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Trypanosoma cruzi: Characterization of an Intracellular Epimastigote-like Form
Experimental Parasitology 92, 263–274 (1999) Article ID expr.1999.4423, available online at http://www.idealibrary.com on
Trypanosoma cruzi: Characterization of an Intracellular Epimastigote-like Form
M. Almeida-de-Faria, E. Freymu¨ller,* W. Colli, and M. J. M. Alves Departamento de Bioquı´mica, Instituto de Quı´mica, and *Centro de Microscopia Eletroˆnica, UNIFESP/EPM, Universidade de Sa˜o Paulo, C.P. 26077, 05599-970 Sa˜o Paulo, Brazil
Almeida-de-Faria, M., Freymu¨ller, 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 338C. 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 6 0.7 mm) than the extracellular epimastigote (25.2 6 2.1 mm), 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. q 1999 Academic Press Index Descriptors and Abbreviations: Trypanosoma cruzi; intracellular epimastigotes; EE, extracellular epimastigotes derived from axenic
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
culture medium; IE, intracellular epimastigotes derived from tissue cultured cells; BSA, bovine serum albumin; FCS, fetal calf serum; LIT medium, liver infusion and tryptose-containing medium; Mab, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBSG, PBS supplemented with 5% glucose, pH 8.0; SDS, sodium dodecyl sulfate.
INTRODUCTION
Trypanosoma cruzi, the etiologic agent of Chagas’ disease, has a complex life cycle characterized by several developmental forms present in vertebrate and invertebrate hosts. Based mainly on morphological criteria, such as the spindle shape of the parasite, and on the position of the kinetoplast relative to the nucleus and the flagellar emergency region, three forms are classically described: (1) amastigotes, an intracellular dividing form possessing an incipient flagellum and present in the cytoplasm of mammalian cells; (2) trypomastigotes, an infective, flagellated and nondividing form present in vertebrate and invertebrate hosts; and (3) epimastigotes, an extracellular flagellated, noninfective and dividing stage present typically in the invertebrate host (cf. Zingales and Colli 1985). Trypomastigotes escape from the phagocytic vacuole shortly after cell entry and differentiate into amastigotes. Optical microscopic observations made long ago based solely on morphology suggested the presence of ˜ an intracellular epimastigote-like form (Romana and Meyer
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264 1942; Meyer and De Oliveira 1948; Morais Rego 1956). Recently, the existence of the intracellular epimastigote-like forms in less than 1% of all infected cells has been reported, using for detection a specific monoclonal antibody to epimastigotes (Faucher et al. 1995). Few studies elucidating the intracellular cycle of the T. cruzi infection have been made. Usually, the literature refers to the intracellular amastigote–trypomastigote transition in the cytoplasm of the host cell without mentioning the possible existence of an epimastigote intermediate, probably due to the scant evidence in support of its existence. Due to the scarce characterization of the suggested intracellular epimastigote-like stage, a thorough study of that form has been made, taking into account morphometric, biological, and biochemical parameters as well as ultrastructural localization of antigens. These parameters have been compared with those from bona fide epimastigote forms grown in axenic liquid medium.
MATERIALS AND METHODS
Cells. Epimastigote forms of T. cruzi CL-14 clone, a clone derived from the CL strain (Brener and Chiari 1963) were grown in LIT medium (liver infusion–tryptose) at 288C (Camargo 1964; Castellani et al. 1967) (EE forms, for extracellular epimastigotes). CHO-K1 cells were maintained in RPMI medium, supplemented with 10% heatinactivated newborn calf serum, at 378C with 5% CO2. Intracellular epimastigotes (IE), trypomastigotes, and other intracellular stages were obtained by infection of CHO-K1 cells with trypomastigotes. Host cells were infected in the presence of 2% newborn calf serum (Andrews and Colli 1982) and maintained at 338C, as the intracellular morphogenesis of that strain is temperature sensitive, as described (Brener et al. 1976). Under these conditions, the intracellular epimastigotes appear around day 5 post-infection and trypomastigotes are found in the extracellular medium from the 7th to the 10th day. When necessary, the infected cells were disrupted in a Potter and the parasites separated by centrifugation (480g, 5 min) from host cell debris. Parasites were pelleted by centrifugation, resuspended in RPMI medium, and centrifuged on 1 ml of lymphoprep for 10 min at 4300g (Nycomed Pharma AS). Separation of T. cruzi forms by DEAE–cellulose. A mixture of intracellular parasites (1 3 109) in phosphate-buffered saline supplemented with 5% glucose (PBSG), pH 8.0, was overlayed on a 10-ml DEAE–cellulose column and eluted with the same buffer (Lanham and Godfrey 1970). Fractions of 1 ml were collected and the amount of each parasite form was determined by microscopic observation. Elution of epimastigotes from the column was partially achieved by elution with the same buffer with the pH lowered to 7.0. Complement mediated lysis. Parasites (1 3 107/ml) in DME medium were incubated with or without (control) 10% fresh human serum. After 30 min at 378C, the number of intact parasites was counted. Monoclonal and polyclonal antibodies. Monoclonal antibodies
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were obtained as previously described (Alves et al. 1986), except that extracts from epimastigotes grown in LIT medium were employed to immunize Balb/c mice. One monoclonal antibody (3C5 Mab) was selected based on its reactivity with intra- and extracellular epimastigotes by indirect immunofluorescence. The antibody isotype was determined as IgG2b, using an isotyping kit (Boehringer Mannheim Biochemicals). WIC-29.26, an anti-carbohydrate monoclonal antibody specific for epimastigotes (Snary et al. 1981), was kindly provided by Dr. G. A. M. Cross (Rockefeller University) and 2C2 monoclonal antibody (Andrews et al. 1987) was kindly provided by Dr. N. W. Andrews (Yale University). Indirect immunofluorescence. Parasites or infected CHO-K1 cells were washed three times in RPMI medium without serum and fixed in 2% paraformaldehyde in PBS, pH 7.4, for 1 h at room temperature. The cells were incubated with the antibodies in the desired dilution (1:30 for 3C5; 1:500 for 2C2; 1:100 for WIC-29.26) for 30 min at 378C in a wet chamber, washed in PBS, incubated with fluoresceinlabeled goat anti-mouse IgG for 30 min at 378C, washed in PBS, and photographed on a Nikon Microphot Fx microscope. Electron microscopy. The infected cells were washed in RPMI medium without serum and fixed in a solution of 0.5% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid in 0.1 M cacodylate buffer, pH 7.3. After 60 min, the cells were washed in the same buffer, postfixed for 1 h in 1% OsO4, 0.8% potassium ferricyanide, and 5 mM CaCl2, in 0.1 M cacodylate buffer, pH 7.3, and embedded in Araldite. Ultrathin sections were prepared for morphological analysis. For the immunolabeling experiments, parasites were fixed in 0.1% glutaraldehyde, 4% paraformaldehyde, and 2% picric acid in 0.1 M cacodylate buffer, pH 7.3, washed in the same buffer, embedded in 10% gelatin, cut into 1-mm2 pieces, and maintained overnight at 48C in 2.3 M sucrose. After freezing in liquid nitrogen, the samples were dehydrated in methanol by cryosubstitution at 2908C for 48 h and embedded in Lowicryl HM20 resin at 2458C. Ultrathin sections were collected on 300-mesh nickel grids, incubated for 30 min with 0.05 M glycine in PBS, followed by 30 min in PBS containing 5% bovine serum albumin (BSA), 0.1% cold water fish skin, and 5% normal serum. The material was incubated overnight with the primary antibody (10 mg/ml) in PBS–BSA, washed with PBS–BSA, and incubated for 60 min at room temperature with anti-IgG mouse antibody (1:20) coupled to colloidal gold particles (10 nm). After this step, the grids were washed with PBS and distilled water, stained with uranyl acetate, and observed in a Jeol 1200 EXII transmission electron microscope. Control grids were made incubating grids with anti-epimastigote polyclonal antibody or with colloidal gold-anti-mouse IgG only. Organelle positioning in parasites. Acidic vacuoles were detected by incubation of 108/ml epimastigotes with 3 mM acridine orange for 15 min at 378C, as described (Docampo et al. 1995), and observed under the fluorescence microscope. Detergent-extracted cytoskeleton was submitted to negative staining for observation in the transmission electron microscope (Robinson et al. 1995). Epimastigotes from acellular culture or intracellular epimastigotes were deposited on formvar carbon-coated grids and treated with a mixture of 1% Nonidet-P40, 1 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 mM leupepstatin, 20 mM E64, and 2 mM benzamidine in PEME buffer (100 mM piperazineN,N8-bis(2-ethane sulphonic acid, pH 6.9, 2 mM N,N,N8,N8- tetracetic acid, 1 mM MgS04, 0.1 mM ethylenediaminetetraacetic acid) for 3 min. The samples were fixed in 2.5% glutaraldehyde in PEME buffer, stained with a saturated aqueous solution of uranyl acetate and observed in a transmission electron microscope. Eighteen micrographs of cytoskeletons from epimastigotes grown in axenic medium and 34 from
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two distinct preparations of intracellular epimastigotes were employed to measure the following parameters: (L) parasite body length, (NP) distance between the center of the nucleus and the parasite posterior extremity, (NK) distance between the center of the nucleus and the kinetoplast, (KP) distance between the kinetoplast and the parasite posterior extremity, (KA) distance between the kinetoplast and the parasite anterior extremity; (NK/KP) and (NP/KP) ratios have also been calculated. Parasite reactivity with lectins. Reactivity of parasites (3 3 107/ ml) with lectins was verified by direct fluorescence with wheat germ agglutinin (WGA) and peanut agglutinin (6 mg/ml) or by agglutination (concanavalin A). Paraformaldehyde (2%)-fixed parasites were incubated with fluorescein-labeled lectins for 30 min at 378C, washed with PBS, and mounted on a slide for direct fluorescence observation (Gazzinelli et al. 1991). Fixed parasites (1 3 108) were incubated with concanavalin A (12 mg/ml) for 30 min at 378C, washed with PBS, and mounted on a slide (Alves and Colli 1974).
