EXPERIMENTAL
PARASITOLOGY
Trypanosoma
72, 43-53 (1991)
cruzi: Shedding of Surface Membrane Vesicles
Antigens
as
MARINEI F. GON~ALVES,* EUFROSINA S. UMEzAwA,t ALWANDRO M. KATZIN,$ WANDERLEY DE SOUZA,§ MARIA JULIA M. ALVES,* BIANCA ZINGALES,* AND WALTER COLLI* *Departamento de Bioquimica, Instiruto de Quimica, Universidade de 560 Pa&o, Caixa Postal 20780, CEP 01498, Scio Paulo, Brazil: flnstituto de Medicina Tropical, Faculdade de Medicinn, Universidade de Srio Paulo, Av. Dr. Eneas de Carvalho Aguiar, 470, Sdo Paula, Brazil; $Departamento de Parasitologia, Instituto de Ciencias Biomhdicas, Unirpersidade de Scio Paulo, Caixa Postal 4365, CEP 05508, Sdo Paula, Brazil; and §Laboratdrio de Microscopia Eletronica e Ultra-Estrutura Celular, Institute de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. Ilha do Fundao. CEP 21941, Rio de Janeiro, Brazil CONCALVES, M. F.. UMEZAWA, E. S., KATZIN, A. M., DE SOUZA, W., ALVES, M. J. M.. ZINGALES, B., AND COLLI, W. 1990. Trypanosoma cruzic Shedding of surface antigens as
membrane vesicles. Experimental Parasitology 72, 43-53. Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CAI, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter. ‘T:’ 1991 Academic PESS. IK INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Parasite hemoflagellate; Shedding; Surface antigens; Vesicles; Bovine serum albumin (BSA); Dulbeccos’s modified Eagle medium (DMEM); Fetal calf serum (FCS); Hyperimmune serum (HIS); Polyacrylamide gel electrophoresis (PAGE): Sodium dodecyl sulfate (SDS); Trichloroacetic acid (TCA); Tissue culture trypomastigotes (Tct)
patients, suggesting that these components are the product of an active infection (Freilij et al. 1987). The occurrence of parasite antigens in tissues and organs in which intracellular parasites cannot be visualized has been reported in the chronic phase of experimental infection (Ben Younes-Chennoufi et al. 1988). Also, it has been proposed that the adsorption of T. cruzi antigens to uninfected cells may lead to damage of these cells by the immune response directed to the parasite (Ribeiro dos Santos and Hudson 1980; Williams et al. 1985). Furthermore, the parasite constituents can represent a persistent antigenic stimulation believed to occur in the chronic phase when parasites are not detected in most of the cases. This antigenic burden could play a
Circulating antigens have been demonstrated in sera from Trypanosoma cruziinfected animals (Dzbtnski 1974; De Siqueira er al. 1979; Araujo 1982) and from human patients in the acute and chronic phases of Chagas’ disease (Araujo et al. 1981; Marcipar et al. 1982; Kahn et al. 1983; Freilij et al. 1987). Several reports also describe the presence of parasite antigens in the urine from acutely infected mice and dogs (Bongertz et al. 1981) and from patients with acute, congenital, and chronic Chagas’ disease (Freilij et al. 1987; Katzin et al. 1989a, b). Moreover, whenever parasitemia becomes negative during the course of specific treatment, T. cruzi antigens cannot be detected in the urine in most of the 43
0014-4894/91 $3.00 Copyright B 1991 by Academic Press. Inc. All rights of reproductmn in any form reserved.
44
GONCALVES ET AL.
major role in the pathology of Chagas’ disease (Ben Younes-Chennoufi er al. 1988). The source of circulating and excreted antigens as well as tissue-adsorbed T. cruzi components has not been established. Nonetheless, it can be predicted that these antigens are originated from either parasite lysis or an active release of some specific antigens. Both processes could also occur concomitantly. In the present report we demonstrate that T. cruzi actively releases surface antigens by a spontaneous process which probably involves plasma membrane vesiculation. Tc-85, and 85kDa surface glycoprotein involved in the invasion of cells by the parasite (Alves et al. 1986), is one of these antigens. We also show that trypomastigotes from different strains have different shedding capacities. MATERIALS
AND METHODS
Parasite swains and cu/rure. Tissue culture trypomastigotes (tct) from the Y (Silva and Nussensweig 1953), the CA1 (Gonzalez Cappa et al. 1980). the RA (Gonzalez Cappa et al. 1981), and the YuYu (Filardi and Brener 1987) strains from T. cruzi were obtained from monolayers of LLC-MK2 cells (Andrews and Colli 1982). Antibodies. A polyclonal hyperimmune serum (HIS) against methoxypsoralen-inactivated tct (Y strain) was obtained (Andrews et al. 1985). A monoclonal antibody (HIAIO) that recognizes an 85kDa glycoprotein (Tc-85) of the trypomastigote surface (Katzin and Colli 1983)was prepared as described (Alves et al. 1986). Metabolic labeling wirh [‘5S]merhionine und radioiodinarion ofparasites. Parasites were incubated for 2
hr at 37°C in Dulbecco’s modified Eagle medium (DMEM)-methionine free, 2% fetal calf serum (FCS) in the presence of 25 uCi/ml [‘*S]methionine (1200 Gil mmol; Amersham, U.K.), as described (Zingales er al. 1982). Tissue culture trypomastigotes (Y strain) were radioiodinated with Na13’I (IPEN, Sao Paulo) using Iodogen (Pierce Chemical Co.) as catalyst (Zingales 1984). Labeling
with [3Hjuridine
and [‘Hjpalmiric
acid.
Tissue culture trypomastigotes (5 x lO’/ml) were incubated for 4 hr at 37°C in DMEMJ% dialysed FCS, containing either 5,6-[3H]uridine (40.5 Wmmol; New England Nuclear) or 9,10(n)-[3H]palmitic acid (50 Gil
mmol; Amersham, U.K.). Either precursor was added at 50 $X/ml. Shedding essays. Labeled parasites were washed twice in DMEM-O.5% FCS and resuspended at IO8 cells/ml in the same medium containing 20 mM N-2-hydroxyethylpiperazine-W-2 ethanesulfonic acid (Hepes). The suspension was maintained at 37°C (unless otherwise stated) in a humidified atmosphere with 5% COz. Aliquots (10’ cells) were removed at different times and centrifuged at 2500g for 5 min. The supernatants and the sedimented parasites were precipitated independently with cold tricholoroacetic acid (TCA) at 10% final concentration. In both cases, the precipitates were collected on 0.22~pm nitrocellulose filters and washed with 30 ml of 5% TCA. The dried filters were either counted in a liquid spectrometer (LKB, 1214Rackbeta) for [35S]methionine incorporation or in a gamma counter (Beckman/gamma 5500) for 13’1labeling. The quantification of the liberated polypeptides was calculated as the percentage (%) of TCA-precipitated radioactivity in the supematant medium in relation to the total radioactivity (pellet plus supernatant). The amounts referred to in the text represent the cumulative measurement at each experimental point. Immunoprecipirurion. The identification of cell surface antigens was done as described (Zingales et al. 1982). Labeled living parasites (5 x IO’) were incubated with 0.5 ml of DMEM containing HIS at a 1:25 dilution. Incubation was performed for 1 hr at 4°C with occasional agitation. Parasites were then washed to remove unbound antibody and lysed with a lysing buffer containing 1% Nonidet P-40, 10 mM Tris-HCI, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 n-&f N-tosyl-t-lysylchloromethyl ketone, 0.25 ug/ml leupeptin, and 2.8 units/ml aprotinin. Immunocomplexes present in the 10,OOOgsupernatant were precipitated with 100~1of a 10% suspension of heat-killed and formalin-fixed Sraphylococcus aureus (Cowan I strain) (Kessler 1975). Samples were processed as described (Zingales et al. 1982). The identification of total antigens liberated into the medium was done as described below. A twofold concentrated lysing buffer was added to the supematant obtained after centrifugation of parasites. The sample was precleared to remove nonspecific adsorbing material by incubation (2 hr at 4°C) with normal rabbit serum and 70 ~1 of a 10% suspension of S. aureus. Samples were then incubated overnight at 4°C with either HIS or HlAlO monoclonal antibody. After 30 min incubation with 70 ul of a 10% suspension of 5. aureus, the samples were processed as described previously (Zingales et al. 1982).The immunoprecipitated antigens were counted either in a gamma counter or in a scintillation spectrometer. Gel elecrrophoresis. Unidimensional gel electrophoresis was performed according to Laemmli (1970) on
T.
