Early and late molecular and morphologic changes that
occur during the in vitro transformation of Trypanosoma
cruzi metacyclic trypomastigotes to amastigotes

 

VÍCTOR T CONTRERAS, MARÍA C NAVARRO, ANA R DE LIMA, FRANCY DURAN, ROSA ARTEAGA AND YUNAIMY FRANCO

Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Bárbula, Valencia, Estado Carabobo, Venezuela.

 

ABSTRACT

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37° C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37° C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.

Key terms: Amastigogenesis; differentiation; macromolecular changes; metacyclic trypomastigotes; Trypanosoma cruzi.

Corresponding Author: Víctor T. Contreras. c/o VLN 1500, PO BOX 025685, Miami, FL 33102-5685. Telephone-Fax: (582) 41-8673342. e-mail: convictu@cantv.net.ve

Received: December 13, 2001. In revised form: January 30, 2002. Accepted: January 31, 2002

INTRODUCTION

Trypanosoma cruzi, the etiological agent of Chagas' disease, undergoes a complex biological cycle involving at least four major stages: epimastigotes, metacyclic trypomastigotes, bloodstream trypomastigotes and amastigotes in two types of host: an insect vector and a vertebrate (Kollien and Schaub, 2000; Tyler and Engman, 2001). It is transmitted as the infective metacyclic form to the mammalian host via the insect vector's feces. The primary amastigogenesis of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. This differentiation process is a fundamental step in the life cycle of the parasite because in addition to guaranteeing the survival of the parasite in the new host, the amastigotes are as virulent as trypomastigotes in a murine experimental model (Ley et al., 1988).

To study the mechanism by which the T. cruzi trypomastigotes perform their morphological transformations, it becomes necessary to obtain stage-specific molecular or biochemical patterns. In the Leishmania species, another group of trypanosomatids, studying the complete developmental cycle of Leishmania mexicana in axenic culture showed that the morphological changes observed as the developmental sequence proceed from stage to stage were accompanied by appropriate changes in biochemical properties (Bates, 1994).

Most of the information showing ultra-structural, antigenic, and physiological changes during the amastigogenesis process came from the studies made with tissue culture-derived trypomastigotes (Andrews et al, 1987; Tomlinson et al, 1995). Although the trypomastigote bloodstream can appear to be similar in morphology and share some biological properties with metacyclic forms, they are functionally different with respect to their commitment to the production of an amastigote stage. Thus, while 100% of trypomastigote-amastigote transformation in axenic medium at low pH was obtained beginning with tissue culture-derived trypomastigotes (Tomlinson et al, 1995), the metacyclic trypomastigotes under identical conditions do not differentiate, but rather undergo cell death over a 24 h period (Tyler and Engman, 2001). Aside from extensive inter-strain antigenic polymorphism, the metacyclic trypomastigotes display stage-specific antigens that are different from the tissue culture-derived trypomastigotes and their mode of interaction with host cells is not identical (Burleigh and Andrews, 1995; Yoshida et al, 1997). It is known that as the transformation from trypomastigotes to amastigotes occurs, significant changes in morphology are accompanied by the acquisition of new surface molecules in the molecular weight range of 70-92 kDa (Barros et al, 1997). However, no studies comparing the biochemical patterns of trypomastigotes-derived and metacyclic-derived amastigotes were found.

Several authors have reported the long term routine maintenance of T. cruzi extracellular amastigotes in axenic culture by successive transfer in fresh medium (Villalta and Kierzenbaum 1982; Rondinelli et al, 1988; Engel and Dvorak, 1988). Engel and Dvorak (1988) devised a protocol for the production and maintenance of T. cruzi amastigotes in culture at 27° C based upon changing the culture medium at pre-selected intervals. Their data indicated that the amastigote population was composed of predominantly G₁ cells at the end of two DNA synthetic cycles. They stated that the change of culture medium initiates a unidirectional response in amastigotes consisting of the inhibition of cell transformation, stimulation of DNA synthesis, and cell division. It has been pointed out that metacyclic trypomastigotes is characteristic of true cell cycle arrest and could be considered to be resident in G₀ until triggered to re-enter the cell cycle upon host cell invasion (Tyler and Engman, 2001).

The in vitro study of the changes that occur in proteins, glycoproteins and stage-specific antigens of T. cruzi during the morphological transformation of the metacyclic trypomastigotes into amastigotes should provide significant indications of how the parasite adapts to its mammalian host environment. However, it has been difficult to follow the molecular events associated with these morphological changes because there is no system that allows the in vitro production of high yields of extra-cellular T. cruzi amastigotes from metacyclic trypomastigotes.