RESULTS
Detection of intracellular epimastigotes (IE) with 3C5 Mab. In order to characterize IE forms, a monoclonal antibody (3C5 Mab) has been raised. This Mab reacts with typical epimastigote forms from axenic cultures and with the intracellular epimastigotes, but not with trypomastigotes, as judged by immunofluorescence (Figs. 1A–1H). Although a faint reactivity could be observed with amastigote forms, the reactivity of 3C5 Mab was highly intense with the IE forms, typically found in a short and defined period of time (5th–6th day), just before the appearance of trypomastigotes. Mechanically released epimastigote-like forms showed a motility pattern very similar, although somewhat slower, to that of epimastigotes and clearly different from the other defined stages or intermediate transient forms. Probably as a result of multiple and asynchronous infections, cultured cells frequently contained a large amount of IE forms recognized by the antibody and coexisting with the amastigote, trypomastigote, and undefined transient developmental stages (not shown). Similar epimastigote-like forms were seen in the intracellular development of the classical Y strain. Mab 3C5 recognizes 34-, 48-, 60-, 80-, and 97-kDa polypeptides in Western blots of EE extracts (Fig. 2, lane 1) and 34-, 48-, and 60-kDa polypeptides when IE extracts were employed, in addition to a faint band in the 130-kDa region. At least some of the antigens are localized on the cell surface, as suggested by immunoelectron microscopy of EE (Fig. 3A) and IE (Fig. 3B) forms. The background seen in Fig. 3 may be a result of shedding of parasite molecules since neighboring cells of the same preparation that have not been invaded by the parasite did not show any gold labeling.
FIG. 1. Specific labeling of intracellular and extracellular T. cruzi epimastigotes by indirect immunofluorescence with 3C5 Mab. (A, C, E, G) Fluorescence microscopy; (B, D, F, H) phase-contrast microscopy; (A, B) epimastigotes inside CHO-K1 cells, 6th day post-infection; (C, D) epimastigotes from LIT medium; (E, F) trypomastigotes; (G, H) amastigotes, isolated from infected cultured cells. Bars, 10 mm.
Morphological characterization of the intracellular epimastigotes. The morphology of IE was studied by transmission electron microscopy of ultrathin sections (Fig. 4).
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FIG. 2. Antigens recognized by 3C5 Mab. Extracts from epimastigotes grown in LIT medium (1) and from intracellular epimastigotes (2) were analyzed by Western blots after SDS–PAGE and incubation with 3C5 Mab. Molecular masses (kDa) are indicated on the left.
The kinetoplast appears as a slightly concave disk localized between the nucleus and the anterior end of the parasite body with the dense material tightly packed. The nucleus with a spherical shape and a typical nuclear membrane is localized in the central region of the cell (Figs. 4B and 4C), with condensed chromatin localized at the periphery and in the central region of the nucleus, in addition to an evident nucleolus (Fig. 4C). Furthermore, the cytostome, a welldescribed structure found in different stages of T. cruzi (Milder and Deane 1969), is also present in IE (Fig. 4B), as well as membranous and multivesicular structures which are localized in the posterior portion of the parasite (Fig. 4B) and similar to reservosomes, previously described in EE forms (Soares and De Souza 1988). A similar morphology is found in EE forms shown in Fig. 4A for comparison. Acidic vacuoles, detected by incubation with acridine orange, were abundant and distributed through the whole body (not shown), as described previously for epimastigotes (DoCampo et al. 1995). The parameters analyzed in IE and EE forms were similar, such as the morphology of the mitochondrion and its localization throughout the whole body, the round nucleus with a large and central nucleolus and abundant chromatin, and the rod-like structure of the kinetoplast (Figs. 4A–4C). Since the relative positions of organelles are morphological parameters employed to distinguish among stages of T. cruzi, the position of organelles in IE and EE forms was measured in cytoskeleton preparations submitted to negative staining and transmission electron microscopy (Fig. 5 and Table 1). Total lengths of 25.2 6 2.1 and 5.4 6 0.7 mm were measured for EE and IE, respectively. The length of the cell body, without the flagellum, was measured
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as 18.5 mm for the EE and 3.4 mm for the IE population. The distance between the kinetoplast and the nucleus (NK) in relation to the distance between the kinetoplast and the posterior extremity (KP) gave a mean of 0.27 and 0.37 mm for EE and IE forms, respectively. The distance between the nucleus and the posterior extremity (NP) in relation to the distance between the kinetoplast and the posterior extremity (KP) was 0.66 and 0.64 mm for EE and IE, respectively (Table 1). It is important to stress that the position of the kinetoplast in relation to the nucleus varies when several individuals of the population are compared (cf. Figs. 3B and 4B), as found in the EE population, most likely reflecting asynchrony in the differentiation of the population members. The cell surface of intracellular epimastigotes. The cell surfaces of both epimastigote forms were also compared as to their surface charges, sensitivity to complement-mediated lysis, and reactivity with lectins and monoclonal antibodies. IE forms react with the fluorescein-labeled lectins peanut agglutinin (Fig. 6A) and WGA (Fig. 6C) and are agglutinated by concanavalin A (Fig. 6E), suggesting the presence of galactose, mannose, and (GlcNAc)2/NeuNAc on their surface. Although axenic-derived epimastigotes react with the same lectins (Figs. 6B, 6D, and 6F), quantitative differences were apparent, mainly with peanut agglutinin. With the latter, IE (Fig. 6A) reacted much stronger than EE (Fig. 6B). This result suggests that terminal galactose from surface glycoproteins are relatively more exposed, reflecting heterogeneity in surface carbohydrate expression. Since epimastigotes from axenic medium and trypomastigotes from T. cruzi have differences in the negative surface charge, allowing the separation of these forms on DEAE– cellulose (cf. De Souza 1984), binding of IE to the column was then checked in a mixed population of intracellular forms (amastigotes, IE, and trypomastigotes). The majority of amastigotes and trypomastigotes were found in the washing solution (PBSG, 5% glucose-phosphate-buffered saline, pH 8.0), while the IE forms remained bound to the column (Fig. 7), as did the EE forms in a control experiment performed in parallel (not shown). The results strongly indicate that IE have a surface negative charge equivalent to the EE forms and different from amastigotes or trypomastigotes. As epimastigotes are lysed by complement, in contrast to trypomastigotes, lysis of IE by serum as complement source was then verified. Incubation of epimastigotes with fresh normal human serum for 30 min at 378C resulted in 90 and 100% lysis of IE and EE forms, respectively. Absence of total lysis in IE forms may reflect some heterogeneity within the population. A parallel experiment done with amastigotes revealed that only 26% of the population was lysed by complement.
