Trypanosoma cruzi: a specific surface marker for the amastigote form

Among the known life cycle stages of Trypanosoma cruzi only the amastigote form bound lactoferrin (LF), a glycoprotein produced by neutrophils. This capacity was readily demonstrable by indirect immunofluorescence in amastigotes derived from mice, a mammalian cell culture, or grown in an axenic medium. No LF binding was detectable on trypomastigotes from blood or mammalian cells, insect-derived metacyclics or epimastigotes, or on epimastigotes grown in Warren's medium. Serum levels of LF were increased in mice acutely infected with T. cruzi, and amastigotes from the spleens of these animals were found to have the glycoprotein on their surface. The amastigote LF receptor may have biological significance in parasite-host interaction since mononuclear phagocytes also express a LF receptor, and treatment of these cells with LF has been shown to increase their capacities to take up and kill T. cruzi amastigotes in vitro. The LF receptor is the first marker for T. cruzi amastigotes for which a naturally occurring ligand has been described.     

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.     

Separation of Individual Stages of Trypanosoma cruzi Grown in Cell Culture by Continuous Free-Flow Electrophoresis

The separation of extracellular protozoan parasites from host cells based on a difference in surface charge has been described. However, with Trypanosoma cruzi no method exists for the isolation of pure parasite stages from heterogeneous mixtures. Studies on the electrophoresis of mixed stage populations confirm significant surface charge density differences exist among epimastigotes, trypomastigotes, and amastigotes. In ascending order of electronegativity, amastigotes have the lowest charge density, try-pomastigotes next, followed by epimastigotes. A technique has been developed for the separation of purified populations of parasites based on these charge differences using a continuous free-flow electrophoresis apparatus. The separated populations are morphologically intact and maintain their infectivity to mice. This separation method is applicable for preparative and analytical isolation of pure stages of T. cruzi for biochemical and immunological studies.     

Growth of Isolated Amastigotes of Trypanosoma cruzi in Cell-Free Medium1

Amastigotes of Trypanosoma cruzi were purified from overlays of infected Vero cell cultures by centrifugation over a discontinuous gradient of metrizamide. Pure amastigote preparations were usually recovered from the pellet under the layer of specific gravity 1.086. The isolated amastigotes grew in cell-free ML-15HA medium. Growth rate for the different strains of T. cruzi were in the order Y > Tulahuan > CL. The generation time of amastigotes in ML-15HA medium was 16.8, 18.0, and 26.4 h for the Y, Tulahuen, and CL strains, respectively, in the presence of 5% CO₂) and 16.8, 31.2, and 36.4 h, respectively, in the absence of CO₂. Intracellular amastigotes did not differ ultrastructurally from amastigotes from either the density-gradient fractionation or culture in cell-free medium.     

Purification of Tissue Forms (Amastigotes) of Trypanosoma cruzi by Immunoaffinity Chromatography1

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Scpharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Trypanosoma cruzi: Cell Biological Behavior of Epimastigote and Amastigote Forms in Axenic Culture

A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G₁ cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.     

Trypanosoma cruzi: cell biological behavior of epimastigote and amastigote forms in axenic culture

A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G1 cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.     

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.     

All <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> developmental forms present lysosome-related organelles

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.     

Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco-protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against 7. cruzi.     

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co‐opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host–parasite interaction.     

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T. cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Trypanosoma cruzi: Nucleotide and vitamin requirements of growing epimastigotes

Using a defined culture medium it was shown that Trypanosoma cruzi epimastigotes (strains Y, Ma, and F1) do not require exogeneous nucleotides for continuous cultivation. Biochemical determinations carried out on parasites grown in the presence or absence of exogenous nucleotides revealed no differences in intracellular nucleotide concentrations. This suggests that T. cruzi epimastigotes have the capacity for de novo nucleotide synthesis. Choline and folic acid were necessary only for high yields of T. cruzi, suggesting that epimastigotes can partially satisfy their vitamin requirements.     

Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10⁷ amastigotes—with typical shape and positive for the amastigote marker Ssp4—from 5x10⁶ infected Vero cells (48h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.     

Stage-Regulated GFP Expression in Trypanosoma cruzi: Applications from Host-Parasite Interactions to Drug Screening

Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.     

Trypanosoma cruzi: morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C.Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C.No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr.Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.     

Trypanosoma cruzi: Antigenic invariance of the cell surface glycoprotein

The cell surface antigens of Trypanosoma cruzi have been studied for evidence of antigenic variation. The majority of the cell surface antigens found on epimastigotes were also present on trypomastigote and amastigote forms. Serum absorption studies and peptide mapping of the major cell surface glycoprotein from a series of clones and strains of Trypanosoma cruzi failed to find evidence of antigenic variation. Differences found between geographically distinct strains of Trypanosoma cruzi were minor and not associated with the major glycoprotein. Components present in normal mouse serum were capable of binding to the surface of Trypanosoma cruzi and these components could interfere in subsequent radioimmune assays, particularly with bloodstream derived trypomastigotes.     

Inositol 1,4,5‐trisphosphate receptor regulates replication,differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi

In animals, inositol 1,4,5‐trisphosphate receptors (IP₃Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca²⁺ release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP₃R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP₃R genes expressing T. cruzi IP₃R (TcIP₃R) exhibited IP₃‐mediated Ca²⁺ release from the ER, and demonstrated receptor binding to IP₃. TcIP₃R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP₃R gene loci led to the death of the parasite, suggesting that IP₃R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP₃R displayed defects in growth, transformation and infectivity, indicating that TcIP₃R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP₃R exhibited a reduction of disease symptoms, indicating that TcIP₃R is an important virulence factor. Combined with the fact that the primary structure of TcIP₃R has low similarity to that of mammalian IP₃Rs, TcIP₃R is a promising drug target for Chagas disease.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.