Trypanosoma cruzi: interaction with vertebrate cells in vitro. IV. Environmental temperature effects

The effects of 4 environmental temperatures (29, 32, 35, and 38 C) on the interaction between Trypanosoma cruzi and bovine embryo skeletal muscle cells were quantified. Three aspects of the interaction (penetration of host cells by trypomastigotes, the lag period prior to the reproductive phase, and the reproductive phase) were markedly affected by temperature. There was a linear increase in the number of trypomastigotes penetrating cells in the 29–35 C range. Temperatures above 35 C can be considered supraoptimal as no further increase in the rate of penetration occurred. The lag period decreased linearly as temperature increased in the 29–35 C range; at 38 C, the lag period was markedly shortened. The doubling time of amastigotes increased linearly as temperature increased in the 32–38 C range; at 29 C, the doubling time was markedly lengthened. At all temperatures, parasites reproduced for 9 generations before cell rupture. The changes in lag period and doubling time complemented each other in the 32–38 C range. Thus, there was essentially no change in the overall length of the intracellular cycle which lasted 6.1 to 6.5 days. At 29 C, however, the cycle was lengthened to 8.9 days. Thermodynamic analysis revealed marked differences, characterized by a negative activation energy and negative enthalpy, between the reproductive phase of parasites within vertebrate cells and the vertebrate cells themselves. However, the thermodynamic parameters of parasites reproducing extracellularly in liquid medium and intracellularly were the same.     

Trypanosoma cruzi: interaction with vertebrate cells in vitro. 2. Quantitative analysis of the penetration phase

A technique is described for quantifying the in vitro penetration of vertebrate cells by trypomastigotes of Trypanosoma cruzi. It was found that the parasites are distributed among host cells in a manner described by the negative binomial distribution. The rate at which trypomastigotes penetrate bovine embryonic skeletal muscle cells (BESM) decreased exponentially in time in this system. The rate of the exponential decrease was dependent upon the concentration of parasites, being faster for more concentrated suspensions of trypomastigotes. A significantly lower penetration rate of canine kidney and HeLa cells was found when compared to bovine embryonic skeletal muscle cells. Within a single population of BESM cells, the smaller cells were penetrated more rapidly than the larger ones per unit cell area.     

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

Trypanosoma cruzi: Comparative fatty acid metabolism of the epimastigotes and trypomastigotes in vitro

In vitro studies on the fatty acid metabolism of the epimastigotes and trypomastigotes of Trypanosoma cruzi showed the following: (1) Trypomastigotes demonstrated the ability to convert labeled palmitic acid to CO₂. Epimastigotes either did not convert this fatty acid to CO₂ or did so at a very low rate. (2) Trypomastigotes incorporated palmitic acid into neutral lipids, but, at a rate less than that of the epimastigotes. (3) While epimastigotes readily incorporated palmitic acid into phospholipid lipids, trypomastigote forms seemed to lack this ability.     

The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.     

Inefficient Complement System Clearance of Trypanosoma cruzi Metacyclic Trypomastigotes Enables Resistant Strains to Invade Eukaryotic Cells

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.     

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.     

Trypanosoma cruzi: Quantification and Analysis of the Infectivity of Cloned Stocks1

The infection of bovine embryo skin and muscle cells by trypomastigotes of four Trypanosoma cruzi clones (CA-I/71, /72, Miranda/76, /80) was quantified. Stable and reproducible intra-isolate differences were observed; an almost 70-fold difference in infectivity occurred between clones. The CA-I/71 clone was not susceptible to N-acetyl-D-glucosamine at a concentration that inhibits the infection of vertebrate cells by Ernestina and Y-strain parasites. Eight other monosaccharides that are common constitutents of vertebrate cell surface glycoproteins also failed to inhibit the infection of vertebrate cells by the CA-I/71 clone.     

Down Regulation of NO Signaling in Trypanosoma cruzi upon Parasite-Extracellular Matrix Interaction: Changes in Protein Modification by Nitrosylation and Nitration

Background

Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas'' disease in humans, to components of the extracellular matrix (ECM) is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling.

Methodology/Principal Findings

Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins) or by the biotin switch method (S-nitrosylated proteins) and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation.

Conclusions/Significance

For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake.

Epimastigotes and trypomastigotes of Trypanosoma cruzi, obtained from liquid cultures, have vesicles and multivesicular structures in their cytoplasm. Horseradish peroxidase (HRP) was used as a tracer to study the uptake of protein by these two forms. In epimastogotes HRP is ingested by a process of pinocytosis which occurs through the cytostome. Trypomastigotes do not have a cytostome, and pinocytosis occurs through the flagellar pocket region. The pinocytotic vesicles can fuse with each other to form large multivesicular structures that are more abundant in epimastigotes than in trypomastigotes. The cell membrane as well as the membranes of the pinocytotic vesicles and the large multivesicular structure have carbohydrates, as detected by the periodic acid-thiosemicarbazide-silver proteinate technique. Intramembranous particles were observed by using the freeze-fracture technique. The cell membrane has many particles, whereas the membranes of the vesicles and multivesicular structure have few or no particles.     

Biochemical Requirements for Intracellular Invasion by Trypanosoma cruzi: Protein Synthesis1

The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.     

Lectin receptors in Trypanosoma cruzi an N-acetyl-d-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the ¹³¹I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent M_r 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}$ inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}\text{-}\text{specific}$ receptor activity.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

A Monoclonal Antibody to Alpha Tubulin Recognizes Host Cell and Trypanosoma cruzi Tubulins1

ABSTRACT. A mouse monoclonal anti-α-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines–J774 macrophages, BSC-1, and Vero cells–infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.     

The MASP Family of Trypanosoma cruzi: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection

Background

Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.

Methods

By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.

Findings and Conclusions

We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.     

MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host.

Methodology/Principal Findings

In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption.

Conclusions/Significance

The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi.     

Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.     

Inhibition of HSP90 in Trypanosoma cruzi induces a stress response but no stage differentiation

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite Leishmania donovani. We investigated the role of HSP90 in the related parasite Trypanosoma cruzi by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G₁ phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in T. cruzi and serves as a feedback inhibitor in the cellular stress response. In contrast to L. donovani cells, however, T. cruzi cells treated with GA do not begin to differentiate into relevant life cycle stages.