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FIG. 3. Immunoelectron microscopy of epimastigotes of T. cruzi. Thin sections of epimastigotes incubated initially with 3C5 Mab and subsequently with gold-labeled anti-mouse IgG. Gold particles are seen on the surface plasma membrane (arrows) of intracellular epimastigotes (B) and of epimastigotes grown in axenic medium (A). N, nucleus; K, kinetoplast. Bar, 1 mm.
To further characterize the IE forms, their reactivity with stage-specific antibodies was checked by indirect immunofluorescence. The antibodies employed were: WIC-29.26 Mab, which recognizes a carbohydrate epitope present in the 72-kDa glycoprotein from EE forms (Snary et al. 1981); H1A10 Mab, a trypomastigote-specific antibody that reacts with an 85-kDa glycoprotein (Alves et al. 1986); and 2C2
Mab, an amastigote-specific antibody that defines the Ssp4 epitope (Andrews et al. 1987). Both IE and EE forms were recognized by diluted (1:100) WIC-29.26, but not, as expected, by H1A10 Mab (Table 2). A faint reactivity of IE forms with diluted (1:500) 2C2 Mab was observed in comparison with the strong reactivity observed with amastigotes. In contrast, no reactivity was observed with EE forms,
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FIG. 4. Transmission electron microscopy of epimastigote forms. (A) Epimastigotes grown in LIT medium; (B, C) intracellular epimastigotes. N, Nucleus; n, nucleolus; *condensed chromatin; K, kinetoplast; F, flagellum; arrow: mitochondrion; arrowhead: cytostome. Bar, 0.5 mm.
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FIG. 5. Transmission electron microscopy of T. cruzi epimastigotes submitted to detergent extraction, fixation, and negative staining. (A) Epimastigote grown in LIT medium; (B) intracellular epimastigote. N, nucleus; K, kinetoplast. Bar: (A), 2 mm; (B), 1 mm.
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TABLE 1 Morphological Parameters from Axenic Culture-Derived Epimastigotes (EE) and Intracellular Epimastigotes (IE) from Trypanosoma cruzi Parameters (mm) L KA KP NK NP NK/KP NP/KP
EE 18.5 12.2 6.8 1.9 4.5 0.27 0.66
6 6 6 6 6 6 6
IE 5.2 3.7 0.9 0.4 0.9 0.07 0.07
3.42 1.53 2.16 0.81 1.40 0.37 0.64
6 6 6 6 6 6 6
0.56 0.34 0.39 0.18 0.29 0.08 0.07
Note. L, total body length; NP, nucleus-posterior extremity; NK, nucleus-kinetoplast; KA, kinetoplast-anterior extremity; KP, kinetoplast-posterior extremity. Results are means plus or minus standard deviation of 18 (EE) and 34 (IE) independent measurements.
even at higher concentrations of the antibody. It is interesting to point out that EE forms displayed a stronger reactivity with antibodies WIC-29.26 and 3C5 compared to IE forms. These differences, which are predominantly of a quantitative nature, indicate that EE and IE differ slightly in their surface chemical constitution. Development of intracellular epimastigotes in axenic medium. The possibility that IE forms could grow in axenic medium was verified. Intracellular epimastigotes (5 3 106 parasites/ml) were inoculated into LIT medium, normally employed for the growth of EE forms, and the number of parasites was recorded every day. An exponential curve was observed for 5–6 days after the inoculum, followed by a stationary phase, with the parasites reaching a density around 3–5 3 107 parasites/ml. The growth behavior of IE was very similar to that of EE (Fig. 8).
DISCUSSION
The presence of an intracellular epimastigote-like form was described for the first time in tissue culture cells by Meyer and De Oliveira in 1948. Subsequently, rarely has that issue been approached and most of the time it is restricted to only the description of epimastigotes inside cells without further characterization, probably due to their low amounts and appearance for short periods of time. As shown here, these transient forms share some characteristics with amastigotes and trypomastigotes but their predominant properties are those from epimastigotes, including the overall cell shape
and, thus, they have been called intracellular epimastigotelike forms. The development of monoclonal antibody 3C5 Mab allowed the detection of epimastigotes as a transient form between the amastigote and the trypomastigote stages around the 5th–6th day of infection, a period in which trypomastigotes begin to appear in a normal infection of tissue-cultured cells. This antibody reacts with metacyclic trypomastigotes which arise from epimastigote differentiation in axenic media (not shown) and faintly with amastigotes but does not react with tissue-culture-derived trypomastigotes, thus allowing the detection of intracellular epimastigotes (Fig. 1). The intracellular epimastigotes show morphological characteristics encountered in the usual epimastigotes, including cell shape, relative position of organelles, and point of emergence of the flagellum, despite being almost five times shorter than the extracellular epimastigotes (5.4 6 0.7 and 25.2 6 2.1 mm, respectively). Intracellular epimastigotes showed a kinetoplast localized anterior to the nucleus with a characteristic organization of the kinetoplast DNA, a roundshaped nucleus with condensed chromatin at the periphery and a nucleolus in the center, as described for epimastigotes (De Souza 1984). Intracellular epimastigotes can easily be distinguished from amastigotes by the cell shape and the exteriorized flagellum and from trypomastigotes by the cell shape and relative position of the nucleus and kinetoplast, as trypomastigotes show an elongated nucleus localized at the posterior region of the body with a dispersed chromatin and a kinetoplast with a more rounded form. The cytostome, related with endocytosis of nutrients (Soares and De Souza 1988) and present in amastigotes and epimastigotes, but not in trypomastigotes (Milder and Deane 1969), is also found in IE forms (Fig. 4B). Multimembranous structures similar to reservosomes were encountered in the posterior region of IE as it is found in epimastigotes but not in trypomastigotes or amastigotes (Soares and De Souza 1991; Soares et al. 1992). Acidic vacuoles detected by acridine orange are also present in intracellular epimastigotes (not shown). IE and EE forms have some other common characteristics in addition to similar morphology. Under the conditions employed, both forms have similar surface charge and bind to DEAE–celullose, in contrast to trypomastigotes or amastigotes which do not bind to the resin (Kreier et al. 1977; ´ Souto-Padron et al. 1984). Also, IE forms are lysed by normal human serum, a well-known property of epimastigotes, since trypomastigotes and amastigotes are resistant to complement-mediated lysis in the absence of antibodies (Muniz and Borrielo 1945; Nogueira et al. 1975; Kipnis et al. 1981; Krettli and Brener 1976; Rimoldi et al. 1988). Quantitative differences on the cell surface of axenicderived and intracellular epimastigotes were found when
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FIG. 6. Surface carbohydrates in epimastigotes. Reactivity of IE (A, C, E) and EE (B, D, F) forms were analyzed by incubation of parasites with fluorescein-labeled lectins (A–D) or by agglutination of the parasites with Concanavalin A (E, F). (A, B) Peanut agglutinin; (C, D) wheat germ agglutinin; (E, F) Concanavalin A. Bar, 20 mm.
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FIG. 8. Growth curves of intracellular epimastigotes and extracellular epimastigotes in LIT medium at 288C. Symbols: (m) EE; (v) IE.