In the present work, we have studied the kinetics of transformation of amastigotes from metacyclic trypomastigotes as well as the changes that occur in proteins, glycoproteins, and antigens during the in vitro metacyclic trypomastigote-amastigote transformation of T. cruzi.

 

MATERIALS AND METHODS

Parasites and stages

T. cruzi. EPm6 was obtained and kept in the laboratory as previously described (Contreras et al, 1994). Metacyclic trypomastigotes were induced in vitro using TAU3AAG medium as previously described (Contreras et al, 1985b; Bonaldo et al, 1988). In vitro-produced, metacyclic trypomastigotes were purified using DEAE-52 (diethylaminoethyl-cellulose) columns equilibrated with buffer PSG (0.073M NaCl; 0.005M sodium phosphate; 1% Glucose, pH 8.0) of ionic strength (I) 0.181 (De Sousa, 1983).

In vitro amastigogenesis

DEAE-52-purified metacyclic trypomastigotes were concentrated by centrifugation and resuspended in cold MEM medium without serum (1.0 to 1.5 x 10⁹ parasites/ml). One ml of resuspended parasites was transferred to sterile plastic tissue culture flasks (175 cm²; Falcon Labware, Oxnar, CA) containing 14 ml of MEMTAU medium pH 5.8, which consists of a 1:1 mixture of TAU3AAG medium and MEM 10% FBS (Fetal Bovine Serum) medium, supplemented with 70 mM Sucrose, 20 µg/ml bovine or human Hemoglobin, 200 U/ml Penicillin, 200 µg/ml Streptomycin, and 20 mM MES (2[N-morpholinoethanesulfonic] acid, hydrate), followed by incubation at 37° C without agitation in a 5% CO₂ atmosphere for 1, 2 or three days referred to as the pre-incubation phase. After three days of pre-incubation, the parasites were centrifuged and resuspended in PSG (I = 0.145), washed once, and applied to the top of a DEAE-52 column previously equilibrated with PSG (I = 0.145). Parasites were eluted with 20 ml of PSG (I = 0.145) at room temperature. Aliquots of eluates containing 100% metacyclic trypomastigotes were concentrated by centrifugation (8,000 xg, 10 min, 4° C) and resuspended in fresh MEMTAU medium. Parasite suspensions with final concentrations of ~4 x 10⁷ metacyclics/ml were transferred to sterile culture flasks and incubated for 1, 2, 3 or 4 days under the same conditions described above, referred to as the re-incubation phase. The in vitro differentiation was monitored by counting cells in a hematocytometer and by differential counting in Giemsa-stained smears.

 

Production of T. cruzi developmental stages for antigenic and SDS-PAGE analysis

4 x 10⁸ metacyclic trypomastigotes (day 0) were washed twice with PBS (0.15M NaCl; 0.02M sodium phosphate, pH 7.2) and concentrated by centrifugation in Eppendorff tubes (12,000 xg, 5 min 4° C). Wet masses of parasites (14 mg) were stored at -70° C for later use. Untransformed metacyclic trypomastigotes pre-incubated for 1, 2 and 3 days and purified through DEAE-cellulose columns (I = 0.145) were washed, concentrated, and stored as above.

Differentiating cells from 3-day purified metacyclic trypomastigotes, re-incubated in fresh MEMTAU medium for 1, 2, 3 and 4 days and not purified through DEAE-cellulose were processed as described.

 

Lysis of parasites

Wet masses of purified metacyclic trypomastigotes (day 0), pre-incubated (days 1, 2 and 3) and re-incubated (days 1,2,3 and 4) were suspended (25 mg/100 µl) in lysis solution (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 1 mM Iodoacetamide, 1 mM 1,10-phenanthroline, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 mM TLCK (tosyllysylchloromethylketone), 0.5 mM TPCK (tosylphenyl-alanylchloromethylketone), 0.1 mM E-64 (L-trans-epoxysuccinylleucylamido(4-guanidino)-butane), 25 µM Leupeptin, Antipain 10 µg/ml, 0.5% NP-40), frozen quickly in dry ice-ethanol, thawed in a 37° C water bath and vigorously vortexed for 3 min at room temperature. After three cycles of freezing and thawing, the broken parasites were centrifuged at 12,000 xg for 5 min at 4° C. The supernatants were collected and stored in aliquots, which were either used immediately or frozen for storage at -70° C in 5% glycerol. The protein contents of the supernatants were quantified using the Coomassie Plus assay (Pierce, Rockford, Illinois).