Cruzi:
SHEDDING
8% polyacrylamide gels containing 0.1% SDS (SDSPAGE). After electrophoresis, the gels were fixed, stained, and destained, processed for fluorography for [35S]methionine samples, dried, and exposed to X-ray films. Molecular mass markers (Sigma) were myosin, P-galactosidase, phosphorylase B, bovine serum albumin, and ovalbumin. Gelfiltrarion. The material liberated by 1.6 x lo8 labeled parasites in 0.2 ml of culture medium was chromatographed on a Sepharose 4B (Pharmacia Fine Chemicals) column (0.8 X 48 cm). The column was eluted with 10 n&f phosphate buffer, pH 7.2, 150 m&f NaCI, 0.02% NaN, at room temperature. Fractions of 0.5 ml were collected. Electron microscopy. The parasites were collected by centrifugation and fixed for 60 min at room temperature in a solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, washed in phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed in a Jeol 100 CX electron microscope. For the immunocytochemical localization of antigens, the parasites were processed as described previously (Abuin et al. 1989). For negative staining the samples were placed on formvar and carbon-coated grids, treated with 1% phosphotungstic acid, dried. and observed.
4.5
OF ANTIGENS
TABLE I Shedding of Polypeptides by Trypomastigotes of Trypanosoma cruzi under Different Conditions” Temperature (“Cl
Addition
4 37 37 37 37 37 37
0.596 FCS 0.5% FCS 5 % FCS 10 % FCS 4 % HIS 3.5 mg/ml BSA
Liberated polypeptides mb 2.9 14.5 14.2 14.3 15.8 15.0 12.9
a [“S]Methionine-labeled trypomastigotes (Y strain) were incubated in DMEM for 3 hr under the indicated conditions. b The percentage of liberated polypeptides was calculated as described under Material and Methods.
within the experimental error, were found; (ii) the same values for the TCA-precipitable radioactivity of supernatant aliquots were obtained before and after filtration by a 0.22~pm nitrocellulose membrane; (iii) parasites were prelabeled with [3H]uridine RESULTS and then placed in fresh culture medium, [35S]Methionine-labeled trypomastigotes under the same conditions of the shedding (Y strain) liberate polypeptides into the me- assays. Aliquots were withdrawn at differdium. This phenomenon was observed in ent times and TCA-precipitable radioactivthe absence or presence of bovine serum ity was measured in the supernatant. Apalbumin (BSA) or different concentrations proximately 2% of the total incorporated of FCS (Table I). Furthermore, it was ver- [3H]uridine was found in the medium at all ified that substitution of FCS by a rabbit experimental times. Furthermore, it was verified that parasites are fully motile and HIS, elicited by living trypomastigotes, does not alter the extent of shedding, sug- viable after the shedding assay, since they gesting that the process is independent of retain the same infection capacity of cell the binding of specific antibodies to the par- monolayers (experiments were performed asite surface. Also the kinetics of shedding according to Zingales et al. 1982, data not is independent of the presence of HIS (not shown). In order to characterize the nature of the shown). The incubation of trypomastigotes at 4°C promoted 80% inhibition of the re- polypeptides released into the culture melease of polypeptides observed at 37”C, dium as to their molecular mass and their showing that the shedding process is tem- cellular localization, trypomastigotes were either labeled with [35S]methionine (Fig. perature dependent (Table I). Several controls were performed in order 1A) or surface radioiodinated (Fig. IB). to rule out the occurrence of parasite lysis The pattern of the labeled polypeptides asduring the incubation period: (i) parasites sociated with the parasites before (lanes a, were counted under a light microscope af- a’) and after 3 hr shedding (lanes b, b’) was ter the shedding assays and no differences, compared by SDS-PAGE to the pattern of
46
GONCALVES ET AL.
Quantification of TCA-precipitable [35S]methionine-labeled components shed in the 205culture medium indicates that the total 116amount is dependent on the strain (Fig. 2). 116S?STTaking as reference the endpoint measurement after 4 hr observation, it is concluded 6666that the YuYu, Y, CAl, and RA strains lib454t)erate, respectively, 20, 11,7.5, and 4.5% of the total labeled polypeptides, under the experimental conditions employed. The nature of the antigens shed by the FIG. 1. Polypeptides from trypomastigotes (Y strain) released into the culture medium. Parasites strains was also analyzed by immunoprewere labeled with [35S]methionine (A) or ‘311-Iodogen cipitation using the HIS against trypo(B). Polypeptide pattern of parasites before shedding mastigotes. The four strains released poly(a, a’) and after 3 hr shedding at 37°C (b, b’) and of peptides bearing common epitopes recogpolypeptides released in the culture medium (DMEM nized by the HIS (Fig. 3A). The patterns - 0.5% FCS) (c, c’). Antigens immunoprecipitated with HIS from total parasites (d, d’) and from the cul- were similar, showing mainly the presence ture medium (e, e’). Molecular mass markers (kDa) are of polypeptides ranging from 70 to 150 kDa. shown on the left. Comparison of this pattern with that obtained by immunoprecipitation of living tct the components recovered into the culture of the four strains with the same antiserum medium (lanes c, c’). In both experiments (data not shown) suggests that most of the (Fig. 1A and 1B) polypeptides of 70 to 150 kDa that had been lost by the parasites were recovered in the incubation medium. In contrast, very few polypeptides below this molecular mass could be detected in the medium (Fig. 1, lanes c, c’). This observation reinforces the conclusion that parasites are not lysed under the shedding conditions. The experiments performed with the radioiodinated trypomastigotes (Fig. 1B) allowed the conclusion that the parasites release surface polypeptides. The evidence that the components released into the medium are the main surface antigens of the trypomastigote form was obtained in immunoprecipitation experiments, where living labeled parasites and I 2 3 1 the corresponding supernatant medium obHOURS tained after 3 hr shedding were incubated FIG. 2. Kinetics of polypeptide liberation by four with a HIS against trypomastigotes. The strains of T. cruzi. Tissue culture trypomastigotes antigenic patterns are very similar in both were labeled with [35S]methionine for 2 hr, washed, cases (Fig. lA, lanes d, e and Fig. IB, lanes and placed in DMEM - 0.5% FCS at 37°C. Aliquots were withdrawn at different times and TCA-precipd’, e’). itable radioactivity was measured in the supematant The liberation of polypeptides observed and in the sedimented parasites. The percentage of initially with the Y strain was also verified liberated radioactivity was determined as described for tct of the YuYu, CAl, and RA strains. under Materials and Methods. B a’ b’
Aobcde
C’
d’
e’
205-
io
T. cruzi: SHEDDING OF ANTIGENS A,
RA
,,
CA1
,.