FIG. 7. Intracellular epimastigotes are retained on DEAE– Cellulose. Elution of fractions 1–18 was performed with PBSG, pH 8.0, and fractions 19–23 were eluted with PBSG, pH 7.0. Symbols: (m) trypomastigotes; (v) amastigotes; (m) epimastigotes.
their reactivities with lectins and/or monoclonal antibodies were compared (Table 2 and Fig. 6). Intracellular epimastigotes are less reactive with concanavalin A, with dilute WIC29.26 Mab, a carbohydrate-specific antibody (Snary et al. 1981), or with 3C5 Mab. On the other hand, intracellular epimastigotes react strongly with peanut agglutinin, specific for b-D-Gal(1-3)-GalNAc. Interestingly, 2C2, a monoclonal
TABLE 2 Trypanosoma cruzi Stages and Reactivity to Antibodies Trypanosoma cruzi developmental forms (clone CL-14) Antibody
EE
IE
AMA
TRYP
3C5 Mab WIC-29.26 Mab H1A10 Mab 2C2 Mab Anti-EE NMS
111 11 2 2 111 2
11 6 2 6 111 2
1 6 2 111 1 2
2 2 11 ND 1 2
Note. EE, Epimastigotes grown in LIT medium; IE, intracellular epimastigotes; AMA, intracellular amastigotes; TRYP, tissue-culturederived trypomastigotes; anti-EE, polyclonal antibody raised against EE extracts; NMS, normal mouse serum. Reactivity: very strong, 111; strong, 11; positive 1; faint, 6; negative, 2.
antibody that recognizes a carbohydrate-containing protein (Andrews et al. 1987) and does not react with EE forms, reacts with intracellular epimastigotes, although faintly compared with the fluorescence displayed by amastigotes. Intracellular epimastigotes showed an exponential growth phase (day 2 to day 6) after inoculation in axenic medium, followed by a stationary phase, with typical IE forms normally found in division. Interestingly, preliminary observations showed that intracellular epimastigotes, similarly to EE forms, did not invade the nonphagocytic CHO-K1 cultured cell line in contrast to trypomastigotes or even amastigotes (Nogueira and Cohn 1976; Umezawa et al. 1985; Ley et al. 1988; Mortara 1991). However, the invasive properties of the transient epimastigote-like forms deserve further studies since their synchronic development may become a powerful tool to determine the time of appearance of surface antigens responsible for host cell invasion. Altogether (Table 3), the results indicate that the intracellular epimastigote has the predominant characteristics of a true epimastigote despite some quantitative differences on the cell surfaces of IE and EE, as detected by the different intensities in the reactivity with lectins and stage-specific antibodies. Notwithstanding, the presence of the 2C2 epitope—albeit considerably less represented compared to amastigotes—indicates that characteristics proper to amastigotes still remain in the intracellular epimastigote-like form. Although the data herein presented corroborate the notion that epimastigotes do appear inside host cells as a transition form in the amastigote–trypomastigote transformation, their presence inside mammalian cells during natural infections was never reported. This is probably due to the fact that in
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TABLE 3 Summary of the Transient Intracellular Epimastigote Characteristics Probes and properties Reactivity with Mab: 3C5 WIC-29.26 H1A10 2C2 Cytostome Invasiveness Flagellum emergence from the cell body Morphometric ratios Surface charge Complement lysis Growth in LIT medium Kinetoplast position and appearance
Comments
IE behavior
Reacts preferentially and intensely with epimastigotes Reacts with carbohydrate epitopes from 72-kDa epimastigote-specific surface glycoprotein Reacts with peptide epitope from the trypomastigote-specific glycoprotein Tc-85 Reacts with Ssp-4, an 84-kDa-specific glycoprotein from amastigotes Present in amastigotes and extracellular epimastigotes Capacity to interionize into nonphagocytic vertebrate cells —
Intense Positive
IE are shorter than epimastigotes from LIT medium — In the absence of antibodies — —
Epimastigote Epimastigote Epimastigote Epimastigote Epimastigote
vitro infections are usually heavier in view of the multiplicities of infection employed and relatively more synchronous than in vivo infections, thus facilitating the observation of the intracellular epimastigotes in the narrow period of the cycle in which they appear. Epimastigotes were consistently found in the lumen of the anal glands of opossums, but not within the stratified epithelium or the striated muscular layer that surrounds the gland (Deane et al. 1984). Considering that amastigote-like forms (spheromastigotes) were described in the intestinal tract of the invertebrate host (cf. De Souza 1984), it is tempting to suppose that the parasite has a continuous differentiation cycle independent of the host (vertebrate or invertebrate), which follows a pattern represented by the sequence trypomastigote–amastigote– epimastigote–trypomastigote. The predominance of a given stage and the period in which it remains as such would be dictated mostly by the environmental conditions. Due to the natural asynchrony by which a T. cruzi population evolves and to the sudden changes in the environment, molecules which are preferentially assigned to a given stage may appear at any other stage as a reflection of the continuous gradient of morphological transitions.
ACKNOWLEDGMENTS This work was financed by a thematic grant from Fundac¸a˜o de ` Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP 95/04562-3) to W.C. and M.J.M.A. and is part of a doctoral degree thesis of M.
Negative Faint Present Noninvasive Epimastigote simile simile simile simile simile simile
Almeida-de-Faria, a fellow from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). The assistance of Dr. A. F. Ribeiro and W. Caldeira from the Instituto de Biocieˆncias, USP, with the confocal microscope is deeply acknowledged.
REFERENCES
Alves, M. J. M., and Colli, W. 1974. Agglutination of Trypanosoma cruzi by Concanavalin A. Journal of Protozoology 21, 575–578. Alves, M. J. M., Abuin, G., Kuwajima, V. Y., and Colli, W. 1986. Partial inhibition of trypomastigotes entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi. Molecular and Biochemical Parasitology 21, 75–82. Andrews, N. W., and Colli, W. 1982. Adhesion and interiorization of Trypanosoma cruzi in mammalian cells. Journal of Protozoology 29, 264–269. Andrews, N. W., Hong, K. S., Robbins, E. S., and Nussenzweig, V. 1987. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Experimental Parasitology 64, 474–484. Brener, Z., and Chiari, E. 1963. Variac¸o˜es morfolo´gicas observadas em diferentes amostras de Trypanosoma cruzi. Revista do Instituto de Medicina Tropical de Sa˜o Paulo 5, 220–224. Brener, Z., Golgher, R., Bertelli, M. S., and Teixeira, J. A. 1976. Straindependent thermosensitivity influencing intracellular differentiation of Trypanosoma cruzi in cell culture. Journal of Protozoology 23, 147–150. Camargo, E. P. 1964. Growth and differentiation in Trypanosoma cruzi.
274 I. Origin of metacyclic trypanosomes in liquid media. Revista do Instituto de Medicina Tropical de Sa˜o Paulo 6, 93–100. Castellani, O., Ribeiro, L. V., and Fernandes, J. F. 1967. Differentiation of Trypanosoma cruzi in culture. Journal of Protozoology 4, 447– 451. Deane, M. P., Lenzi, H. L., and Jansen, A. 1984. Trypanosoma cruzi: Vertebrate and invertebrate cycles in the same mammal host, the opossum Didelphis marsupialis. Memo´rias do Instituto Oswaldo Cruz 79, 513–515. De Souza, W. 1984. Cell biology of Trypanosoma cruzi. International Review of Cytology 86, 197–283. DoCampo, R., Scott, D. A., Vercesi, A., and Moreno, S. N. J. 1995. Intracellular Ca21 storage in acidocalcisomes of Trypanosoma cruzi. Biochemical Journal 310, 1005–1012. Faucher, J. F., Baltz, T., and Petry, K. G. 1995. Detection of an “epimastigote-like” intracellular stage of Trypanosoma cruzi. Parasitology Research 81, 441–443. Gazinelli, R. T., Romanha, A. J., Fontes, G., Chiari, E., Gazzinelli, G., and Brener, Z. 1991. Distribution of carbohydrates recognized by the lectins Euonymus europeus and Concanavalin A in monoxenic and heteroxenic trypanosomatids. Journal of Protozoology 38, 320–325. Kipnis, T. L., David, J. R., Alper, C. A., Sher, A., and Dias da Silva, W. 1981. Enzymatic treatment transforms trypomastigotes of Trypanosoma cruzi into activators of alternative complement pathway and potentiates their uptake by macrophages. Proceedings of the National Academy of Sciences, USA 78, 602–605. Kreier, J. P., Al-Abbassy, S. N., and Seed, T. M. 1977. Trypanosoma cruzi—Surface change characteristics of cultured epimastigotes, trypomastigotes and amastigotes. Revista do Instituto de Medicina Tropical de Sa˜o Paulo 19, 10–20. Krettli, A. U., and Brener, Z. 1976. Protective effects of specific antibodies in Trypanosoma cruzi. Journal of Immunology 116, 755–760. Lanham, S. M., and Godfrey, D. G. 1970. Isolation of salivarian trypanosomes from man and other mammals using DEAE–cellulose. Experimental Parasitology 28, 521–534. Ley, V., Andrews, N. W., Robbins, E. S., and Nussenzweig, V. 1988. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. Journal of Experimental Medicine 168, 649–659. Meyer, H., and De Oliveira, M. X. 1948. Cultivation of Trypanosoma cruzi in tissue cultures: A four year study. Parasitology 39, 91–94. Milder, R., and Deane, P. M. 1969. The cytostome of Trypanosoma cruzi and T. conorhini. Journal of Protozoology 16, 730–737. Morais Reˆgo, S. F. 1956. Soˆbre o encontro de formas tissulares do Trypanosoma cruzi Chagas 1909 no sangue circulante do camundongo branco (Mus musculus). Folia Clinica et Biologica 26, 17–44.
