Trypanosoma cruzi: A Specific Surface Marker for the Amastigote Form1

ABSTRACT. 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.     

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.     

Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.     

Trypanosoma cruzi: characterization of an intracellular epimastigote-like form.

Almeida-de-Faria, M., Freymüller, E., Colli, W., and Alves, M. J. M. 1999. Trypanosoma cruzi: Characterization of an intracellular epimastigote-like form. Experimental Parasitology 92, 263-274. A detailed study of transient epimastigote-like forms as intermediates in the differentiation of Trypanosoma cruzi amastigotes to trypomastigotes inside the host cell cytoplasm was undertaken using the CL-14 clone grown in cells maintained at 33 degrees C. Several parameters related to these forms have been compared with epimastigotes and other stages of the parasite. Consequently, the designation of intracellular epimastigotes is proposed for these forms. Despite being five times shorter (5.4 +/- 0.7 micrometer) than the extracellular epimastigote (25.2 +/- 2.1 micrometer), the overall morphology of the intracellular epimastigote is very similar to a bona fide epimastigote, when cell shape, position, and general aspect of organelles are compared by transmission electron microscopy. Epimastigotes from both sources are lysed by human complement and bind to DEAE-cellulose, in contrast to amastigotes and trypomastigote forms. A monoclonal antibody (3C5) reacts with both epimastigotes either isolated from axenic media or intracellular and very faintly with amastigotes, but not with trypomastigotes. Some differences of a quantitative nature are apparent between the two epimastigote forms when reactivities with lectins or stage-specific antibodies are compared, revealing the transient nature of the intracellular epimastigote. The epitope recognized by 3C5 monoclonal antibody reacts slightly more intensely with extracellular than with intracellular epimastigotes, as detected by immunoelectron microscopy. Also a very faint reaction of the intracellular epimastigotes was observed with monoclonal antibody 2C2, an antibody which recognizes a glycoprotein specific for the amastigote stage. Biological parameters as growth curves in axenic media and inhability to invade nonphagocytic tissue-cultured cells are similar in the epimastigotes from both origins. It is proposed that the epimastigote-like forms are an obligatory transitional stage in the transformation of amastigotes to trypomastigotes with a variable time of permanency in the host cell cytoplasm depending on environmental conditions.     

Active entry of bloodstream forms of Trypanosoma cruzi into macrophages.

The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi when not able to differentiate into amastigotes. Their uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.     

Trypanosoma cruzi: adrenergic modulation of cyclic AMP role in proliferation and differentiation of amastigotes in vitro

Trypanosoma cruzi amastigotes, obtained from the supernatant of J774G-8 macrophage cultures infected with Y strain trypomastigotes, proliferated and differentiated into epimastigotes in Warren medium at 28-29 C. The basal level of adenosine 3':5'-monophosphate (cAMP) in recently harvested amastigotes was 0.12 pmole/10(7) cells, which could be increased in a dose-dependent manner to 0.62 pmole/10(7) cells with 1 mM of the adrenergic ligand isoproterenol plus 0.5 mM isobutyl methylxanthine. Isoproterenol inhibited [3H]thymidine incorporation into amastigote DNA, as well as the proliferation of amastigotes and newly transformed epimastigotes. Because dibutyryl cAMP had the same effect as isoproterenol on the cells, the experimental results suggest a role for cAMP, modulated by adrenergic ligands, in the control of proliferation and differentiation of amastigotes.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

L-proline is essential for the intracellular differentiation of Trypanosoma cruzi

Using as the host cell, a proline-requiring mutant of Chinese hamster ovary cell (CHO-K1), it was possible to arrest the differentiation of amastigote forms of Trypanosoma cruzi at the intermediate intracellular epimastigote-like stage. Complete differentiation to the trypomastigote stage was obtained by addition of L-proline to the medium. This effect was more pronounced using the T. cruzi CL-14 clone that differentiates fully at 33 degrees C (permissive temperature) and poorly at 37 degrees C (restrictive temperature). A synchronous differentiation of T. cruzi inside the host-cell is then possible by temperature switching in the presence of proline. It was found that differentiation of intracellular epimastigotes and trypomastigote bursting were proline concentration dependent. The intracellular concentration of proline was measured as well as the transport capacity of proline by each stage of the parasite. Amastigotes have the highest concentration of free proline (8.09 +/- 1.46 mM) when compared to trypomastigotes (3.81 +/- 1.55) or intracellular epimastigote-like forms (0.45 +/- 0.06 mM). In spite of having the lowest content of intracellular free proline, intracellular epimastigotes maintained the highest levels of L-proline transport compared to trypomastigotes and intracellular amastigotes, providing evidence for a high turnover for the L-proline pool in that parasite stage. This is the first report to establish a relationship between proline concentration and intracellular differentiation of Trypanosoma cruzi in the mammalian host.     

Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3'-untranslated region position-dependent cis-element and an untranslated region-binding protein

The genome of Trypanosoma cruzi contains tandem arrays of alternating genes encoding amastin and tuzin. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the protozoan parasite, and tuzin is a G-like protein. We demonstrated previously that the amastin-tuzin gene cluster is polycistronically transcribed to an equal extent in all parasite life cycle stages. The steady state level of amastin mRNA, however, is 68-fold more abundant in amastigotes than in epimastigotes. Here we show that the half-life of amastin mRNA is 7 times longer in amastigotes than in epimastigotes. Linker replacement experiments demonstrate that the middle one-third of the 630-nucleotide 3'-untranslated region (UTR) is responsible for the amastin mRNA up-regulation. This positive effect is dependent on the distance of the 3'-UTR segment from the stop codon and the polyadenylation site as well as on its orientation. A protein or protein complex more abundant in amastigotes than in epimastigotes binds to this minimally defined 3'-UTR segment and may be involved in its regulatory function.     

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.     

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.     

Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.     

Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi

Acid phosphatase activity in Trypanosoma cruzi was found to be located on the external surface of the plasma membranes. Both specific activity and activity per cell of this bound enzyme were significantly higher in the cells of amastigote (an intracellular form) than that of trypomastigote (a bloodstream form) and epimastigote (culture form). During the transformation of epimastigotes to amastigotes in vitro the activity of surface acid phosphatase was elevated concomitant with the increase in population of amastigotes. These results were interpreted as that the elevated enzyme activity is required for the intracellular parasitization of this organism or is a consequence of the morphological transformation.     

Growth of isolated amastigotes of Trypanosoma cruzi in cell-free medium

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 greater than Tulahuen greater than 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.     

Trypanosoma cruzi: amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells

It is generally accepted that Trypanosoma cruzi trypomastigotes represent the infective forms of the etiological agent of Chagas' disease. However, the invasive capacity of amastigotes and their ability to sustain a complete infective cycle in mammalian cultured cells and hosts has been recently demonstrated. In order to compare the process of cell invasion by these different infective forms, I examined the interactions of trypomastigotes and amastigotes with HeLa cells using a new and simple method that improves parasite-cell interactions and significantly reduces incubation periods. T. cruzi forms were centrifuged onto HeLa cells grown on coverslips and parasite-cell interactions were examined by fluorescence and scanning electron microscopy. As expected, it was observed that all parasite forms attach and eventually enter the cells. However, whereas trypomastigotes preferentially invade HeLa cells at the edges, as has recently been demonstrated for other cell types, the initial steps of amastigote-HeLa cell interaction involve binding and entangling of the parasite to surface microvilli. Thus, different T. cruzi infective forms interact with different cell surface structures that could express different receptors at the HeLa cell membrane.