Y
,, YuYu
,
B , RAwCAl,,
(4141414
*05-
444
Y .,YuS,
4
205-
116ST-
116-
66-
66-
45-
45-
sr-
FIG. 3. Release of surface antigens in four strains of T. cruzi. Tissue culture trypomastigotes labeled with [3’S]methionine were incubated in DMEM - 0.5% FCS at 37°C. Aliquots were withdrawn at 1 and 4 hr incubation (respectively 1 and 4 in the figure). The resulting supernatant was immunoprecipitated with the HIS (A) or the HlAlO monoclonal antibody (B). Molecular mass markers (kDa) are shown on the left.
shed antigens were originally located on the parasite surface and that the whole set of surface antigens were liberated. No qualitative differences in the pattern of the polypeptides liberated after 1 or 4 hr of incubation were observed for each strain (Fig. 3A). We have also analyzed the presence of a trypomastigote-specific antigen (Tc-85) in the supernatant of the four strains, using the HlAlO monoclonal as a probe (Alves ef al. 1986). Figure 3B shows that the RA, CA1 , and Y strains liberate an antigen of 85 kDa, whereas the YuYu strain sheds two components of 89 and 85 kDa recognized by HlAlO (cf. Abuin et al. 1989). The observation that tct of four strains of T. cruzi show the ability to liberate the whole set of surface antigens into the medium led us to investigate the mechanism by which these polypeptides were released. Molecules may be shed either independently or as plasma membrane vesicles. To investigate the latter hypothesis, the material released after 3 hr incubation in DMEM-OS% FCS by 1.6 x 10’ 13?S]methionine-labeled tct (Y strain) was chromatographed on a Sepharose 4B column. The eluted radioactivity was followed by counting an aliquot of each fraction be-
47
fore (total radioactivity) and after TCA precipitation. Two peaks of total radioactivity (peaks I and II) were obtained (Fig. 4A). However, only peak I remained after TCA precipitation, indicating that peak II contains mainly free [35S]methionine. Peak II shows the same elution profile of radioiodinated protein A (V,,) and [35S]methionine used to calibrate the column. The remaining aliquots of the fractions corresponding to peaks I and II were pooled independently, concentrated, and subjected to SDS-PAGE (Fig. 4B). All components released into the medium by tct (Y strain) were recovered in peak I (Fig. 4B, lane I), whereas no bands were seen in peak II (Fig. 4B, lane II). These experiments indicate that most of the parasite polypeptides were shed as small vesicles. In order to confirm this hypothesis, tct of the Y strain were labeled with [3H]palmitic acid, under conditions previously established to label the different classes of parasite lipids (Couto et al. 1985) and the shed material was chromatographed on the same Sepharose 4B column. A
0
VP&
30
35
40
FRACTION
45
50
55
I6
60
NUMBER
FIG. 4. (A) Sepharose 4B-chromatography. The polypeptides liberated by [3sS]methionine-labeled trypomastigotes (Y strain) were chromatographed on a Sepharose-4B column. The total (0) and TCAprecipitable (A) radioactivity was determined in each fraction. V,, and VP, indicate, respectively, the positions where Dextran Blue and radioiodinated Protein A/[35S]methionine are eluted. (B) Analysis by SDSPAGE of the pooled fractions of peaks I and II obtained in (A). Molecular mass markers (kDa) are shown on the left.
48
GONCALVES
The eluted radioactivity followed the same pattern depicted in Fig. 4A (results not shown). This experiment allowed the conclusion that the material eluted in peak I contains both proteins and lipids, thus reinforcing the conclusion that shedding occurs by vesiculation. Since surface antigens are the polypeptides preferentially shed by the parasite, these vesicles would represent, at least in part, plasma membrane vesicles. Parasites induced to shed were analyzed by electron microscopy (Fig. 5). Many of the parasites examined showed the presence of small vesicles mainly associated with the flagellar membrane, although they were also seen in association with the membrane which encloses the cell body and in the proximity of the parasite. In most of the cases, the vesicles were small, with a diameter of about 20 nm, although large vesicles, with a diameter of 80 nm were occasionally observed. The vesicles were also seen in trypomastigotes fixed immediately after collection from the supernatant of infected cell monolayers; however, they were much more frequent when the parasites were incubated for some hours, as here described. Also, the vesicles were more abundant when the parasites were incubated at 37°C than at 4”C, in agreement with the results shown in Table I. Gold particles, indicative of the presence of the antigen Tc-85 recognized by the monoclonal antibody HlAlO, were seen in association with the vesicles (Fig. 5), confirming the data from Fig. 3B. DISCUSSION
T. cruzi trypomastigotes spontaneously release polypeptides into the culture medium. The shedding is time (Fig. 2) and temperature dependent (Table I) and is independent of the presence of fetal calf serum or albumin added into the medium, as judged by the total amount of radioactivity analyzed after 3 hr incubation (Table I). Under the same experimental conditions,
ET AL.
epimastigotes do not liberate polypeptides, at least to an appreciable extent. Binding of antibodies to T. cruzi trypomastigotes, but not to epimastigotes, has been reported to cause capping and shedding of the antigenantibody complexes in a temperaturedependent fashion (Kloetzel and Deane 1977; Schmuxiis et al. 1978). The data here presented clearly indicate that the presence of a hyperimmune serum raised against the parasite does not have any influence on the total amount of released polypeptides (Table I) or on the rate of the shedding process (not shown). The experiments however do not exclude that some antigens may have a differential rate of shedding or that a crosslinking of molecules has to occur before shedding, as happens with the variant surface antigens of Trypanosoma congolense (Frevert and Reinwald 1988). Polypeptides shed by the parasite have a molecular mass between 70 and 150 kDa, with a main cluster in the 70-110 kDa region. This general pattern is apparently the same as reported before (Rimoldi et al. 1988). The loss of parasite polypeptides correlates well with their appearance in the culture medium, as can be seen in Fig. 1. This is more evident in the case of the higher molecular mass polypeptides, since the 120- to I50-kDa polypeptides present in the supernatant after the shedding assay were no longer found in the parasite. The fact that the shedding of the 70- to 1lo-kDa polypeptides is only partial could be due to a higher concentration of these polypeptides and/or to different rates of shedding. The pattern of the 13’1-labeled released polypeptides is almost identical to that obtained for the parasites before shedding, suggesting the surface origin of these components (Fig. lB, lanes a’, c’). This hypothesis is reinforced by the immunoprecipitation experiments which show that the antigens recognized by a hyperimmune serum were present on the living parasite and in the medium (Fig. lB, lanes d’ and e’). This phenomenon initially observed for
T.
cm&
SHEDDING
OF ANTIGENS
FIG. 5. Thin sections of trypomastigotes incubated under conditions of shedding. A-D, vesicles are seen in association with the plasma membrane lining the cell body and the flagellum (arrows). A, ~25,000; B, x3S,OOO;C, ~40,000; D, ~13,500. E and F, thin sections of trypomastigotes initially incubated in the presence of the monoclonal antibody HlAlO and subsequently in the presence of gold-labeled anti-mouse IgG before glutaraldehyde fixation. Gold particles are seen on the parasite cell surface as well as in association with vesicles (arrows). E, x33,000; F, ~55,000.
49
50
GONCALVES
tct of the Y strain has been found to occur in tct of the YuYu, RA, and CA1 strains (Figs. 2 and 3). For each strain the pattern of the antigens recovered in the supernatant after 1 or 4 hr shedding is qualitatively identical (Fig. 3A), indicating that the polypeptides are rapidly released and that no extensive degradation occurs. However, differences in the pattern of the released antigens recognized by the HIS are observed among the strains, confirming the microheterogeneity of antigens among strains and clones of T. cruzi described before (Zingales and Colli 1985; Alves ef al. 1986; Abuin et al. 1989). A specific antigen (Tc-85) related to the invasion of cultured mammalian cells by the parasite (Alves ef al. 1986) is also released by the trypomastigotes from the four strains (Fig. 3B). An interesting observation is that the amount of released polypeptides varies among strains, with a striking difference when the YuYu strain is compared to the others (Fig. 2). This could be a consequence of a higher metabolic activity of the YuYu strain observed under the experimental conditions or some other intrinsic characteristic of that strain. In fact, it was found that the incorporation rate of [35S]methionine into TCA-precipitable material is threefold higher for the YuYu strain when compared to the others (data not shown). It has to be pointed out that the total amount of protein per cell is very similar in the four strains. Chromatography of the parasite supernatant obtained after the shedding assay on a Sepharose 4B column (Fig. 4) suggests that polypeptides are released as plasma membrane vesicles. The data was confirmed by electron microscopy (Fig. 5) that also shows the association of an antigen (Tc-85) with the vesicles (Figs. SE and 5F). Membrane vesicles on the surface of trypomastigotes undergoing in vitro transformation to amastigotes have been observed upon incubation of the infective stage for periods longer than 24 hr (Andrews et uI.