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Mortara, R. A. 1991. Trypanosoma cruzi: Amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells. Experimental Parasitology 73, 1–14. Muniz, J., and Borrielo, A. 1945. Estudo sobre a ac¸a˜o lı´tica de diferentes ´ soros sobre as formas de cultura e sangu¨ıcolas do Schizotrypanum cruzi. Revista Brasileira de Biologia 5, 563–571. Nogueira, N., Bianco, C., and Cohn, Z. 1975. Studies on the selective lysis and purification of Trypanosoma cruzi. Journal of Experimental Medicine 142, 224–229. Nogueira, N., and Cohn, Z. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. The Journal of Experimental Medicine 143, 1402–1420. Rimoldi, M. T., Sher, A., Hieny, S., Lituchy, A., Hammer, C. H., and Joiner, K. 1988. Developmentally regulated expression by Trypanosoma cruzi of molecules that accelerate the decay of complement C3 convertases. Proceedings of the National Academy of Sciences, USA 85, 193–197. Robinson, D. R., Sherwin, T., Ploubidou, A., Byard, E. H., and Gull, K. 1995. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. Journal of Cell Biology 128, 1163–1172. Roman˜a, C., and Meyer, H. 1942. Estudo do ciclo evolutivo de Schizo˜ ´ trypanum cruzi em cultura de tecidos e embriao de galinha. Memorias do Instituto Oswaldo Cruz 37, 19–27. Snary, D., Ferguson, M. A. J., Scott, M., and Allen, A. K. 1981. Cell surface antigens of Trypanosoma cruzi: Use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein. Molecular Biochemical Parasitology 3, 343–356. Soares, M. J., and De Souza, W. 1988. Cytoplasmic organelles of trypanosomatids: A cytochemical and stereological study. Journal of Submicroscopic Cytology and Pathology 20, 349–361. Soares, M. J., and De Souza, W. 1991. Endocytosis of gold-labeled proteins and LDL by Trypanosoma cruzi. Parasitology Research 77, 461–468. Soares, M. J., Souto-Padro´n, T., and De Souza, W. 1992. Identification of a large prelysosomal compartment in the pathogenic protozoon Trypanosoma cruzi. Journal of Cell Science 102, 157–167. Souto-Padro´n, T., De Carvalho, T. U., Chiari, E., and De Souza, W. 1984. Further studies on the cell surface charge of Trypanosoma cruzi. Acta Tropica 41, 215–225. Umezawa, E. S., Milder, R. V., and Abrahamsohn, I. A. 1985. Trypanosoma cruzi amastigotes: Development in vitro and infectivity in vivo of the forms isolated from spleen and liver. Acta Tropica 42, 25–32. Zingales, B., and Colli, W. 1985. Trypanosoma cruzi: Interaction with host cells. In “Current Topics in Microbiology and Immunology” (Hudson, L., Ed.), Vol. 117, pp. 129–152. Springer Verlag, Berlin-Heidelberg. Received 19 January 1999; accepted with revision 1 April 1999
Trypanosoma cruzi: Characterization of an Intracellular Epimastigote-like Form
M. Almeida-de-Faria, E. Freymu¨ller,* W. Colli, and M. J. M. Alves Departamento de Bioquı´mica, Instituto de Quı´mica, and *Centro de Microscopia Eletroˆnica, UNIFESP/EPM, Universidade de Sa˜o Paulo, C.P. 26077, 05599-970 Sa˜o Paulo, Brazil
Almeida-de-Faria, M., Freymu¨ller, 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 338C. 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 6 0.7 mm) than the extracellular epimastigote (25.2 6 2.1 mm), 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. q 1999 Academic Press Index Descriptors and Abbreviations: Trypanosoma cruzi; intracellular epimastigotes; EE, extracellular epimastigotes derived from axenic
0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
culture medium; IE, intracellular epimastigotes derived from tissue cultured cells; BSA, bovine serum albumin; FCS, fetal calf serum; LIT medium, liver infusion and tryptose-containing medium; Mab, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBSG, PBS supplemented with 5% glucose, pH 8.0; SDS, sodium dodecyl sulfate.
INTRODUCTION
Trypanosoma cruzi, the etiologic agent of Chagas’ disease, has a complex life cycle characterized by several developmental forms present in vertebrate and invertebrate hosts. Based mainly on morphological criteria, such as the spindle shape of the parasite, and on the position of the kinetoplast relative to the nucleus and the flagellar emergency region, three forms are classically described: (1) amastigotes, an intracellular dividing form possessing an incipient flagellum and present in the cytoplasm of mammalian cells; (2) trypomastigotes, an infective, flagellated and nondividing form present in vertebrate and invertebrate hosts; and (3) epimastigotes, an extracellular flagellated, noninfective and dividing stage present typically in the invertebrate host (cf. Zingales and Colli 1985). Trypomastigotes escape from the phagocytic vacuole shortly after cell entry and differentiate into amastigotes. Optical microscopic observations made long ago based solely on morphology suggested the presence of ˜ an intracellular epimastigote-like form (Romana and Meyer
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264 1942; Meyer and De Oliveira 1948; Morais Rego 1956). Recently, the existence of the intracellular epimastigote-like forms in less than 1% of all infected cells has been reported, using for detection a specific monoclonal antibody to epimastigotes (Faucher et al. 1995). Few studies elucidating the intracellular cycle of the T. cruzi infection have been made. Usually, the literature refers to the intracellular amastigote–trypomastigote transition in the cytoplasm of the host cell without mentioning the possible existence of an epimastigote intermediate, probably due to the scant evidence in support of its existence. Due to the scarce characterization of the suggested intracellular epimastigote-like stage, a thorough study of that form has been made, taking into account morphometric, biological, and biochemical parameters as well as ultrastructural localization of antigens. These parameters have been compared with those from bona fide epimastigote forms grown in axenic liquid medium.