 

Analysis of polypeptides and glycopeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins were analyzed by electrophoresis on a linear gradient of 7-15% polyacrylamide gels (Laemmli, 1970). Wells were loaded with 7 µg of protein. The resulting protein patterns were stained with a combined Coomassie Blue-Silver staining procedure (De Moreno et al, 1985) for proteins and combined Periodic Acid-Alcian Blue-Glutaraldehyde-Silver (PAABGS) stain procedure for glycoproteins (Dubray and Bezard, 1982; Moller et al, 1993; Moller and Poulsen, 1995). For glycoproteins, two types of markers were run in the same gel: the broad molecular weight range was used to estimate molecular weight and control the reversal of silver staining (unstaining) of proteins and a mixture (1 µg/µl) of proteoglycans and glycoproteins (6 µl/well). The following glycoconjugates were used: Chondroitin 6-Sulfate from shark cartilage (type C, Sigma C-4384), Mucin from bovine sub maxillary glands (type I-S, Sigma M-3895), Fetuin from fetal calf serum (Sigma F-2379), and Bovine a-Acid Glycoprotein (orosomucoid, Sigma G-9014). The gels were then scanned in a Bio-Rad Imaging Densitometer, model GS-690, and their profiles were analyzed using the Bio-Rad Molecular Analyst^®/PC 1.2 software package.

Antisera production and Western blot analysis

Metacyclic trypomastigote stage-specific antiserum (anti-META) was prepared as previously described (Contreras et al, 1998). Antiserum directed against extra-cellularly metacyclic-derived amastigotes (anti-EMA) was prepared in New Zealand rabbits as follows. 20 mg (wet weight) of parasites were suspended in 2 ml of Freud's complete adjuvant and injected into the rabbits at 8-day intervals, first subcutaneously in the back, and then twice intramuscularly. After two weeks, the rabbits were intravenously inoculated three times at 48-hour intervals with 2 mg of freeze-thawed parasites in saline solution. Two weeks after the last inoculation, the immune response was assayed by indirect immunofluorescense with the homologous antigen. Three or four weeks after the last inoculation, the rabbits were bled and the antiserum obtained was kept in aliquots at -70° C.

Western blot analysis of purified metacyclic trypomastigotes (day 0), pre-incubated (days 1, 2 and 3) and reincubated (days 1, 2, 3 and 4) were performed essentially as previously described (Contreras et al, 1998). 4 µg of protein was applied to the lanes on a linear gradient of SDS-7-15% polyacrylamide gels. Pre-stained, wide-range (6.5-205 kDa, Sigma) molecular weight standards were included in each gel as markers. After the electrophoresis, the gels were equilibrated in transfer buffer [0.3M Trizma base (Sigma), 1.44% glycine (Sigma) and 20% methanol (Fisher, Pittsburgh, PA)] and transferred to nitrocellulose membrane (Pierce, Rockford) at 100 Volts for 2h in a Bio-Rad Trans Blot^® SD unit. The blotted gels were blocked in TBS (10 mM Tris/HCl, 150 mM NaCl, pH 7.2) containing 5% non-fat milk powder (TBSm). The membranes were incubated overnight at 4° C with a 1: 5,000 dilution of anti-META serum or 1:8,000 dilution of anti-EMA serum. After 6 washes with TBS, membranes were incubated (2h at 37° C) with 1:10,000 dilution of peroxidase-conjugate goat anti-rabbit total immunoglobulins and developed using the Luminol chemiluminescense system according to the protocol supplied by the manufacturer (Super-Signal, Pierce). Finally, the blots were wrapped in plastic wrap and luminographed using AGFA films and DuPont Cronex-Plus intensifying screens. The immunoblots were then scanned and their profiles were analyzed as described above.