ET AL.
1987). In the present case, however, for all strains analyzed no morphological changes of the trypomastigotes were apparent during the observation period (up to 4 hr). Thus, blebbing of membrane surface components appears to be an early, spontaneous, and continuous event associated with trypomastigote metabolism. The release of antigens as vesicles has been demonstrated in other parasites, such as Schistosoma mansoni (Kalapotakis et al. 1988), Plasmodium falciparum (Wilson and Bartholomew 1975), and T. congolensis (Frevert and Reinwald 1988). In the latter case, long filopodia structures (60-80 nm in diameter, 30 pm in length) were observed only when the parasite was exposed to substances which induce crosslinking of the variant surface antigens. For T. cruzi epimastigotes the formation and release of small plasma membrane vesicles (0.5 pm in diameter) could be induced after incubation with crosslinking reagents or acid pH (Franc0 da Silveira et al. 1979) and exposure to phorbol esters (De Siqueira and De Souza 1987). Although the evidence here presented points to the release of vesicles from the surface of trypomastigotes from T. cruzi, the data do not exclude that some antigens may be released by a different mechanism and/or that the same antigen could be released by different mechanisms. In fact, the presence of glycophosphatidylinositolanchored (GPI) proteins in metacyclic trypomastigotes of T. cruzi has been described (Schenkman et al. 1988). Furthermore, enzymes that solubilize the membrane form of the variant surface glycoprotein of african trypanosomes were detected in either epimastigote or metacyclic forms. However, the evidence suggests that besides phospholypase C-like enzymes, T. cruzi possesses other classes of hydrolases which could be involved in mechanisms of solubilization of surface antigens (Schenkman et al. 1988). Although shedding of plasma membrane
51
T. CWZi: SHEDDING OF ANTIGENS
vesicles of trypomastigotes was observed in vitro, the occurrence and the meaning of this process in viva is currently unclear. The presence of T. cruzi antigens has been described in the urine and blood of chagasic patients or experimentally infected animals, either in the acute or in the chronic phases of the disease (Dzbenski 1974; Araujo 1982; Freilij et al. 1987; Katzin er al. 1989a, b; Affranchino et al. 1989). Furthermore, the occurrence of parasite components on tissues and organs of infected animals has been verified (Ben Younes-Chennoufi ef al. 1988). These components could derive either from parasite lysis or from the shedding process here described. If, on one hand, the amount of antigens in body fluids of patients is within the limits of detection, on the other, very few circulating parasites are observed in the chronic phase of the disease. Thus, it is tempting to speculate that an active process of polypeptide liberation by the parasite could be occurring. The circulating antigens either free or in the form of immunocomplexes could play an important role in the immunopathology of Chagas’ disease, contributing to processes of autoaggression or immunosuppression (cf. Brener 1987; Petry and Eisen 1989). If this assumption is true it follows that variations found in the degree of tissue aggression among chagasic patients may in part be due to differences in the ability to shed of T. cruzi strains. This hypothesis finds support in our in vitro observation of different shedding capabilities shown by the four strains of T. cruzi studied. The knowledge of the intimate mechanisms of surface antigen shedding in T. cruzi could contribute to the understanding of phenomena such as immunosuppression and autoaggression in Chagas’ disease. ACKNOWLEDGMENTS This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, FAPESP, CNPq(E.S.U.),andFINEP(W.C.,M.J.M.A.,B.Z.).
REFERENCES ABUIN, G., COLLI, W., DE SOVZA, W., AND Arvss, M. J. M. 1989. A surface antigen of Trvpanosoma cruzi involved in cell invasion (Tc 85) is heterogeneous in expression and molecular constitution. MOlecular and Biochemical Parasitology 35, 229-238. AFFRANCHINO, J. L., IBAIIIEZ, C. F., LUQUETTI, A. U., RASSI, A., REYES, M. B., MACINA, R. A., ASLUNDI. L., PETERSON, V., AND FRASCH, A. C. C. 1989. Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas’ disease. Molecular and Biochemical Parasitology 34, 221-228. 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, 7542. ANDREWS, N. W., AND COLLI, W. 1982. Adhesion and interiorization of Trypanosoma cruzi in mammalian cells. Journal of Protozoology 29, 26&269. ANDREWS, N. W., ALVES, M. J. M., SCHUMACHER, R. I., AND COLLI, W. 1985. Trypanosoma cruzi: Protection in mice immunized with 8-methoxypsoralen-inactivated trypomastigotes. Experimental Parasitology 60, 255-262. 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. ARAUJO, F. G., CHIARI. E., AND DIAS, J. C. P. 1981. Demonstration of Trypanosoma cruzi antigen in serum from patients with Chagas’ disease. Lancet 1, 246349. ARAUJO, F. G. 1982. Detection of circulating antigens of Trypanosoma cruzi by enzyme immunoassay. Annals of Tropical Medicine and Parasitology 76, 25-36. BEN YOUNES-CHENNOUFI. A., HONTEBEYRIEJOSKOWICZ, M.. TRICOTTET. V., EISEN. H., REYNES. M., AND SAID, G. 1988. Persistence of Trypanosoma cruzi antigens in the inflammatory lesions of chronically infected mice. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 7783. BONGERTZ, V., HUNGERER. K., AND GALVAO CASTRO, M. 1981. Trypanosoma cruzi circulating antigens. Memdrias do Institute Oswaldo Cruz 76, 7182. BRENER, Z. 1987. Pathogenesis and immunopathology of chronic Chagas’ disease. Memdrias do Institute Oswaldo Cruz 82, 205-213. COUTO. A. S., ZINGALES, B., LEDERKREMER, R. M., AND COLLI. W. 1985. Trypanosoma cruzi: Meta-
52
GONCALVES
bolic labeling of trypomastigote sialoglycolipids. Experientia 41, 736-738. DE CARVALHO, T. U., AND DE SOUZA, W. 1987. Effect of phorbol-12-myristate-13 acetate (PMA) on the fine structure of Trypanosoma cruzi and its interaction with activated and resident macrophages. Parasitological Research 14, 11-17. DE SIQUEIRA, A. F., FERRIOLI FILHO, F., AND DOMINGUES RIBEIRO, R. 1979. Aspectos imunitarios iniciais observados em ratos infectados por Trypanosoma cruzi. A circulacao de antigenos soluveis e as modificacdes do complemento serico dos animais em dias sucessivos da infeccao. Revista Brasileira de Pesquisa Medica e Biologica lZ(Supp1. l), 75-79. DZBBNSKI, T. H. 1974. Exoantigens of Trypanosoma cruzi in vivo. Tropenmedizin und Parasitologie 25, 48549 1. FILARDI, L. S., AND BRENER, Z. 1987. Susceptibility and natural resistance of Trypanosoma cruzi strains to drug used clinically in Chagas’ disease. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 755-759. FRANCO DA SILVEIRA, J., ABRAHAMSON, P. A., AND COLLI, W. 1979. Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi. Biochimica et Biophysics Acta 550, 222-232. FREILU, H. L., CORRAL, R. S., KATZIN, A. M., AND GRINSTEIN, S. 1987. Antigenuria in infants with acute and congenital Chagas’ disease. Journal of Clinical Microbiology 25, 133-137. FREVERT, V., AND REINWALD, E. 1988. Formation of tilopodia in Trypanosoma congolense by crosslinking the variant surface antigen. Journal of UltraStructure and Molecular Structure Research 99, 124136. GONZALEZ CAPPA, S. M., CHIALE, P., DEL PRADO, G. E., KATZIN, A. M., MARTINI, G. W., ISOLA, E. D., ORREGO, L. A., AND SEGURA, E. 1980. Aislamiento de una cepa de Ttypanosoma cruzi de un paciente con miocardiopatia chagasica cronica y su caracterizacion biologica. Medicina (Buenos Aires) 4O(Suppl. l), 63-68. GONZALEZ CAPPA, S. M., KATZIN, A. M., ANASCO, N., AND LAJMANOVICH, S. 1981. Comparative studies of infectivity and surface carbohydrates of several strains of Trypanosoma cruzi. Medicina (Buenos Aires) 41, 549-555. KAHN, T., CORRAL, R., FREILIJ, H., AND GRINSTEIN, S. 1983. Detection of circulating immune complexes, antigens and antibodies by enzyme-linked immunosorbent assay in human Trypanosoma cruzi infection. IRCS Medical Science 11, 670-671. KALAPOTHAKIS, E., HIRST, E., AND EVANS, W. H. 1988. Perturbations of the topography of the tegument of adult Schistosoma mansoni. A scanning