MATERIALS AND METHODS
Cells. Epimastigote forms of T. cruzi CL-14 clone, a clone derived from the CL strain (Brener and Chiari 1963) were grown in LIT medium (liver infusion–tryptose) at 288C (Camargo 1964; Castellani et al. 1967) (EE forms, for extracellular epimastigotes). CHO-K1 cells were maintained in RPMI medium, supplemented with 10% heatinactivated newborn calf serum, at 378C with 5% CO2. Intracellular epimastigotes (IE), trypomastigotes, and other intracellular stages were obtained by infection of CHO-K1 cells with trypomastigotes. Host cells were infected in the presence of 2% newborn calf serum (Andrews and Colli 1982) and maintained at 338C, as the intracellular morphogenesis of that strain is temperature sensitive, as described (Brener et al. 1976). Under these conditions, the intracellular epimastigotes appear around day 5 post-infection and trypomastigotes are found in the extracellular medium from the 7th to the 10th day. When necessary, the infected cells were disrupted in a Potter and the parasites separated by centrifugation (480g, 5 min) from host cell debris. Parasites were pelleted by centrifugation, resuspended in RPMI medium, and centrifuged on 1 ml of lymphoprep for 10 min at 4300g (Nycomed Pharma AS). Separation of T. cruzi forms by DEAE–cellulose. A mixture of intracellular parasites (1 3 109) in phosphate-buffered saline supplemented with 5% glucose (PBSG), pH 8.0, was overlayed on a 10-ml DEAE–cellulose column and eluted with the same buffer (Lanham and Godfrey 1970). Fractions of 1 ml were collected and the amount of each parasite form was determined by microscopic observation. Elution of epimastigotes from the column was partially achieved by elution with the same buffer with the pH lowered to 7.0. Complement mediated lysis. Parasites (1 3 107/ml) in DME medium were incubated with or without (control) 10% fresh human serum. After 30 min at 378C, the number of intact parasites was counted. Monoclonal and polyclonal antibodies. Monoclonal antibodies
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were obtained as previously described (Alves et al. 1986), except that extracts from epimastigotes grown in LIT medium were employed to immunize Balb/c mice. One monoclonal antibody (3C5 Mab) was selected based on its reactivity with intra- and extracellular epimastigotes by indirect immunofluorescence. The antibody isotype was determined as IgG2b, using an isotyping kit (Boehringer Mannheim Biochemicals). WIC-29.26, an anti-carbohydrate monoclonal antibody specific for epimastigotes (Snary et al. 1981), was kindly provided by Dr. G. A. M. Cross (Rockefeller University) and 2C2 monoclonal antibody (Andrews et al. 1987) was kindly provided by Dr. N. W. Andrews (Yale University). Indirect immunofluorescence. Parasites or infected CHO-K1 cells were washed three times in RPMI medium without serum and fixed in 2% paraformaldehyde in PBS, pH 7.4, for 1 h at room temperature. The cells were incubated with the antibodies in the desired dilution (1:30 for 3C5; 1:500 for 2C2; 1:100 for WIC-29.26) for 30 min at 378C in a wet chamber, washed in PBS, incubated with fluoresceinlabeled goat anti-mouse IgG for 30 min at 378C, washed in PBS, and photographed on a Nikon Microphot Fx microscope. Electron microscopy. The infected cells were washed in RPMI medium without serum and fixed in a solution of 0.5% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid in 0.1 M cacodylate buffer, pH 7.3. After 60 min, the cells were washed in the same buffer, postfixed for 1 h in 1% OsO4, 0.8% potassium ferricyanide, and 5 mM CaCl2, in 0.1 M cacodylate buffer, pH 7.3, and embedded in Araldite. Ultrathin sections were prepared for morphological analysis. For the immunolabeling experiments, parasites were fixed in 0.1% glutaraldehyde, 4% paraformaldehyde, and 2% picric acid in 0.1 M cacodylate buffer, pH 7.3, washed in the same buffer, embedded in 10% gelatin, cut into 1-mm2 pieces, and maintained overnight at 48C in 2.3 M sucrose. After freezing in liquid nitrogen, the samples were dehydrated in methanol by cryosubstitution at 2908C for 48 h and embedded in Lowicryl HM20 resin at 2458C. Ultrathin sections were collected on 300-mesh nickel grids, incubated for 30 min with 0.05 M glycine in PBS, followed by 30 min in PBS containing 5% bovine serum albumin (BSA), 0.1% cold water fish skin, and 5% normal serum. The material was incubated overnight with the primary antibody (10 mg/ml) in PBS–BSA, washed with PBS–BSA, and incubated for 60 min at room temperature with anti-IgG mouse antibody (1:20) coupled to colloidal gold particles (10 nm). After this step, the grids were washed with PBS and distilled water, stained with uranyl acetate, and observed in a Jeol 1200 EXII transmission electron microscope. Control grids were made incubating grids with anti-epimastigote polyclonal antibody or with colloidal gold-anti-mouse IgG only. Organelle positioning in parasites. Acidic vacuoles were detected by incubation of 108/ml epimastigotes with 3 mM acridine orange for 15 min at 378C, as described (Docampo et al. 1995), and observed under the fluorescence microscope. Detergent-extracted cytoskeleton was submitted to negative staining for observation in the transmission electron microscope (Robinson et al. 1995). Epimastigotes from acellular culture or intracellular epimastigotes were deposited on formvar carbon-coated grids and treated with a mixture of 1% Nonidet-P40, 1 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 mM leupepstatin, 20 mM E64, and 2 mM benzamidine in PEME buffer (100 mM piperazineN,N8-bis(2-ethane sulphonic acid, pH 6.9, 2 mM N,N,N8,N8- tetracetic acid, 1 mM MgS04, 0.1 mM ethylenediaminetetraacetic acid) for 3 min. The samples were fixed in 2.5% glutaraldehyde in PEME buffer, stained with a saturated aqueous solution of uranyl acetate and observed in a transmission electron microscope. Eighteen micrographs of cytoskeletons from epimastigotes grown in axenic medium and 34 from
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two distinct preparations of intracellular epimastigotes were employed to measure the following parameters: (L) parasite body length, (NP) distance between the center of the nucleus and the parasite posterior extremity, (NK) distance between the center of the nucleus and the kinetoplast, (KP) distance between the kinetoplast and the parasite posterior extremity, (KA) distance between the kinetoplast and the parasite anterior extremity; (NK/KP) and (NP/KP) ratios have also been calculated. Parasite reactivity with lectins. Reactivity of parasites (3 3 107/ ml) with lectins was verified by direct fluorescence with wheat germ agglutinin (WGA) and peanut agglutinin (6 mg/ml) or by agglutination (concanavalin A). Paraformaldehyde (2%)-fixed parasites were incubated with fluorescein-labeled lectins for 30 min at 378C, washed with PBS, and mounted on a slide for direct fluorescence observation (Gazzinelli et al. 1991). Fixed parasites (1 3 108) were incubated with concanavalin A (12 mg/ml) for 30 min at 378C, washed with PBS, and mounted on a slide (Alves and Colli 1974).
RESULTS
Detection of intracellular epimastigotes (IE) with 3C5 Mab. In order to characterize IE forms, a monoclonal antibody (3C5 Mab) has been raised. This Mab reacts with typical epimastigote forms from axenic cultures and with the intracellular epimastigotes, but not with trypomastigotes, as judged by immunofluorescence (Figs. 1A–1H). Although a faint reactivity could be observed with amastigote forms, the reactivity of 3C5 Mab was highly intense with the IE forms, typically found in a short and defined period of time (5th–6th day), just before the appearance of trypomastigotes. Mechanically released epimastigote-like forms showed a motility pattern very similar, although somewhat slower, to that of epimastigotes and clearly different from the other defined stages or intermediate transient forms. Probably as a result of multiple and asynchronous infections, cultured cells frequently contained a large amount of IE forms recognized by the antibody and coexisting with the amastigote, trypomastigote, and undefined transient developmental stages (not shown). Similar epimastigote-like forms were seen in the intracellular development of the classical Y strain. Mab 3C5 recognizes 34-, 48-, 60-, 80-, and 97-kDa polypeptides in Western blots of EE extracts (Fig. 2, lane 1) and 34-, 48-, and 60-kDa polypeptides when IE extracts were employed, in addition to a faint band in the 130-kDa region. At least some of the antigens are localized on the cell surface, as suggested by immunoelectron microscopy of EE (Fig. 3A) and IE (Fig. 3B) forms. The background seen in Fig. 3 may be a result of shedding of parasite molecules since neighboring cells of the same preparation that have not been invaded by the parasite did not show any gold labeling.
FIG. 1. Specific labeling of intracellular and extracellular T. cruzi epimastigotes by indirect immunofluorescence with 3C5 Mab. (A, C, E, G) Fluorescence microscopy; (B, D, F, H) phase-contrast microscopy; (A, B) epimastigotes inside CHO-K1 cells, 6th day post-infection; (C, D) epimastigotes from LIT medium; (E, F) trypomastigotes; (G, H) amastigotes, isolated from infected cultured cells. Bars, 10 mm.
Morphological characterization of the intracellular epimastigotes. The morphology of IE was studied by transmission electron microscopy of ultrathin sections (Fig. 4).
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FIG. 2. Antigens recognized by 3C5 Mab. Extracts from epimastigotes grown in LIT medium (1) and from intracellular epimastigotes (2) were analyzed by Western blots after SDS–PAGE and incubation with 3C5 Mab. Molecular masses (kDa) are indicated on the left.