 

RESULTS

In vitro transformation of metacyclic trypomastigotes into amastigotes

Table I shows the effect of the preincubation and re-incubation in MEMTAU medium on the capacity of T. cruzi metacyclic trypomastigotes to transform into amastigotes at 37° C. Note that during the first 2 days of pre-incubation the metacyclic trypomastigotes did not undergo a statistically significant transformation to amastigotes (2.1 ± 0.5). On the first day of pre-incubation, almost all of the metacyclic trypomastigotes display a lengthening and a thinning of the entire organism, showing a small-sized kinetoplast, usually very far from its pointed posterior end (Fig 1B, C, D). This morphology was notably kept in parasites pre-incubated by two or three days (Fig 1E, F, G, respectively). When these parasites were re-incubated in fresh MEMTAU medium, distinct morphological changes and progressive shortening was observed in the metacyclic trypomastigote population during the first day of culture (Fig 1H, I, J). The changes were complete after 2-4 days of incubation, when a homogeneous population of typical amastigotes appeared (Fig 1M, N; Table I).

Figure 1- Giemsa-stained smears of Trypanosoma cruzi incubated in MEMTAU medium at 37° C. TAU3AAG-purified parasites before of the pre-incubation phase shows a typical morphology of metacyclic trypomastigotes (A). Pre-incubated metacyclic trypomastigotes from day 1 (B,C,D), 2 (E,F), or 3 (G) show a progressive lengthening at the posterior end, an overall thinning of the parasites, nuclei (n) elongated and a small-sized kinetoplast (k) very far from its pointed posterior end. Re-incubated metacyclic trypomastigotes from day 1 (H, I, J) and 2 (K) show broadening of the parasite body with a terminal kinetoplast . Re-incubated parasites from day 3 (L, M) and 4 (N) show a progressive shortening of the entire organism, nuclei condensed and transformation into forms resembling amastigotes. Note that pre-incubated metacyclic trypomastigotes release thin processes (trails) from the posterior end and along the parasite body (arrowheads, Fig 1B-G). Parasites were visualized by light microscopy on Giemsa-stained smears.

TABLE I

Effect of the pre-incubation and re-incubation in MEMTAU medium in the capacity of
T. cruzi metacyclic trypomastigotes to transform into amastigotes at 37° C

Time (days)		Percentage of forms (X ±SD)
	Metacyclic	Differentiation	Amastigote
0	100	0	100
1(a)	98.0 ± 0.6	1.3 ± 0.2	1.3 ± 0.2
2(a)	95.0 ± 0.7	3.1 ± 0.3	2.1 ± 0.5
3(a)	78.0 ± 3.1	10.9 ± 1.7	11.2 ± 2.1
1(b)	57.1 ± 5.3	26.4 ± 3.3	16.5 ± 4.2
2(b)	29.7 ± 2.3	22.0 ± 2.3	48.3 ± 3.3
3(b)	9.6 ± 1.3	6.5 ± 1.1	83.9 ± 3.5
4(b)	4.1 ± 0.7	8.2 ± 4.9	87.7 ± 6.3
X ±SD: Means and standard deviation
(a) Metacyclic trypomastigotes from TAU3AAG medium purified through DEAE-cellulose columns (I =0.181) pre-incubated in MEMTAU medium for 1, 2, or 3 days. (b) Metacyclic trypomastigotes pre-incubated for 3 days purified through DEAE-cellulose columns (I =0.145) and re-incubated in fresh MEMTAU medium for 1, 2, or 3 days. Differential cell counting was performed either in a Neubauer chamber or in permanent smears stained with Giemsa. Data correspond to at least five different duplicated experiments.

 

Analysis by SDS-PAGE of polypeptides and glycopeptides

In order to investigate whether different biochemical events might be occurring in pre-incubated and re-incubated parasites, we characterized the differentiation process in terms of proteins and glycoproteins. The protein profile displayed by metacyclic trypomastigotes pre-incubated for 1 day shows significant changes, represented by the disappearance of the silver-stained cluster, (Fig 2, triangle opening at right of lane 0, vs. lane 1 Pre-incubated), appearance of at least nine bands of high molecular weight, above the 116 kDa marker, another nine bands of medium molecular weight between the 116 and 55 kDa markers, and eight bands of low molecular weight below the 55 kDa marker, which are identified by short bars at right of lane 1, Pre-incubated (Fig. 2). This pattern is notably conserved in metacyclic trypomastigotes pre-incubated for 2 and 3 days; although a progressive lowering in intensity of the silver-stained polypeptides above the 66 kDa marker can be observed (Fig. 2, lanes 2 and 3, Pre-incubated).