ET AL. electron microscope study. Brazilian Journal of Medical and Biological Research 21, 961-969. KATZIN, A. M., AND COLLI, W. 1983. Lectin receptors in Ttypanosoma cruzi: An N-acetyl-o-ghrcosamine containing surface glycoprotein specific for the trypomastigote stage. Biochimica et Biophysics Acta 727, 403-411. KATZIN, A. M., MARCIPAR, A., FREILIJ, H., CORRAL, R., AND YANOVSKY, J. F. 1989a. Rapid determination of Trypanosoma cruzi urinary antigens in human chronic Chagas disease by agglutination test. Experimental Parasitology 68, 208-215. KATZIN, A. M., ALVES, M. J. M., ABUIN, G., AND COLLI, W. 1989b. Antigenuria in chronic chagasic patients detected by a monoclonal antibody raised against Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 341-343. KESSLER, S. W. 1975. Rapid isolation of antigens from cells with a Staphylococcal protein A antibody adsorbent: Parameters of the interaction of antibodyantigens complexes with protein A. Journal of Immunology 115, 1617-1624. KLOETZEL, J., AND DEANE. M. P. 1977. Presence of immunoglobulins on the surface of bloodstream Trypanosoma cruzi. Capping during differentiation in culture. Revista do Instituto de Medicina Tropical de Sao Paul0 19, 397-402. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MARCIPAR, A., BARNES, S.. LENTWOJT, E., AND BROWN, G. 1982. Immunoenzyme determination of antibody-bound soluble antigens of Trypanosoma cruzi. Applied Biochemistry and Biotechnology 7, 459-462. PETRY, K., AND EISEN, H. 1989. Chagas’ disease: A model for the study of autoimmune diseases. Parasitology Today 5, 111-l 16. RIBEIRO DOS SANTOS, R., AND HUDSON, L. 1980. Trypanosoma cruzi: Binding of parasite antigens to mammalian cell membranes. Parasite Immunology 2, l-10. RIMOLDI, M. T., SHER, A., HEINY, 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 Science USA 85, 193-197. SCHENKMAN, S., YOSHIDA, N., AND ALMEIDA. M. L. C. 1988. Glycophosphatidylinositol-anchored proteins in metacyclic trypomastigotes of Trypanosoma cruzi. Molecular and Biochemical Parasitology 29, 141-152. SCHMUNIS, G. A., SZARFMAN, A., LANGEMBACH, T., AND DE SOUZA, W. 1978. Induction of capping in blood-stage trypomastigotes of Trypanosoma cruzi
T. cruzi:
SHEDDING
by human anti-Tvpanosoma cruzi antibodies. Infection and Immunity
20, 561-569.
SILVA, L. H. P.. AND NUSSENSWEIG,V. 1953. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clinica et Biologica (S. Paula) 20, 191-207. WILLIAMS. G. T., FIELDER, L., SMITH, H., AND HUDSON, L. 1985. Adsorption of Trypanosoma cruzi proteins to mammalian cells in vitro. Acta Tropica 42, 33-38.
WILSON, R. J. M., AND BARTHOLOMEW.1975. The release of antigens by Plasmodium falciparum. Parasitology
71, 183-192.
ZINGALES, B. 1984. Labeling of trypanosomatids surface antigens by lactoperoxidase and Iodo-Gen. In
OF ANTIGENS
53
“Genes and Antigens of Parasites” (C. M. Morel, Ed.), pp. 333-338, Funda9Bo Oswald0 Cruz, Rio de Janeiro, Brash. ZINGALES,B., ANDREWS,N. W.. KUWAJIMA. V. Y., AND COLLI, W. 1982. Cell surface antigens of Trypanosoma cruzi: Possible correlation with the interiorization process in mammalian cells. Molecular Biochemistry and Parasitology 6, 1l-124. ZINGALES, B., AND COLLI, W. 1985. Trypanosoma cruzi: Interaction with host cells. In “Current Topics in Microbiology and Immunology” (L. Hudson, Ed.), Vol. 117, pp. 129-152, Springer-Verlag, Berlin, Heidelberg. Received 8 February 1990; accepted with revision 6 June 1990
PARASITOLOGY
Trypanosoma
72, 43-53 (1991)
cruzi: Shedding of Surface Membrane Vesicles
Antigens
as
MARINEI F. GON~ALVES,* EUFROSINA S. UMEzAwA,t ALWANDRO M. KATZIN,$ WANDERLEY DE SOUZA,§ MARIA JULIA M. ALVES,* BIANCA ZINGALES,* AND WALTER COLLI* *Departamento de Bioquimica, Instiruto de Quimica, Universidade de 560 Pa&o, Caixa Postal 20780, CEP 01498, Scio Paulo, Brazil: flnstituto de Medicina Tropical, Faculdade de Medicinn, Universidade de Srio Paulo, Av. Dr. Eneas de Carvalho Aguiar, 470, Sdo Paula, Brazil; $Departamento de Parasitologia, Instituto de Ciencias Biomhdicas, Unirpersidade de Scio Paulo, Caixa Postal 4365, CEP 05508, Sdo Paula, Brazil; and §Laboratdrio de Microscopia Eletronica e Ultra-Estrutura Celular, Institute de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. Ilha do Fundao. CEP 21941, Rio de Janeiro, Brazil CONCALVES, M. F.. UMEZAWA, E. S., KATZIN, A. M., DE SOUZA, W., ALVES, M. J. M.. ZINGALES, B., AND COLLI, W. 1990. Trypanosoma cruzic Shedding of surface antigens as
membrane vesicles. Experimental Parasitology 72, 43-53. Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CAI, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter. ‘T:’ 1991 Academic PESS. IK INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Parasite hemoflagellate; Shedding; Surface antigens; Vesicles; Bovine serum albumin (BSA); Dulbeccos’s modified Eagle medium (DMEM); Fetal calf serum (FCS); Hyperimmune serum (HIS); Polyacrylamide gel electrophoresis (PAGE): Sodium dodecyl sulfate (SDS); Trichloroacetic acid (TCA); Tissue culture trypomastigotes (Tct)
patients, suggesting that these components are the product of an active infection (Freilij et al. 1987). The occurrence of parasite antigens in tissues and organs in which intracellular parasites cannot be visualized has been reported in the chronic phase of experimental infection (Ben Younes-Chennoufi et al. 1988). Also, it has been proposed that the adsorption of T. cruzi antigens to uninfected cells may lead to damage of these cells by the immune response directed to the parasite (Ribeiro dos Santos and Hudson 1980; Williams et al. 1985). Furthermore, the parasite constituents can represent a persistent antigenic stimulation believed to occur in the chronic phase when parasites are not detected in most of the cases. This antigenic burden could play a
Circulating antigens have been demonstrated in sera from Trypanosoma cruziinfected animals (Dzbtnski 1974; De Siqueira er al. 1979; Araujo 1982) and from human patients in the acute and chronic phases of Chagas’ disease (Araujo et al. 1981; Marcipar et al. 1982; Kahn et al. 1983; Freilij et al. 1987). Several reports also describe the presence of parasite antigens in the urine from acutely infected mice and dogs (Bongertz et al. 1981) and from patients with acute, congenital, and chronic Chagas’ disease (Freilij et al. 1987; Katzin et al. 1989a, b). Moreover, whenever parasitemia becomes negative during the course of specific treatment, T. cruzi antigens cannot be detected in the urine in most of the 43
0014-4894/91 $3.00 Copyright B 1991 by Academic Press. Inc. All rights of reproductmn in any form reserved.
