Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.     

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis.     

Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence-verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with dye reversal. We identified eight distinct categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of chagasic cardiomyopathy and provide new targets for intervention.     

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.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

The innate immune response to Trypanosoma cruzi infection

Trypanosoma cruzi infection is a major public health problem in Latin America. The host innate immune system plays a pivotal role in the recognition of T. cruzi infection and the subsequent development of adaptive immunity. In this review, we focus on the TLR-dependent and -independent innate immune responses to T. cruzi.     

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°C, and slightly decreased at 40°C relative to its level at 29°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°C and 40°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°C, in addition, an increase in the total amount of this template is observed in the cells.     

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.     

Structure and expression of three gp82 gene subfamilies of Trypanosoma cruzi

The glycoprotein gp82 is a GPI-anchored cell surface protein of Trypanosoma cruzi and is involved in cell invasion. Gp82 is encoded by multiple genes. To investigate the genetic basis of its biological function, we analyzed structure and expression of gp82 multigene family members in the Peruvian and Guatemalan strains. Three major groups of gp82 genes (A, B and C) were categorized by analyzing multiple DNA clones from the genomic PCR products. Within each group, 95–97% homology was observed, whereas between the groups, homology was 67–79%. The copy numbers of groups A, B and C as determined by real-time PCR were 18, 8 and 7 copies, respectively, in the Peru-2 strain. Significant elevation of the mRNA expression levels (5–10 times more) of all the subfamily genes was observed in the metacyclic stage compared with the epimastigote stage. When we focused on the binding motif sequence reported previously, we found substantial difference between that of A and C. However, the peptide inhibition invasion assay showed no functional difference. Taken together, we demonstrated that three subfamilies of gp82 were in the genome of T. cruzi and maintained their functional structure, and that the mRNA expressions of those genes were equally controlled in a stage-specific manner.     

Trypanosoma cruzi induces changes in cardiac connexin43 expression

Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.     

Mismatch repair in Trypanosoma brucei: heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.     

Resistance to Trypanosoma cruzi infection in relation to the timing of IgG humoral response

The Biozzi "high" (BH) and "low" (BL) responder mice (Selection III) differed in their susceptibility to Trypanosoma cruzi. The BH strain responded quickly to the infection, similar to the reaction of (CBA X C57B1/10)F1 mice but in contrast to the susceptible BL strain. We suggest that the IgG response mounted by the host during the prepatent period of the infection is crucial to the outcome of the infection.     

Trypanosoma cruzi: immunosuppressed response to different antigens in the infected mouse.

Trypanosoma cruzi: Immunosuppressed response to different antigens in the infected mouse. Experimental Parasitology45, 190–199. Trypanosoma cruzi infection in mice results in functional changes in the normal immunological responses to heterologous antigens. An immunosuppression of the 19 and 7S antibody response is observed in infected animals against both a particulate antigen and against soluble antigens. Furthermore, the immune response to the soluble T-independent antigens, DNP-Ficoll and LPS, was also similarly impaired when antigen was administered to trypanosome-infected animals. The suppression of the immune response to these antigens does not seem to involve an alteration in the macrophage, as evidenced by a normal uptake and handling of soluble ¹³¹I-labeled HSA and by a normal immune response when antigen-exposed peritoneal macrophages from trypanosome-infected mice were transferred to normal mice. These data support the concept that T. cruzi induces an immunosuppression to both T-dependent and T-independent antigens and that the depression observed is not due to an alteration in macrophage function.