 

Figure 2- Protein analyses of Trypanosoma cruzi differentiating cells incubated in MEMTAU at 37° C medium by SDS-PAGE (7-15%) revealed with a combined Coomassie Blue-Silver stain procedure. Lane 0 refers to metacyclic trypomastigotes from TAU3AAG medium. "Pre-incubated" indicates purified metacyclic trypomastigotes (I = 0.145) after 1, 2 or 3 days in MEMTAU medium. "Re-incubated" refers to parasites incubated for 1, 2, 3 or 4 days in fresh MEMTAU medium that derived from 3-day purified metacyclic trypomastigotes. Mr corresponds to the relative mobility of the protein markers in kilodaltons (kDa). Each well was loaded with 7 µg of the protein.

Parasites re-incubated for 1, 2, 3 and 4 days share a number of bands with purified-metacyclic trypomastigotes from 3-days of pre-incubation (Fig. 2, lanes 1, 2, 3 and 4, Re-incubated vs. lane 3, Pre-incubated). However, the comparison of purified-metacyclic trypomastigotes from 3-day-pre-incubated and 2-day-re-incubated parasites shows some differences, represented by a strong increase in the intensity of bands of high molecular weight, above the 116 kDa marker and the appearance of at least 6 major polypeptides of medium molecular weight, between the 97 and 55 kDa markers (Fig. 2, short bars at right of lane 2 Re-incubated). The comparison of the proteic profile displayed by the departure stage corresponds to metacyclic trypomastigote from a chemically-defined medium at 27° C (Fig 2 lane 0) and the arrival amastigotes stage (EMA), represented by differentiating cells from MEMTAU medium at 37° C (Fig. 2, lane 4 Re-incubated) to make evident a higher polypeptides complexity in latter.

To determine total glycoproteins of the parasites during the in vitro differentiation, an equivalent gel as obtained in Figure 2 was visualized by the PAABGS procedure as shown in Figure 3. Lane 0 shows the glycoprotein's profile displayed by TAU3AAG-purified metacyclic trypomastigotes at 27° C, which present an intense silver-stained cluster of glycopolypeptides with a molecular mass ranking from 95 to 62 kDa (Fig. 3, triangle opening at left of lane 0), three glycopeptides in the region of the 55 kDa marker, and four barely detectable glycopeptides below of the 45 kDa marker, identified by short bars at left of lane 0, Figure 3. The metacyclic trypomastigotes pre-incubated for 1 or 2 days display a not well-resolved glycoprotein profiles showing a smear of highly glycosilated products with a molecular mass ranking from 135 to 55 kDa (Fig. 3 triangle opening at left of lane 1 Pre-incubated), whose intensity decreases and is resolved as sharp bands on the third day of pre-incubation. A comparison of 0 and 3-day pre-incubated patterns (Fig. 3, lane 0 vs. lane 3 Pre-incubated) showed significant changes represented by the appearance of at least 6 well-resolved bands above the 116 kDa marker, 8 dark bands between the 116 and 55 kDa markers, and 4 bands of different width and intensity below the 55 kDa marker, identified by short bars at the left of lane 3, Pre-incubated, Figure 3.

The parasites re-incubated for 1 day also displayed a less resolved profile, showing an intensely silver-stained cluster of glycopeptides no resolved ranking from 97 to 62 kDa (Fig. 3, triangle opening to the right of lane 1 Re-incubated), which is rather resolved as sharp bands on the second day of re-incubation (Fig. 3, lane 2 Re-incubated). The comparison of 3-day-pre-incubated and 2-day-re-incubated patterns shows significant changes represented by a rearrangement of glycopeptides (66/62/60 triplet, Fig. 3 triangle opening at right of lane 2 Re-incubated) and the appearance of two glycopeptides between 66 and 55 kDa markers (60 and 56 kDa) (Fig. 3 short bar at right of lane 3 Re-incubated). This last profile is remarkably conserved and a progressive resolution of the glycopeptides can be seen, according to the morphological transformation progress (Fig. 3 lanes 2 to 4 Re-incubated; Table I).

 

Western blot analysis of antigens.