44
GONCALVES ET AL.
major role in the pathology of Chagas’ disease (Ben Younes-Chennoufi er al. 1988). The source of circulating and excreted antigens as well as tissue-adsorbed T. cruzi components has not been established. Nonetheless, it can be predicted that these antigens are originated from either parasite lysis or an active release of some specific antigens. Both processes could also occur concomitantly. In the present report we demonstrate that T. cruzi actively releases surface antigens by a spontaneous process which probably involves plasma membrane vesiculation. Tc-85, and 85kDa surface glycoprotein involved in the invasion of cells by the parasite (Alves et al. 1986), is one of these antigens. We also show that trypomastigotes from different strains have different shedding capacities. MATERIALS
AND METHODS
Parasite swains and cu/rure. Tissue culture trypomastigotes (tct) from the Y (Silva and Nussensweig 1953), the CA1 (Gonzalez Cappa et al. 1980). the RA (Gonzalez Cappa et al. 1981), and the YuYu (Filardi and Brener 1987) strains from T. cruzi were obtained from monolayers of LLC-MK2 cells (Andrews and Colli 1982). Antibodies. A polyclonal hyperimmune serum (HIS) against methoxypsoralen-inactivated tct (Y strain) was obtained (Andrews et al. 1985). A monoclonal antibody (HIAIO) that recognizes an 85kDa glycoprotein (Tc-85) of the trypomastigote surface (Katzin and Colli 1983)was prepared as described (Alves et al. 1986). Metabolic labeling wirh [‘5S]merhionine und radioiodinarion ofparasites. Parasites were incubated for 2
hr at 37°C in Dulbecco’s modified Eagle medium (DMEM)-methionine free, 2% fetal calf serum (FCS) in the presence of 25 uCi/ml [‘*S]methionine (1200 Gil mmol; Amersham, U.K.), as described (Zingales er al. 1982). Tissue culture trypomastigotes (Y strain) were radioiodinated with Na13’I (IPEN, Sao Paulo) using Iodogen (Pierce Chemical Co.) as catalyst (Zingales 1984). Labeling
with [3Hjuridine
and [‘Hjpalmiric
acid.
Tissue culture trypomastigotes (5 x lO’/ml) were incubated for 4 hr at 37°C in DMEMJ% dialysed FCS, containing either 5,6-[3H]uridine (40.5 Wmmol; New England Nuclear) or 9,10(n)-[3H]palmitic acid (50 Gil
mmol; Amersham, U.K.). Either precursor was added at 50 $X/ml. Shedding essays. Labeled parasites were washed twice in DMEM-O.5% FCS and resuspended at IO8 cells/ml in the same medium containing 20 mM N-2-hydroxyethylpiperazine-W-2 ethanesulfonic acid (Hepes). The suspension was maintained at 37°C (unless otherwise stated) in a humidified atmosphere with 5% COz. Aliquots (10’ cells) were removed at different times and centrifuged at 2500g for 5 min. The supernatants and the sedimented parasites were precipitated independently with cold tricholoroacetic acid (TCA) at 10% final concentration. In both cases, the precipitates were collected on 0.22~pm nitrocellulose filters and washed with 30 ml of 5% TCA. The dried filters were either counted in a liquid spectrometer (LKB, 1214Rackbeta) for [35S]methionine incorporation or in a gamma counter (Beckman/gamma 5500) for 13’1labeling. The quantification of the liberated polypeptides was calculated as the percentage (%) of TCA-precipitated radioactivity in the supematant medium in relation to the total radioactivity (pellet plus supernatant). The amounts referred to in the text represent the cumulative measurement at each experimental point. Immunoprecipirurion. The identification of cell surface antigens was done as described (Zingales et al. 1982). Labeled living parasites (5 x IO’) were incubated with 0.5 ml of DMEM containing HIS at a 1:25 dilution. Incubation was performed for 1 hr at 4°C with occasional agitation. Parasites were then washed to remove unbound antibody and lysed with a lysing buffer containing 1% Nonidet P-40, 10 mM Tris-HCI, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 n-&f N-tosyl-t-lysylchloromethyl ketone, 0.25 ug/ml leupeptin, and 2.8 units/ml aprotinin. Immunocomplexes present in the 10,OOOgsupernatant were precipitated with 100~1of a 10% suspension of heat-killed and formalin-fixed Sraphylococcus aureus (Cowan I strain) (Kessler 1975). Samples were processed as described (Zingales et al. 1982). The identification of total antigens liberated into the medium was done as described below. A twofold concentrated lysing buffer was added to the supematant obtained after centrifugation of parasites. The sample was precleared to remove nonspecific adsorbing material by incubation (2 hr at 4°C) with normal rabbit serum and 70 ~1 of a 10% suspension of S. aureus. Samples were then incubated overnight at 4°C with either HIS or HlAlO monoclonal antibody. After 30 min incubation with 70 ul of a 10% suspension of 5. aureus, the samples were processed as described previously (Zingales et al. 1982).The immunoprecipitated antigens were counted either in a gamma counter or in a scintillation spectrometer. Gel elecrrophoresis. Unidimensional gel electrophoresis was performed according to Laemmli (1970) on
T.
Cruzi:
SHEDDING
8% polyacrylamide gels containing 0.1% SDS (SDSPAGE). After electrophoresis, the gels were fixed, stained, and destained, processed for fluorography for [35S]methionine samples, dried, and exposed to X-ray films. Molecular mass markers (Sigma) were myosin, P-galactosidase, phosphorylase B, bovine serum albumin, and ovalbumin. Gelfiltrarion. The material liberated by 1.6 x lo8 labeled parasites in 0.2 ml of culture medium was chromatographed on a Sepharose 4B (Pharmacia Fine Chemicals) column (0.8 X 48 cm). The column was eluted with 10 n&f phosphate buffer, pH 7.2, 150 m&f NaCI, 0.02% NaN, at room temperature. Fractions of 0.5 ml were collected. Electron microscopy. The parasites were collected by centrifugation and fixed for 60 min at room temperature in a solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, washed in phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed in a Jeol 100 CX electron microscope. For the immunocytochemical localization of antigens, the parasites were processed as described previously (Abuin et al. 1989). For negative staining the samples were placed on formvar and carbon-coated grids, treated with 1% phosphotungstic acid, dried. and observed.
4.5
OF ANTIGENS
TABLE I Shedding of Polypeptides by Trypomastigotes of Trypanosoma cruzi under Different Conditions” Temperature (“Cl
Addition
4 37 37 37 37 37 37
0.596 FCS 0.5% FCS 5 % FCS 10 % FCS 4 % HIS 3.5 mg/ml BSA
Liberated polypeptides mb 2.9 14.5 14.2 14.3 15.8 15.0 12.9
a [“S]Methionine-labeled trypomastigotes (Y strain) were incubated in DMEM for 3 hr under the indicated conditions. b The percentage of liberated polypeptides was calculated as described under Material and Methods.
within the experimental error, were found; (ii) the same values for the TCA-precipitable radioactivity of supernatant aliquots were obtained before and after filtration by a 0.22~pm nitrocellulose membrane; (iii) parasites were prelabeled with [3H]uridine RESULTS and then placed in fresh culture medium, [35S]Methionine-labeled trypomastigotes under the same conditions of the shedding (Y strain) liberate polypeptides into the me- assays. Aliquots were withdrawn at differdium. This phenomenon was observed in ent times and TCA-precipitable radioactivthe absence or presence of bovine serum ity was measured in the supernatant. Apalbumin (BSA) or different concentrations proximately 2% of the total incorporated of FCS (Table I). Furthermore, it was ver- [3H]uridine was found in the medium at all ified that substitution of FCS by a rabbit experimental times. Furthermore, it was verified that parasites are fully motile and HIS, elicited by living trypomastigotes, does not alter the extent of shedding, sug- viable after the shedding assay, since they gesting that the process is independent of retain the same infection capacity of cell the binding of specific antibodies to the par- monolayers (experiments were performed asite surface. Also the kinetics of shedding according to Zingales et al. 1982, data not is independent of the presence of HIS (not shown). In order to characterize the nature of the shown). The incubation of trypomastigotes at 4°C promoted 80% inhibition of the re- polypeptides released into the culture melease of polypeptides observed at 37”C, dium as to their molecular mass and their showing that the shedding process is tem- cellular localization, trypomastigotes were either labeled with [35S]methionine (Fig. perature dependent (Table I). Several controls were performed in order 1A) or surface radioiodinated (Fig. IB). to rule out the occurrence of parasite lysis The pattern of the labeled polypeptides asduring the incubation period: (i) parasites sociated with the parasites before (lanes a, were counted under a light microscope af- a’) and after 3 hr shedding (lanes b, b’) was ter the shedding assays and no differences, compared by SDS-PAGE to the pattern of
46
GONCALVES ET AL.
