Trypanosoma cruzi trypomastigotes from killing by human anti-α-galactosyl

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1 Journal of Cell Science 113, (2000) Printed in Great Britain The Company of Biologists Limited 2000 JCS Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-α-galactosyl antibodies Vera Lucia Pereira-Chioccola 1,2, Alvaro Acosta-Serrano 3, Igor Correia de Almeida 4, *, Michael A. J. Ferguson 4, Thais Souto-Padron 5, Maurício M. Rodrigues 1, Luiz R. Travassos 1 and Sergio Schenkman 1, 1 Department of Microbiologia, Imunologia e Parasitologia, UNIFESP, R. Botucatu 862 8A, , São Paulo, SP, Brazil 2 Laboratory of Xenodiagnosis, Instituto Dante Pazzanese de Cardiologia do Estado de São Paulo , Brazil 3 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA 4 Department of Biochemistry, University of Dundee, Dundee DD1 5EH, UK 5 Instituto de Biofísica, UFRJ, RJ, , Brazil *Present address: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Corresponding author ( sergio@ecb.epm.br) Accepted 24 January; published on WWW 7 March 2000 SUMMARY In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10 7 sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to α-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl 2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-α-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role. Key words: Trypanosoma cruzi, Trans-sialidase, Surface coat, Antibodies, α-galactosyl INTRODUCTION Trypanosoma cruzi, the agent of Chagas disease, is able to acquire sialic acid from the mammalian host due to the presence of its unique surface trans-sialidase (TS) (Schenkman et al., 1994b). The sialic acid is incorporated mostly into mucin-like molecules attached to the parasite membrane through a glycosylphosphatidylinositol anchor (Schenkman et al., 1993). These mucins are formed by short sequences of Thr-, Ser- and Pro-rich polypeptides, containing O-linked N- acetyl-glucosamine as the internal unit of galactosyl-rich oligosaccharides, which can be terminally sialylated (Reyes et al., 1994). The O-linked glycan structure varies according to the parasite developmental stage and strain, and it is the main acceptor site for the TS-mediated sialylation of the parasite (Acosta et al., 1994). In addition to sialic acid, mucin glycans of infective trypomastigotes derived from infected mammalian cells also contain terminal α-galactosyl residues, which are epitopes recognized by lytic antibodies found in chronic Chagasic patients (Almeida et al., 1993). These antibodies are similar but not identical to the well-known natural anti-α-gal antibodies present in large amounts in the sera of healthy humans (Galili et al., 1993). The natural anti-α-gal antibodies react specifically with the carbohydrate structure Galα(1 3)- Galβ(1 4)-GlcNAc (Galili et al., 1988b), and probably arise from the continuous stimulation by the normal intestinal flora (Galili et al., 1988a). On the other hand, the anti-α-gal from patients with Chagas disease (Ch anti-α-gal), although recognizing α-galactosyl epitopes of murine glycoproteins (Towbin et al., 1987; Galili et al., 1988b; Avila et al., 1989), binds with much higher affinity the O-linked oligosaccharide

2 1300 V. L. Pereira-Chioccola and others chains of mucin-like glycoproteins of trypomastigotes (Towbin et al., 1979; Almeida et al., 1993; Almeida et al., 1994b). Chagasic anti-α-gal antibodies agglutinate and destroy mammalian cell-derived trypomastigotes in a complementindependent way (Almeida et al., 1991, 1994a; Gazzinelli et al., 1991). Although several studies have suggested that sialic acids and mucins are involved in the parasite internalization by mammalian cells (Piras et al., 1987), the relative abundance of mucins and of sialic acid on the surface of the parasite indicates that they may have a primary structural and protective role. In the present work, we decided to reevaluate the distribution of mucins and glycosylinositol phospholipids (GIPLs), which are the major