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos.

A cDNA of the sea urchin Strongylocentrotus purpuratus was identified as encoding polyubiquitin and used to detect a single gene with transcripts containing multiple ubiquitin coding units. Polyubiquitin transcripts exist as a 3.2-kb RNA in polyribosomes and as three higher molecular weight RNAs in purified nuclei. The amount of polyubiquitin RNA is essentially constant at 10(4) -10(5) transcripts per embryo during the egg-to-blastula period and then declines during further development. Heat shock elicits a transient increase in the level of polyubiquitin RNA, while Zn(II) ions induce a sustained accumulation, that is influenced by developmental parameters: One round of Zn(II) induction elicits the accumulation of the nuclear 7.6- and 5.6-kb RNAs, as well as the 3.2-kb polysomal RNA; however, a second round of induction yields only the 5.6- and 3.2-kb RNAs, suggestive of a change in pre-mRNA size or processing. Polyubiquitin RNA is expressed equally in ectodermal and mesoendodermal tissues and is induced in both tissue fractions by treatment of pluteus larvae with Zn(II). However, in isolated and cultured tissue fractions, polyubiquitin RNA is not inducible by Zn(II), in contrast to the full inducibility of metallothionein mRNAs. Polyubiquitin RNA induction thus appears to be conditioned by the integrity of the embryo, as well as by previous exposure to inducer.

Trypanosoma cruzi: cloning and characterization of a RAB7 gene.

The small monomeric GTP-binding proteins of the RAB subfamily are key regulatory elements of the machinery that controls membrane traffic in eukaryotic cells. These proteins have been localized to many different intracellular organelles, on both endocytic and exocytic compartments, suggesting that each step of vesicular traffic can involve a specific RAB protein. The presence of conserved amino acid domains in these proteins has allowed the cloning of their genes from several organisms, including yeast, plants, humans, and parasites. In this work we describe the identification, cloning, and characterization of a RAB7 gene homologue in Trypanosoma cruzi (TcRAB7). Our data indicate that this gene is present as a single copy in the T. cruzi genome, located on a 2.25-Mb chromosomal DNA. TcRAB7 is expressed in T. cruzi epimastigotes, metacyclic trypomastigotes, and spheromastigotes. We established transformed cell lines that express two versions of an epitope-tagged TcRAB7 protein: one wild type (pTAG) and one deleted at the C-terminal cysteines (pDeltaCXC). Wild-type TcRAB7 protein (pTAG) appears to be localized exclusively in the membrane fraction, while the mutated TcRAB7 protein (pDeltaCXC) loses the ability to associate with the membrane, showing only cytosolic localization. Also, we produced the recombinant TcRAB7 protein and demonstrated that it binds GTP. The identification of exo- and endocytic machinery components in T. cruzi and their function would provide specific markers of these subcellular compartments, thereby unveiling important aspects of vesicular traffic in this parasite.

Signal transduction in Trypanosoma cruzi: opposite effects of adenylcyclase and phospholipase C systems in growth control.

Fetal calf serum (FCS), which is mitogenic for the pathogenic protozoa T. cruzi, inhibits cAMP production in basal and forskolin-stimulated epimastigotes. It also activates phosphoinositides hydrolysis yielding diacylglycerol and inositol phosphates (Ins-P). Ins-P production is enhanced by AlF4-, GTP or beta-gamma-methylene-GTP, thus implying G proteins mediation in the phenomenon. An enzyme with phospholipase C activity which may be involved in the phospholipid metabolism was partially characterized.

Trypanosoma cruzi: properties of a clone isolated from CL strain.

BALB/c mice injected i.p. with 2 x 10(6) metacyclic forms of CL-14, a clone isolated from the CL strain of Trypanosoma cruzi, did not show parasitemia as evaluated by direct blood microscopy examination, hemoculture and xenodiagnosis. Moreover, new-born mice (1-2 days old) injected with culture- or insect-derived CL-14 trypomastigotes also displayed negative parasitemia. No mortality was observed in either group of animals. However, despite this apparent non-infectivity, mice injected with clone 14 developed high resistance against a lethal challenge with virulent trypomastigotes. All challenged mice survived and the parasitemia was negative. These results indicate that clone 14 is a very good antigen for the study of acquired immunity in T. cruzi infection and, therefore, a potential candidate for the development of a vaccine against this parasite.

Construction of a normalized cDNA library for the Trypanosoma cruzi genome project.

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.

Identification of transcribed sequences (ESTs) in the Trypanosoma cruzi genome project.

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

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.

Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression.

Metallothionein (MT) is shown to be present in sea urchin embryos on the basis of its characteristic properties as a small protein (6-7 Da) of extraordinarily high cysteine content, whose biosynthesis is readily induced by heavy metals. Induction by Zn2+ results in the accumulation of the cysteine-rich MT protein, a 0.8 kb MT mRNA and a 2.9 kb nuclear RNA. The amount of MT mRNA is regulated intrinsically through the course of embryogenesis to the pluteus stage: A maternal MT mRNA is poly(A)-deficient and is polyadenylated after fertilization. New MT mRNA begins to accumulate between the seventh and eighth cell cleavage, reaches a maximum at the mesenchyme blastula stage, decreases during gastrulation, and rises again in the early pluteus stage. "Animalizing" embryos with Zn2+ during early embryogenesis causes a sustained accumulation of MT mRNA to levels greater than 25 times the normal amount. MT mRNA is present in high amount in the ectoderm of the pluteus, but is barely detectable in the mesoderm-endoderm tissue fraction. Treatment of either the pluteus or its isolated tissue fractions with Zn2+ results in the induction of MT mRNA accumulation in the mesoderm-endoderm but not in the already MT mRNA-enriched ectoderm. Furthermore, differences in Zn2+ induction of the MT gene in the blastula and gastrula are consistent with a developmental pattern in which MT gene expression is maintained constitutively at a high level in the ectoderm and at a low level in the mesoderm-endoderm tissues, which are, however, preferentially inducible by Zn2+.

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.

Heat shock proteins in Trypanosoma cruzi: identification and localization of HSP70 and HSP60 proteins and structure of HSP60 genes (brief report).

To identify the members of the HSP70 and HSP60 families of Trypanosoma cruzi, we analysed 35S methionine epimastigote cells by two dimensional Western blot. At 29 degrees C, an HSP70 monoclonal antibody (anti-D. melanogaster) recognized eight isotypes. At least five of these were heat-induced. Polyclonal antibody against the 65 KDa antigen (anti-M. tuberculosis) recognized three isotypes with identical molecular weights, but different microliters. Only one isoform was heat induced. The cellular distribution of HSP70 and HSP60 was studied by immunoelectron microscopy. Anti-HSP70 reactive protein was localized in the cytoplasm, mitochondria and nucleus, while anti-HSP60 protein was found in the mitochondrion and in close association with the kinetoplast. To characterize the HSP60 gene and its proteins, we isolated a genomic T. cruzi clone encoding the HSP60 gene. T. cruzi HSP60 genes could be shown to be organized in 2100 nt tandem arrays. RELP in the HSP60 genes revealed that at least three different types of HSP60 genes were encoded in the T cruzi genome. The predicted open reading frame measured exhibits about 50% identity to other HSP60 described. Expression of these HSP60 genes could not be induced by 2 hours heat shock at 37 degrees C. Post-transcriptional mechanisms may be responsible for HSP60 induction in T. cruzi.