The kinetoplast appears as a slightly concave disk localized between the nucleus and the anterior end of the parasite body with the dense material tightly packed. The nucleus with a spherical shape and a typical nuclear membrane is localized in the central region of the cell (Figs. 4B and 4C), with condensed chromatin localized at the periphery and in the central region of the nucleus, in addition to an evident nucleolus (Fig. 4C). Furthermore, the cytostome, a welldescribed structure found in different stages of T. cruzi (Milder and Deane 1969), is also present in IE (Fig. 4B), as well as membranous and multivesicular structures which are localized in the posterior portion of the parasite (Fig. 4B) and similar to reservosomes, previously described in EE forms (Soares and De Souza 1988). A similar morphology is found in EE forms shown in Fig. 4A for comparison. Acidic vacuoles, detected by incubation with acridine orange, were abundant and distributed through the whole body (not shown), as described previously for epimastigotes (DoCampo et al. 1995). The parameters analyzed in IE and EE forms were similar, such as the morphology of the mitochondrion and its localization throughout the whole body, the round nucleus with a large and central nucleolus and abundant chromatin, and the rod-like structure of the kinetoplast (Figs. 4A–4C). Since the relative positions of organelles are morphological parameters employed to distinguish among stages of T. cruzi, the position of organelles in IE and EE forms was measured in cytoskeleton preparations submitted to negative staining and transmission electron microscopy (Fig. 5 and Table 1). Total lengths of 25.2 6 2.1 and 5.4 6 0.7 mm were measured for EE and IE, respectively. The length of the cell body, without the flagellum, was measured
ALMEIDA-DE-FARIA ET AL.
as 18.5 mm for the EE and 3.4 mm for the IE population. The distance between the kinetoplast and the nucleus (NK) in relation to the distance between the kinetoplast and the posterior extremity (KP) gave a mean of 0.27 and 0.37 mm for EE and IE forms, respectively. The distance between the nucleus and the posterior extremity (NP) in relation to the distance between the kinetoplast and the posterior extremity (KP) was 0.66 and 0.64 mm for EE and IE, respectively (Table 1). It is important to stress that the position of the kinetoplast in relation to the nucleus varies when several individuals of the population are compared (cf. Figs. 3B and 4B), as found in the EE population, most likely reflecting asynchrony in the differentiation of the population members. The cell surface of intracellular epimastigotes. The cell surfaces of both epimastigote forms were also compared as to their surface charges, sensitivity to complement-mediated lysis, and reactivity with lectins and monoclonal antibodies. IE forms react with the fluorescein-labeled lectins peanut agglutinin (Fig. 6A) and WGA (Fig. 6C) and are agglutinated by concanavalin A (Fig. 6E), suggesting the presence of galactose, mannose, and (GlcNAc)2/NeuNAc on their surface. Although axenic-derived epimastigotes react with the same lectins (Figs. 6B, 6D, and 6F), quantitative differences were apparent, mainly with peanut agglutinin. With the latter, IE (Fig. 6A) reacted much stronger than EE (Fig. 6B). This result suggests that terminal galactose from surface glycoproteins are relatively more exposed, reflecting heterogeneity in surface carbohydrate expression. Since epimastigotes from axenic medium and trypomastigotes from T. cruzi have differences in the negative surface charge, allowing the separation of these forms on DEAE– cellulose (cf. De Souza 1984), binding of IE to the column was then checked in a mixed population of intracellular forms (amastigotes, IE, and trypomastigotes). The majority of amastigotes and trypomastigotes were found in the washing solution (PBSG, 5% glucose-phosphate-buffered saline, pH 8.0), while the IE forms remained bound to the column (Fig. 7), as did the EE forms in a control experiment performed in parallel (not shown). The results strongly indicate that IE have a surface negative charge equivalent to the EE forms and different from amastigotes or trypomastigotes. As epimastigotes are lysed by complement, in contrast to trypomastigotes, lysis of IE by serum as complement source was then verified. Incubation of epimastigotes with fresh normal human serum for 30 min at 378C resulted in 90 and 100% lysis of IE and EE forms, respectively. Absence of total lysis in IE forms may reflect some heterogeneity within the population. A parallel experiment done with amastigotes revealed that only 26% of the population was lysed by complement.
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FIG. 3. Immunoelectron microscopy of epimastigotes of T. cruzi. Thin sections of epimastigotes incubated initially with 3C5 Mab and subsequently with gold-labeled anti-mouse IgG. Gold particles are seen on the surface plasma membrane (arrows) of intracellular epimastigotes (B) and of epimastigotes grown in axenic medium (A). N, nucleus; K, kinetoplast. Bar, 1 mm.
To further characterize the IE forms, their reactivity with stage-specific antibodies was checked by indirect immunofluorescence. The antibodies employed were: WIC-29.26 Mab, which recognizes a carbohydrate epitope present in the 72-kDa glycoprotein from EE forms (Snary et al. 1981); H1A10 Mab, a trypomastigote-specific antibody that reacts with an 85-kDa glycoprotein (Alves et al. 1986); and 2C2
Mab, an amastigote-specific antibody that defines the Ssp4 epitope (Andrews et al. 1987). Both IE and EE forms were recognized by diluted (1:100) WIC-29.26, but not, as expected, by H1A10 Mab (Table 2). A faint reactivity of IE forms with diluted (1:500) 2C2 Mab was observed in comparison with the strong reactivity observed with amastigotes. In contrast, no reactivity was observed with EE forms,
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FIG. 4. Transmission electron microscopy of epimastigote forms. (A) Epimastigotes grown in LIT medium; (B, C) intracellular epimastigotes. N, Nucleus; n, nucleolus; *condensed chromatin; K, kinetoplast; F, flagellum; arrow: mitochondrion; arrowhead: cytostome. Bar, 0.5 mm.
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FIG. 5. Transmission electron microscopy of T. cruzi epimastigotes submitted to detergent extraction, fixation, and negative staining. (A) Epimastigote grown in LIT medium; (B) intracellular epimastigote. N, nucleus; K, kinetoplast. Bar: (A), 2 mm; (B), 1 mm.
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TABLE 1 Morphological Parameters from Axenic Culture-Derived Epimastigotes (EE) and Intracellular Epimastigotes (IE) from Trypanosoma cruzi Parameters (mm) L KA KP NK NP NK/KP NP/KP
EE 18.5 12.2 6.8 1.9 4.5 0.27 0.66
6 6 6 6 6 6 6
IE 5.2 3.7 0.9 0.4 0.9 0.07 0.07
3.42 1.53 2.16 0.81 1.40 0.37 0.64
6 6 6 6 6 6 6
0.56 0.34 0.39 0.18 0.29 0.08 0.07
Note. L, total body length; NP, nucleus-posterior extremity; NK, nucleus-kinetoplast; KA, kinetoplast-anterior extremity; KP, kinetoplast-posterior extremity. Results are means plus or minus standard deviation of 18 (EE) and 34 (IE) independent measurements.
even at higher concentrations of the antibody. It is interesting to point out that EE forms displayed a stronger reactivity with antibodies WIC-29.26 and 3C5 compared to IE forms. These differences, which are predominantly of a quantitative nature, indicate that EE and IE differ slightly in their surface chemical constitution. Development of intracellular epimastigotes in axenic medium. The possibility that IE forms could grow in axenic medium was verified. Intracellular epimastigotes (5 3 106 parasites/ml) were inoculated into LIT medium, normally employed for the growth of EE forms, and the number of parasites was recorded every day. An exponential curve was observed for 5–6 days after the inoculum, followed by a stationary phase, with the parasites reaching a density around 3–5 3 107 parasites/ml. The growth behavior of IE was very similar to that of EE (Fig. 8).