Quantification of TCA-precipitable [35S]methionine-labeled components shed in the 205culture medium indicates that the total 116amount is dependent on the strain (Fig. 2). 116S?STTaking as reference the endpoint measurement after 4 hr observation, it is concluded 6666that the YuYu, Y, CAl, and RA strains lib454t)erate, respectively, 20, 11,7.5, and 4.5% of the total labeled polypeptides, under the experimental conditions employed. The nature of the antigens shed by the FIG. 1. Polypeptides from trypomastigotes (Y strain) released into the culture medium. Parasites strains was also analyzed by immunoprewere labeled with [35S]methionine (A) or ‘311-Iodogen cipitation using the HIS against trypo(B). Polypeptide pattern of parasites before shedding mastigotes. The four strains released poly(a, a’) and after 3 hr shedding at 37°C (b, b’) and of peptides bearing common epitopes recogpolypeptides released in the culture medium (DMEM nized by the HIS (Fig. 3A). The patterns - 0.5% FCS) (c, c’). Antigens immunoprecipitated with HIS from total parasites (d, d’) and from the cul- were similar, showing mainly the presence ture medium (e, e’). Molecular mass markers (kDa) are of polypeptides ranging from 70 to 150 kDa. shown on the left. Comparison of this pattern with that obtained by immunoprecipitation of living tct the components recovered into the culture of the four strains with the same antiserum medium (lanes c, c’). In both experiments (data not shown) suggests that most of the (Fig. 1A and 1B) polypeptides of 70 to 150 kDa that had been lost by the parasites were recovered in the incubation medium. In contrast, very few polypeptides below this molecular mass could be detected in the medium (Fig. 1, lanes c, c’). This observation reinforces the conclusion that parasites are not lysed under the shedding conditions. The experiments performed with the radioiodinated trypomastigotes (Fig. 1B) allowed the conclusion that the parasites release surface polypeptides. The evidence that the components released into the medium are the main surface antigens of the trypomastigote form was obtained in immunoprecipitation experiments, where living labeled parasites and I 2 3 1 the corresponding supernatant medium obHOURS tained after 3 hr shedding were incubated FIG. 2. Kinetics of polypeptide liberation by four with a HIS against trypomastigotes. The strains of T. cruzi. Tissue culture trypomastigotes antigenic patterns are very similar in both were labeled with [35S]methionine for 2 hr, washed, cases (Fig. lA, lanes d, e and Fig. IB, lanes and placed in DMEM - 0.5% FCS at 37°C. Aliquots were withdrawn at different times and TCA-precipd’, e’). itable radioactivity was measured in the supematant The liberation of polypeptides observed and in the sedimented parasites. The percentage of initially with the Y strain was also verified liberated radioactivity was determined as described for tct of the YuYu, CAl, and RA strains. under Materials and Methods. B a’ b’
Aobcde
C’
d’
e’
205-
io
T. cruzi: SHEDDING OF ANTIGENS A,
RA
,,
CA1
,.
Y
,, YuYu
,
B , RAwCAl,,
(4141414
*05-
444
Y .,YuS,
4
205-
116ST-
116-
66-
66-
45-
45-
sr-
FIG. 3. Release of surface antigens in four strains of T. cruzi. Tissue culture trypomastigotes labeled with [3’S]methionine were incubated in DMEM - 0.5% FCS at 37°C. Aliquots were withdrawn at 1 and 4 hr incubation (respectively 1 and 4 in the figure). The resulting supernatant was immunoprecipitated with the HIS (A) or the HlAlO monoclonal antibody (B). Molecular mass markers (kDa) are shown on the left.
shed antigens were originally located on the parasite surface and that the whole set of surface antigens were liberated. No qualitative differences in the pattern of the polypeptides liberated after 1 or 4 hr of incubation were observed for each strain (Fig. 3A). We have also analyzed the presence of a trypomastigote-specific antigen (Tc-85) in the supernatant of the four strains, using the HlAlO monoclonal as a probe (Alves ef al. 1986). Figure 3B shows that the RA, CA1 , and Y strains liberate an antigen of 85 kDa, whereas the YuYu strain sheds two components of 89 and 85 kDa recognized by HlAlO (cf. Abuin et al. 1989). The observation that tct of four strains of T. cruzi show the ability to liberate the whole set of surface antigens into the medium led us to investigate the mechanism by which these polypeptides were released. Molecules may be shed either independently or as plasma membrane vesicles. To investigate the latter hypothesis, the material released after 3 hr incubation in DMEM-OS% FCS by 1.6 x 10’ 13?S]methionine-labeled tct (Y strain) was chromatographed on a Sepharose 4B column. The eluted radioactivity was followed by counting an aliquot of each fraction be-
47
fore (total radioactivity) and after TCA precipitation. Two peaks of total radioactivity (peaks I and II) were obtained (Fig. 4A). However, only peak I remained after TCA precipitation, indicating that peak II contains mainly free [35S]methionine. Peak II shows the same elution profile of radioiodinated protein A (V,,) and [35S]methionine used to calibrate the column. The remaining aliquots of the fractions corresponding to peaks I and II were pooled independently, concentrated, and subjected to SDS-PAGE (Fig. 4B). All components released into the medium by tct (Y strain) were recovered in peak I (Fig. 4B, lane I), whereas no bands were seen in peak II (Fig. 4B, lane II). These experiments indicate that most of the parasite polypeptides were shed as small vesicles. In order to confirm this hypothesis, tct of the Y strain were labeled with [3H]palmitic acid, under conditions previously established to label the different classes of parasite lipids (Couto et al. 1985) and the shed material was chromatographed on the same Sepharose 4B column. A
0
VP&
30
35
40
FRACTION
45
50
55
I6
60
NUMBER
FIG. 4. (A) Sepharose 4B-chromatography. The polypeptides liberated by [3sS]methionine-labeled trypomastigotes (Y strain) were chromatographed on a Sepharose-4B column. The total (0) and TCAprecipitable (A) radioactivity was determined in each fraction. V,, and VP, indicate, respectively, the positions where Dextran Blue and radioiodinated Protein A/[35S]methionine are eluted. (B) Analysis by SDSPAGE of the pooled fractions of peaks I and II obtained in (A). Molecular mass markers (kDa) are shown on the left.