surface components of the infective forms. We found that mucins are relatively more abundant in trypomastigotes than in epimastigote forms. Electron microscopic studies revealed a coat enveloping the parasite, which is disrupted upon addition of Ch anti-α-gal, leading to parasite lysis. We therefore studied the effect of mucin sialylation and found that it dramatically increased the resistance of the parasite to Ch anti-α-gal-mediated killing. MATERIALS AND METHODS Parasites Epimastigotes of G (Yoshida, 1983) and Y (Silva and Nussenzweig, 1953) strains of T. cruzi were cultivated in liver infusion-tryptose broth containing 10% fetal bovine serum, 10 µg/ml hemin, at 28 C. Metacyclic trypomastigotes were obtained from cultures in the stationary phase after passage through a DEAE-cellulose column (Acosta et al., 1994). Tissue culture-derived trypomastigotes (Y strain) were obtained from supernatants of infected LLC-MK 2 cells (American Type Culture Collection, Rockville, MD, USA; CCI-7) cultivated at 37 C and 5% CO 2 in low-glucose Dulbecco s modified Eagle s medium (Gibco, Grand Island, NY) (DMEM), containing penicillin, streptomycin and 10% fetal bovine serum. To prepare parasites devoid of sialic acid, serum was replaced by 0.2% bovine serum albumin (BSA) (Ultrapure, Boehringer, Germany) on the third day of infection. Cultivation was continued until the parasites were released from the infected cell. Purification of GIPLs and mucins Tissue culture-derived trypomastigotes and epimastigotes (10 10 ) were freeze-dried and extracted as described (Almeida et al., 1994a; Gazzinelli et al., 1997). Briefly, the lipids were extracted sequentially from freeze-dried parasites with 50 ml of chloroform/methanol (1:1), chloroform/methanol (1:2) and chloroform/methanol/water (10:20:8). The insoluble pellet was dried under N 2 and extracted (three times, for 3 hours each at room temperature) with 10 volumes of 9% butan- 1-ol in water. After each extraction the soluble phase was collected by centrifugation (10 minutes, 2000 g), pooled, concentrated and freeze-dried to produce the F3 fraction. This fraction was dissolved in 500 µl of 9% butan-1-ol and extracted three times with 500 µl of 91% butan-1-ol. Mucins were collected in the aqueous phase and GIPLs in the organic phase. After phase partition, mucins were further purified on Octyl-Sepharose CL-4B (Sigma Co., St Louis, MO, USA) columns, and the purity was assessed by SDS-PAGE and silver staining, and gas chromatography-mass spectroscopy (GC-MS). Mucin and GIPL quantitation The concentrations of mucins and GIPLs were estimated by measuring the myo-inositol content of the glycosylphosphatidylinositol anchor (Ferguson, 1993). Each sample (10 µl) was mixed with 20 µl of 1 µm d 6-myo-inositol, dried and hydrolyzed with 50 µl 6 N HCl (110 C, overnight). Released myo-inositol was quantitated as a trimethylsilyl derivative using a gas chromatograph-mass spectrometer (Hewlett- Packard ), or by electron-spray mass spectrometry (ES-MS) (Camargo et al., 1997). Chagasic anti-α-galactosyl antibodies Chagasic anti-α-gal antibodies (Ch anti-α-gal) were purified from patients with chronic Chagas disease by affinity chromatography on Synsorb 115 (ChemBiomed, Edmonton, Canada), linked to the trisaccharide D-Galα(1 3)-D-Galβ(1 4)-D-GlcNAc (Avila et al., 1989). Nonspecific protein binding was removed by washing the column with 0.5 M NaCl in 0.01 M phosphate-buffered saline (PBS). Then, elution was carried out with 50 mm citric acid, ph 2.8. The IgG fraction was obtained from this fraction after passage through a protein A-Sepharose column followed by acid elution. Acidic eluates were immediately neutralized with 1.5 M Tris-HCl, ph 8.5, and dialyzed against PBS. Anti-recombinant TS sera The recombinant TS was expressed in E. coli (TG1 strain) transformed with the plasmid pts-cat7 (Ribeirão et al., 1997). This plasmid contains a 1.9 kb fragment encompassing the catalytic domain of TS. The expression and purification of this