To determine whether there are changes at the antigen profiles as observed in protein and glycoprotein profiles, Western blots were performed using anti-META and anti-EMA sera (Fig. 4). Anti-META revealed in the departure stage (TAU3AAG-metacyclic trypomastigotes) an antigen pattern characterized by 3 faint bands of Mr 145, 120 and 25 kDa (Fig. 4A, short bars at right of lane 0), 2 clusters of antigens not resolved ranking from 100-85 kDa and 64-55 kDa (Fig 4A triangle opening to the right of lane 0) and 3 major antigenic bands of Mr 47, 40 and 36 kDa (Fig.4A short bars at right of lane 0). While in extracelullar metacyclic-derived amastigotes (EMA) (Fig. 4A, lane 4 Re-incubated), this antiserum revealed a lesser antigenic complexity and a drastic reduction in the pattern's intensity, as expected for a stage-heterologous antiserum. More significant antigenic differences between the departure and arrival stages (Fig. 4A, lane 0 vs. lane 4 Re-incubated) are represented by the appearance of a high molecular weight cluster of antigens above the 116 kDa marker and at least 2 faint bands with Mr of 110 and 72 kDa (Fig. 4A, short bars at right of lane 4) in EMA which is absent in TAU3AAG-metacyclic trypomastigotes.

Figure 3- Glycoprotein analyses of Trypanosoma cruzi differentiating cells incubated in MEMTAU at 37° C medium by SDS-PAGE (7-15%) revealed with a combined Periodic Acid-Alcian Blue-Glutaraldehyde-Silver (PAABGS) stain procedure. Lane 0 refers to metacyclic trypomastigotes from the TAU3AAG medium. "Pre-incubated" indicates purified metacyclic trypomastigotes (I = 0.145) after 1, 2 or 3 days in MEMTAU medium. "Re-incubated" refers to parasites incubated for 1, 2, 3 or 4 days in fresh MEMTAU medium that derived from 3-day purified metacyclic trypomastigotes. Lane "m" shows the molecular weight markers. Lane "s" corresponds to the relative mobility of the glycoprotein markers. Mr corresponds to the relative mobility of the protein markers in kilodaltons (kDa). Each well was loaded with 7 µg of the total protein.

It is noteworthy that the 145 and 120 kDa antigenic bands revealed by this antiserum in TAU3AAG-metacyclic trypomastigotes above of 116 kDa (Fig. 4A, short bars at right of lane 0) were absent in re-incubated parasites and undergoing a progressive lessening in pre-incubated parasites, suggesting that such antigens are metacyclic-specific. In spite of significant changes revealed by anti-META in pre-incubated metacyclic trypomastigotes, the most striking changes occur during the re-incubation phase. Although four major antigenic bands with M_r of 95/80, 66/58, 50/43, 37/35 and 25 kDa, identified by triangles open at right of lane 3 Pre-incubated Figure 4A, seem to be shared by 3-day-preincubated metacyclic trypomastigotes and 1day-re-incubated parasites (Fig 4A lane 1 Re-incubated), an accelerated rearrangement of the bands can be observed arriving to the third day of pre-incubation (Fig. 4A, lane 1 to 3 Re-incubated) suggesting that such antigenic changes might be related with the progressive morphological transformation to amastigotes (Table I).

Western blots using anti-EMA serum showed three different antigen patterns during the differentiation process (Fig. 4B). The first displayed by TAU3AAG-metacyclic trypomastigotes showing at least eight weakened bands with Mr of 170, 145, 120, 82, 62, 52, 47, and 36 kDa (Fig 4B short bars at right of lane 0). A second profile is depicted by untransformed metacyclic trypomastigotes from 1 day-MEMTAU medium (Fig. 4B, lane 1 Pre-incubated), which showed significant differences with the first, represented by the disappearance of the three antigens with Mr of 170, 145 and 120 kDa above the 116 kDa marker, the appearance of five major antigens with Mr of 90/75, 68/60, 57/55, 53/41, 36 kDa and a very faint band of Mr 25, identified by white triangles at right lane 1, Pre-incubated, Figure 4B. This second profile is remarkably conserved in the next days of pre-incubation (Fig. 4B, lanes 2 and 3 Pre-incubated). It is interesting to note that in re-incubated parasites from day 1, the anti-EMA serum essentially revealed major antigens with the same molecular weight as in pre-incubated parasites, while parasites re-incubated after day 2 (Fig. 4B, lanes 2 to 4 Re-incubated) displayed a common third profile, despite an increase in the percentage of amastigotes (Table I). This third profile putative of EMA (Fig. 4B, lane 4 Reincubated) consists of at least 6 bands with Mr of 145, 110, 92/70, 57, 50 and 47 kDa (Fig. 4B, short bars at left of lane 4) and a high molecular weight cluster of antigens above the 170 kDa.