48
GONCALVES
The eluted radioactivity followed the same pattern depicted in Fig. 4A (results not shown). This experiment allowed the conclusion that the material eluted in peak I contains both proteins and lipids, thus reinforcing the conclusion that shedding occurs by vesiculation. Since surface antigens are the polypeptides preferentially shed by the parasite, these vesicles would represent, at least in part, plasma membrane vesicles. Parasites induced to shed were analyzed by electron microscopy (Fig. 5). Many of the parasites examined showed the presence of small vesicles mainly associated with the flagellar membrane, although they were also seen in association with the membrane which encloses the cell body and in the proximity of the parasite. In most of the cases, the vesicles were small, with a diameter of about 20 nm, although large vesicles, with a diameter of 80 nm were occasionally observed. The vesicles were also seen in trypomastigotes fixed immediately after collection from the supernatant of infected cell monolayers; however, they were much more frequent when the parasites were incubated for some hours, as here described. Also, the vesicles were more abundant when the parasites were incubated at 37°C than at 4”C, in agreement with the results shown in Table I. Gold particles, indicative of the presence of the antigen Tc-85 recognized by the monoclonal antibody HlAlO, were seen in association with the vesicles (Fig. 5), confirming the data from Fig. 3B. DISCUSSION
T. cruzi trypomastigotes spontaneously release polypeptides into the culture medium. The shedding is time (Fig. 2) and temperature dependent (Table I) and is independent of the presence of fetal calf serum or albumin added into the medium, as judged by the total amount of radioactivity analyzed after 3 hr incubation (Table I). Under the same experimental conditions,
ET AL.
epimastigotes do not liberate polypeptides, at least to an appreciable extent. Binding of antibodies to T. cruzi trypomastigotes, but not to epimastigotes, has been reported to cause capping and shedding of the antigenantibody complexes in a temperaturedependent fashion (Kloetzel and Deane 1977; Schmuxiis et al. 1978). The data here presented clearly indicate that the presence of a hyperimmune serum raised against the parasite does not have any influence on the total amount of released polypeptides (Table I) or on the rate of the shedding process (not shown). The experiments however do not exclude that some antigens may have a differential rate of shedding or that a crosslinking of molecules has to occur before shedding, as happens with the variant surface antigens of Trypanosoma congolense (Frevert and Reinwald 1988). Polypeptides shed by the parasite have a molecular mass between 70 and 150 kDa, with a main cluster in the 70-110 kDa region. This general pattern is apparently the same as reported before (Rimoldi et al. 1988). The loss of parasite polypeptides correlates well with their appearance in the culture medium, as can be seen in Fig. 1. This is more evident in the case of the higher molecular mass polypeptides, since the 120- to I50-kDa polypeptides present in the supernatant after the shedding assay were no longer found in the parasite. The fact that the shedding of the 70- to 1lo-kDa polypeptides is only partial could be due to a higher concentration of these polypeptides and/or to different rates of shedding. The pattern of the 13’1-labeled released polypeptides is almost identical to that obtained for the parasites before shedding, suggesting the surface origin of these components (Fig. lB, lanes a’, c’). This hypothesis is reinforced by the immunoprecipitation experiments which show that the antigens recognized by a hyperimmune serum were present on the living parasite and in the medium (Fig. lB, lanes d’ and e’). This phenomenon initially observed for
T.
cm&
SHEDDING
OF ANTIGENS
FIG. 5. Thin sections of trypomastigotes incubated under conditions of shedding. A-D, vesicles are seen in association with the plasma membrane lining the cell body and the flagellum (arrows). A, ~25,000; B, x3S,OOO;C, ~40,000; D, ~13,500. E and F, thin sections of trypomastigotes initially incubated in the presence of the monoclonal antibody HlAlO and subsequently in the presence of gold-labeled anti-mouse IgG before glutaraldehyde fixation. Gold particles are seen on the parasite cell surface as well as in association with vesicles (arrows). E, x33,000; F, ~55,000.
49
50
GONCALVES
tct of the Y strain has been found to occur in tct of the YuYu, RA, and CA1 strains (Figs. 2 and 3). For each strain the pattern of the antigens recovered in the supernatant after 1 or 4 hr shedding is qualitatively identical (Fig. 3A), indicating that the polypeptides are rapidly released and that no extensive degradation occurs. However, differences in the pattern of the released antigens recognized by the HIS are observed among the strains, confirming the microheterogeneity of antigens among strains and clones of T. cruzi described before (Zingales and Colli 1985; Alves ef al. 1986; Abuin et al. 1989). A specific antigen (Tc-85) related to the invasion of cultured mammalian cells by the parasite (Alves ef al. 1986) is also released by the trypomastigotes from the four strains (Fig. 3B). An interesting observation is that the amount of released polypeptides varies among strains, with a striking difference when the YuYu strain is compared to the others (Fig. 2). This could be a consequence of a higher metabolic activity of the YuYu strain observed under the experimental conditions or some other intrinsic characteristic of that strain. In fact, it was found that the incorporation rate of [35S]methionine into TCA-precipitable material is threefold higher for the YuYu strain when compared to the others (data not shown). It has to be pointed out that the total amount of protein per cell is very similar in the four strains. Chromatography of the parasite supernatant obtained after the shedding assay on a Sepharose 4B column (Fig. 4) suggests that polypeptides are released as plasma membrane vesicles. The data was confirmed by electron microscopy (Fig. 5) that also shows the association of an antigen (Tc-85) with the vesicles (Figs. SE and 5F). Membrane vesicles on the surface of trypomastigotes undergoing in vitro transformation to amastigotes have been observed upon incubation of the infective stage for periods longer than 24 hr (Andrews et uI.
ET AL.
1987). In the present case, however, for all strains analyzed no morphological changes of the trypomastigotes were apparent during the observation period (up to 4 hr). Thus, blebbing of membrane surface components appears to be an early, spontaneous, and continuous event associated with trypomastigote metabolism. The release of antigens as vesicles has been demonstrated in other parasites, such as Schistosoma mansoni (Kalapotakis et al. 1988), Plasmodium falciparum (Wilson and Bartholomew 1975), and T. congolensis (Frevert and Reinwald 1988). In the latter case, long filopodia structures (60-80 nm in diameter, 30 pm in length) were observed only when the parasite was exposed to substances which induce crosslinking of the variant surface antigens. For T. cruzi epimastigotes the formation and release of small plasma membrane vesicles (0.5 pm in diameter) could be induced after incubation with crosslinking reagents or acid pH (Franc0 da Silveira et al. 1979) and exposure to phorbol esters (De Siqueira and De Souza 1987). Although the evidence here presented points to the release of vesicles from the surface of trypomastigotes from T. cruzi, the data do not exclude that some antigens may be released by a different mechanism and/or that the same antigen could be released by different mechanisms. In fact, the presence of glycophosphatidylinositolanchored (GPI) proteins in metacyclic trypomastigotes of T. cruzi has been described (Schenkman et al. 1988). Furthermore, enzymes that solubilize the membrane form of the variant surface glycoprotein of african trypanosomes were detected in either epimastigote or metacyclic forms. However, the evidence suggests that besides phospholypase C-like enzymes, T. cruzi possesses other classes of hydrolases which could be involved in mechanisms of solubilization of surface antigens (Schenkman et al. 1988). Although shedding of plasma membrane
51
T. CWZi: SHEDDING OF ANTIGENS
vesicles of trypomastigotes was observed in vitro, the occurrence and the meaning of this process in viva is currently unclear. The presence of T. cruzi antigens has been described in the urine and blood of chagasic patients or experimentally infected animals, either in the acute or in the chronic phases of the disease (Dzbenski 1974; Araujo 1982; Freilij et al. 1987; Katzin er al. 1989a, b; Affranchino et al. 1989). Furthermore, the occurrence of parasite components on tissues and organs of infected animals has been verified (Ben Younes-Chennoufi ef al. 1988). These components could derive either from parasite lysis or from the shedding process here described. If, on one hand, the amount of antigens in body fluids of patients is within the limits of detection, on the other, very few circulating parasites are observed in the chronic phase of the disease. Thus, it is tempting to speculate that an active process of polypeptide liberation by the parasite could be occurring. The circulating antigens either free or in the form of immunocomplexes could play an important role in the immunopathology of Chagas’ disease, contributing to processes of autoaggression or immunosuppression (cf. Brener 1987; Petry and Eisen 1989). If this assumption is true it follows that variations found in the degree of tissue aggression among chagasic patients may in part be due to differences in the ability to shed of T. cruzi strains. This hypothesis finds support in our in vitro observation of different shedding capabilities shown by the four strains of T. cruzi studied. The knowledge of the intimate mechanisms of surface antigen shedding in T. cruzi could contribute to the understanding of phenomena such as immunosuppression and autoaggression in Chagas’ disease. ACKNOWLEDGMENTS This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, FAPESP, CNPq(E.S.U.),andFINEP(W.C.,M.J.M.A.,B.Z.).
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