DISCUSSION
The presence of an intracellular epimastigote-like form was described for the first time in tissue culture cells by Meyer and De Oliveira in 1948. Subsequently, rarely has that issue been approached and most of the time it is restricted to only the description of epimastigotes inside cells without further characterization, probably due to their low amounts and appearance for short periods of time. As shown here, these transient forms share some characteristics with amastigotes and trypomastigotes but their predominant properties are those from epimastigotes, including the overall cell shape
and, thus, they have been called intracellular epimastigotelike forms. The development of monoclonal antibody 3C5 Mab allowed the detection of epimastigotes as a transient form between the amastigote and the trypomastigote stages around the 5th–6th day of infection, a period in which trypomastigotes begin to appear in a normal infection of tissue-cultured cells. This antibody reacts with metacyclic trypomastigotes which arise from epimastigote differentiation in axenic media (not shown) and faintly with amastigotes but does not react with tissue-culture-derived trypomastigotes, thus allowing the detection of intracellular epimastigotes (Fig. 1). The intracellular epimastigotes show morphological characteristics encountered in the usual epimastigotes, including cell shape, relative position of organelles, and point of emergence of the flagellum, despite being almost five times shorter than the extracellular epimastigotes (5.4 6 0.7 and 25.2 6 2.1 mm, respectively). Intracellular epimastigotes showed a kinetoplast localized anterior to the nucleus with a characteristic organization of the kinetoplast DNA, a roundshaped nucleus with condensed chromatin at the periphery and a nucleolus in the center, as described for epimastigotes (De Souza 1984). Intracellular epimastigotes can easily be distinguished from amastigotes by the cell shape and the exteriorized flagellum and from trypomastigotes by the cell shape and relative position of the nucleus and kinetoplast, as trypomastigotes show an elongated nucleus localized at the posterior region of the body with a dispersed chromatin and a kinetoplast with a more rounded form. The cytostome, related with endocytosis of nutrients (Soares and De Souza 1988) and present in amastigotes and epimastigotes, but not in trypomastigotes (Milder and Deane 1969), is also found in IE forms (Fig. 4B). Multimembranous structures similar to reservosomes were encountered in the posterior region of IE as it is found in epimastigotes but not in trypomastigotes or amastigotes (Soares and De Souza 1991; Soares et al. 1992). Acidic vacuoles detected by acridine orange are also present in intracellular epimastigotes (not shown). IE and EE forms have some other common characteristics in addition to similar morphology. Under the conditions employed, both forms have similar surface charge and bind to DEAE–celullose, in contrast to trypomastigotes or amastigotes which do not bind to the resin (Kreier et al. 1977; ´ Souto-Padron et al. 1984). Also, IE forms are lysed by normal human serum, a well-known property of epimastigotes, since trypomastigotes and amastigotes are resistant to complement-mediated lysis in the absence of antibodies (Muniz and Borrielo 1945; Nogueira et al. 1975; Kipnis et al. 1981; Krettli and Brener 1976; Rimoldi et al. 1988). Quantitative differences on the cell surface of axenicderived and intracellular epimastigotes were found when
INTRACELLULAR EPIMASTIGOTES OF T. cruzi
271
FIG. 6. Surface carbohydrates in epimastigotes. Reactivity of IE (A, C, E) and EE (B, D, F) forms were analyzed by incubation of parasites with fluorescein-labeled lectins (A–D) or by agglutination of the parasites with Concanavalin A (E, F). (A, B) Peanut agglutinin; (C, D) wheat germ agglutinin; (E, F) Concanavalin A. Bar, 20 mm.
272
ALMEIDA-DE-FARIA ET AL.
FIG. 8. Growth curves of intracellular epimastigotes and extracellular epimastigotes in LIT medium at 288C. Symbols: (m) EE; (v) IE.
FIG. 7. Intracellular epimastigotes are retained on DEAE– Cellulose. Elution of fractions 1–18 was performed with PBSG, pH 8.0, and fractions 19–23 were eluted with PBSG, pH 7.0. Symbols: (m) trypomastigotes; (v) amastigotes; (m) epimastigotes.
their reactivities with lectins and/or monoclonal antibodies were compared (Table 2 and Fig. 6). Intracellular epimastigotes are less reactive with concanavalin A, with dilute WIC29.26 Mab, a carbohydrate-specific antibody (Snary et al. 1981), or with 3C5 Mab. On the other hand, intracellular epimastigotes react strongly with peanut agglutinin, specific for b-D-Gal(1-3)-GalNAc. Interestingly, 2C2, a monoclonal
TABLE 2 Trypanosoma cruzi Stages and Reactivity to Antibodies Trypanosoma cruzi developmental forms (clone CL-14) Antibody
EE
IE
AMA
TRYP
3C5 Mab WIC-29.26 Mab H1A10 Mab 2C2 Mab Anti-EE NMS
111 11 2 2 111 2
11 6 2 6 111 2
1 6 2 111 1 2
2 2 11 ND 1 2
Note. EE, Epimastigotes grown in LIT medium; IE, intracellular epimastigotes; AMA, intracellular amastigotes; TRYP, tissue-culturederived trypomastigotes; anti-EE, polyclonal antibody raised against EE extracts; NMS, normal mouse serum. Reactivity: very strong, 111; strong, 11; positive 1; faint, 6; negative, 2.
antibody that recognizes a carbohydrate-containing protein (Andrews et al. 1987) and does not react with EE forms, reacts with intracellular epimastigotes, although faintly compared with the fluorescence displayed by amastigotes. Intracellular epimastigotes showed an exponential growth phase (day 2 to day 6) after inoculation in axenic medium, followed by a stationary phase, with typical IE forms normally found in division. Interestingly, preliminary observations showed that intracellular epimastigotes, similarly to EE forms, did not invade the nonphagocytic CHO-K1 cultured cell line in contrast to trypomastigotes or even amastigotes (Nogueira and Cohn 1976; Umezawa et al. 1985; Ley et al. 1988; Mortara 1991). However, the invasive properties of the transient epimastigote-like forms deserve further studies since their synchronic development may become a powerful tool to determine the time of appearance of surface antigens responsible for host cell invasion. Altogether (Table 3), the results indicate that the intracellular epimastigote has the predominant characteristics of a true epimastigote despite some quantitative differences on the cell surfaces of IE and EE, as detected by the different intensities in the reactivity with lectins and stage-specific antibodies. Notwithstanding, the presence of the 2C2 epitope—albeit considerably less represented compared to amastigotes—indicates that characteristics proper to amastigotes still remain in the intracellular epimastigote-like form. Although the data herein presented corroborate the notion that epimastigotes do appear inside host cells as a transition form in the amastigote–trypomastigote transformation, their presence inside mammalian cells during natural infections was never reported. This is probably due to the fact that in
273
INTRACELLULAR EPIMASTIGOTES OF T. cruzi
TABLE 3 Summary of the Transient Intracellular Epimastigote Characteristics Probes and properties Reactivity with Mab: 3C5 WIC-29.26 H1A10 2C2 Cytostome Invasiveness Flagellum emergence from the cell body Morphometric ratios Surface charge Complement lysis Growth in LIT medium Kinetoplast position and appearance
Comments
IE behavior
Reacts preferentially and intensely with epimastigotes Reacts with carbohydrate epitopes from 72-kDa epimastigote-specific surface glycoprotein Reacts with peptide epitope from the trypomastigote-specific glycoprotein Tc-85 Reacts with Ssp-4, an 84-kDa-specific glycoprotein from amastigotes Present in amastigotes and extracellular epimastigotes Capacity to interionize into nonphagocytic vertebrate cells —
Intense Positive
IE are shorter than epimastigotes from LIT medium — In the absence of antibodies — —
Epimastigote Epimastigote Epimastigote Epimastigote Epimastigote
vitro infections are usually heavier in view of the multiplicities of infection employed and relatively more synchronous than in vivo infections, thus facilitating the observation of the intracellular epimastigotes in the narrow period of the cycle in which they appear. Epimastigotes were consistently found in the lumen of the anal glands of opossums, but not within the stratified epithelium or the striated muscular layer that surrounds the gland (Deane et al. 1984). Considering that amastigote-like forms (spheromastigotes) were described in the intestinal tract of the invertebrate host (cf. De Souza 1984), it is tempting to suppose that the parasite has a continuous differentiation cycle independent of the host (vertebrate or invertebrate), which follows a pattern represented by the sequence trypomastigote–amastigote– epimastigote–trypomastigote. The predominance of a given stage and the period in which it remains as such would be dictated mostly by the environmental conditions. Due to the natural asynchrony by which a T. cruzi population evolves and to the sudden changes in the environment, molecules which are preferentially assigned to a given stage may appear at any other stage as a reflection of the continuous gradient of morphological transitions.
ACKNOWLEDGMENTS This work was financed by a thematic grant from Fundac¸a˜o de ` Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP 95/04562-3) to W.C. and M.J.M.A. and is part of a doctoral degree thesis of M.
Negative Faint Present Noninvasive Epimastigote simile simile simile simile simile simile
Almeida-de-Faria, a fellow from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). The assistance of Dr. A. F. Ribeiro and W. Caldeira from the Instituto de Biocieˆncias, USP, with the confocal microscope is deeply acknowledged.
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