Figure 4 - Western blot analyses of Trypanosoma cruzi differentiating cells incubated in MEMTAU medium using anti-metacyclic trypomastigotes (Anti-META, Gel A) and anti-extracellularly-derived metacyclic amastigote serum (anti-EMA, Gel B) diluted 1/5,000. Lane 0 refer to metacyclic trypomastigotes from TAU3AAG medium purified through DEAE-52 (I = 0.181). Pre-incubated refers to purified metacyclic trypomastigotes (I = 0.145) after 1, 2 or 3 days in MEMTAU medium. Re-incubated refers to parasites incubated for 1, 2, 3 or 4 days in fresh MEMTAU medium which derived from 3-days-purified metacyclic trypomastigotes. Mr corresponds to the relative mobility of the protein markers in kilodaltons (kDa).

 

DISCUSSION

We have correlated macromolecular and morphological changes during the transformation of T. cruzi-metacyclic trypomastigotes into amastigotes by manipulation of culture conditions. Here we have provided biochemical and antigenic evidence to characterize the in vitro primary amastigogenesis of T. cruzi.

To guarantee that the molecular changes observed during the pre-incubation phase were inherent in the metacyclics per se and not due to forms in differentiation, the analyses were done using DEAE-purified parasites. We could observe that metacyclic trypomastigotes incubated in MEMTAU medium at 37° C displayed a higher polypeptides complexity than metacyclic trypomastigotes from TAU3AAG medium at 27° C (Figs. 2 and 3, lane 1 Pre-incubated, vs. lane 0) suggesting that an accelerated protein synthesis is triggered during the first 24 h of pre-incubation, which does not seem to be maintained in pre-incubated parasites from days 2 and 3, as a progressive lowering in intensity of the high molecular weight bands was observed (Figs. 2 and 3, Pre-incubated). In agreement with this suggestion, a study of the physiological aspect of T. cruzi during heat-shock showed that at 37° C, when heat-shock was applied, the parasites from the 48 h culture did not display the classical response to the heat treatment, since a general increase in RNA and proteins synthesis was observed (De Carvalho et al., 1994).

Although no notable cellular rearrangements were observed during the pre-incubation phase, the surface membrane and some cellular-cytoskeleton components must be actively remodeled during the lengthening at the posterior end of the parasites and the slenderness of the entire parasite (Fig. 1B-G). As described in other studies (Andrews et al, 1987; Barros et al, 1996), we observed by scanning electron microscopy that the lengthening at the posterior end of the metacyclic forms is accompanied by the release of different type of trails (manuscript in preparation). In re-incubation phase, the metacyclic trypomastigotes underwent significant shape and volume changes. The transition from an elongated shape to the final round form must involve selective degradation of cytoskeleton and surface proteins. It is believed that proteases (Rosenthal, 1997) and proteosome activity (González et al, 1996) might be involved in both phases of the primary amastigogenesis. Recently, De Diego et al., (2001) demonstrated that the ubiquitin-proteosome pathway plays an essential role in proteolysis during T. cruzi remodeling. It is worth noting that re-incubated parasites from day 1 display a more intense polypeptide profile than 3-day-preincubated parasites (Fig. 2, lane 1 re-incubated vs. lane 3 pre-incubated), suggesting that the change of culture medium also increases the protein synthesis. This metabolic behavior was previously reported in amastigotes maintained in axenic conditions by changing the culture medium at pre-selected intervals (Engel and Dvorak, 1988). It is worth mentioning that the morphological changes, along with the primary amastigogenesis, occur simultaneously with modifications in the nuclear morphology (Fig. 1) and that it has recently been shown that changes in transcription occur in parallel with modifications in the nuclear structure when proliferative forms of T. cruzi transform into non-proliferative forms (Elias et al., 2001).

The nature of the glycoprotein involved in the primary amastigogenesis is unknown. The results of Table I and Figure 3 seem to indicate that the morphological transformation is associated with changes in their glycoproteins. These changes might be related to the trans-sialidase and mucin-like glicoproteins previously described in T. cruzi (Frash, 2000), as the gene expression these molecules have been shown that are controlled post-transcriptionally during differentiation of T. cruzi (Abuin et al., 1999). Figure 3 shows that parasites pre-incubated for 1 or 2 days display a smear of highly glycosilated products (Fig. 3, triangle opening at left of lane 1, Pre-incubated). One possible explanation of this effect is that sialic acid and macromolecules present in the serum-enriched MEMTAU medium could bind to some receptor on the surface of TAU3AAG-metacyclic trypomastigotes (Khan et al. 1996; Bourguignon et al. 1998), which masks the glycoprotein changes. This proposal is based on the fact that the high molecular weight glycopeptides were maintained, while the intensity of smear decreases and is resolved as sharp bands on the third day of pre-incubation. Another possibility is that the smear is from surface membrane glycoproteins present in the posterior end of the parasite seem to be broken in the third day of pre-incubation (Fig 1F, G). However, more experiments are needed to confirm these hypotheses.

Transformation of T. cruzi from one stage to another is associated with biological property changes and the expression of stage-specific surface molecules. It is known that during the transformation from tissue derived-trypomastigotes to amastigotes, the changes in morphology are accompanied by the appearance of new surface molecules in the molecular weight range of 70-92 kDa (Burgleigh and Andrews, 1995). Although we do not know the degree of antigenic similarity among tissue-derived amastigotes and metacyclic-derived amastigotes; the amastigotes antigens with Mr of 95/72 kDa detected by anti-EMA must be correlated with the acquisition of new surface molecules in the molecular weight range of 92-70 kDa described by other groups (Andrews et al, 1987; Iida and Ley, 1991; Texeira et al, 1994; Burleigh and Andrews, 1995; Barros et al, 1997). The fact that they have been expressed by untransformed metacyclic trypomastigotes from day 1(Fig. 4B, lane 1, Preincubated) demonstrates a dissociation between molecular and the morphological events during the in vitro primary amastigogenesis of T. cruzi. Finally, the antigenic changes revealed by anti-EMA serum (Fig. 4B) resemble the glycopeptidic profiles observed in Figure 3, suggesting that such molecules are related.

An antiserum raised against TAU3AAG metacyclic trypomastigotes must mainly recognize molecules highly immunogenic as the SAPA/trans-sialidase (85-130-200 kDa), the developmentally regulated cruzipain (60-40 kDa) (Cazzulo and Frasch, 1992) and antigens of molecular mass 90, 82, 75 and 35/50 kDa, which have been described as the major surface antigens of T. cruzi metacyclic trypomastigotes (Burgleigh and Andrews, 1995). The molecular mass of antigens revealed by anti-META in TAU3AAG-metacyclic confirms this proposal (Fig. 4A, lane 0). It is noteworthy that anti-META revealed antigenic changes primarily during re-incubation phase, suggesting that the morphological transformation to amastigotes is accompanied by the loss of metacyclic-specific antigens (Table I; Fig. 4A, lane 1 to 4, Re-incubated).

While in nature, primary amastigogenesis occurs within the cells of the mammalian host, the fact that the transformation of T. cruzi metacyclic trypomastigotes to amastigotes, can be mimicked in vitro after successive incubations in MEMTAU medium at 37° C should provide significant clues as to how the parasites adapt to the mammalian host environment. Our results seem to indicate that early and late events occur during the in vitro primary amastigogenesis of T. cruzi. The early events are characterized by an accelerated molecular differentiation without remarkable morphological transformation, while later events are typified by profound morphological and antigenic rearrangements. The first are associated with the pre-incubation phase and the second take action after the parasites are re-incubated in fresh MEMTAU medium. This evidence suggests that the molecular differentiation occurs several days prior to the morphological differentiation, which takes place when the culture medium is changed. Both behaviors have been reported in distinct in vitro-differentiating system used for T. cruzi (Contreras et al, 1985a; Bonaldo et al, 1988; Engel and Dvorak, 1988). Although comparative studies such as those in Figures 2, 3 and 4 with tissue-culture derived trypomastigotes were not performed, it can be supposed that this stage must show changes similar to the ones observed during the re-incubation phase, since our differentiating system mimics environmental conditions (temperature, pH, serum) as reported by others (Villalta and Kierszenbaum, 1982; Engel and Dvorak, 1988; Tomlinson et al., 1995). It would still be premature to assume that the events described here precisely mimic the primary amastigogenesis that occurs within the vertebrate host, but it might be useful in understanding why the metacyclic trypomastigote remains morphologically quiescent for several hours prior to their escape from the parasitophorous vacuole into the host cell cytoplasm (Dvorak, 1975; Burleigh and Andrews, 1995).

ACKNOWLEDGEMENTS

This research was supported by grants from CONICIT S1-97000664 (VTC and ARDL), CONICIT S1-2001000683(MCN, VTC and ARDL), CODECIH FCS-97018 (ARDL and VTC) and FCS-99010 (MCN and VTC). We thank Gregorio Flores, Wilmer Pineda, Johnny Albanesse for their excellent technical assistance.

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