enzyme were previously described in detail (Ribeirão et al., 1997). Groups of A/Sn mice were immunized intraperitoneally in four doses, at 2-week intervals, with 20 µg TS emulsified in a 0.5 mg/ml aluminum hydroxide suspension as adjuvant. Blood was collected 10 days after the last immunization and the sera were assayed for antibodies inhibitory to TS activity (Pereira-Chioccola et al., 1994). Electron microscopy Samples to be analyzed by scanning (SEM) and transmission electron microscopy (TEM) were processed as follow. Trypomastigotes from tissue culture in 0.2% BSA-DMEM were collected by centrifugation (2000 g for 10 minutes), washed twice with PBS, and resuspended in DMEM ( parasites in 50 µl). One of the samples was incubated with 1 mm sialyllactose (SL) (room temperature for 10 minutes). In a second sample, the parasites were incubated with 2 µg /ml Ch antiα-gal (37 C for 30 minutes) and washed with PBS. For SEM, 50 µl of a parasite suspension were placed on glass coverslips (12 mm diameter) previously coated with 0.1% poly-llysine. After 10 minutes, non-adhered parasites were removed by rinsing with PBS, and the coverslips were immersed in 0.5 ml 2% glutaraldehyde, 0.1 M sucrose in 0.1 M sodium cacodylate buffer, ph 7.2 at 4 C for at least 24 hours. The coverslips were then further processed as described previously (Schenkman et al., 1988). Samples were observed in a 25 kv scanning JEOL-JSM 5300 electron microscope. For TEM, parasites were fixed in a suspension containing 2.5% glutaraldehyde, 4% formaldehyde, 0.1 M phosphate buffer, ph 7.2, at 4 C, for 1 hour. After fixation cells were washed twice in PBS containing 5 mg/ml of Ruthenium Red, (Sigma) and postfixed with 1% O so 4, in 0.1 M cacodylate buffer ph 7.2, containing 5 mg/ml Ruthenium Red, for 2 hours in the dark. Samples were washed twice in 0.1 M cacodylate buffer, dehydrated in acetone and embedded in Epon. Ultrathin sections were observed either unstained or stained with uranyl acetate and lead citrate and observed in a ZEISS 900 transmission electron microscope operating at 80 kv. Trypomastigote agglutination and lysis assay Experiments were carried out, in triplicate, in 60-well high profile Terasaki plates (Robbins Scientific Corp., USA). Trypomastigote suspensions (4 µl in DMEM without sialic acid donors; parasites/ml) were pre-treated for 10 minutes at room temperature either with 1 mm SL in DMEM, or with medium alone. Parasites were then incubated with the same volume of a pool of mouse anti-ts

3 The role of T. cruzi sialylated coat 1301 antiserum diluted in normal mouse serum (NMS). Next, 8 µl of Ch anti-α-gal at the indicated concentration was added, and the mixture was further incubated for 30 minutes at 37 C. Alternatively, parasites were incubated with different concentrations of Concanavalin A (Sigma) and MgCl 2. The controls were incubated with DMEM. As the anti-α-gal induces both agglutination and lysis, one independent of the other (see Results), we measure the number of motile and free (non-agglutinated) trypomastigotes in a hemocytometer. This assay is sensitive, requires very little material and provides no background. The results are expressed as percentages (mean ± s.d.) of motile and free parasites relative to controls. RESULTS Density of mucins and GIPLs on epimastigotes and trypomastigote forms We have purified mucins and GIPLs from epimastigote and trypomastigote forms grown in the presence of fetal bovine serum. Both classes of glycoconjugate were recovered almost quantitatively in the 9% butan-1-ol extract of the chloroform/ methanol/water delipidated cells as judged by ES-MS (not shown). The 9% butan-1-ol extract was partitioned between water and butan-1-ol, producing a mucin-containing aqueous phase and a GIPL-containing organic phase. The GIPL fractions were analyzed by ES-MS and found to be free of contaminating phospholipids. The ES-MS spectra were consistent with the known GIPL structures present in epimastigotes (Previato et al., 1990; Lederkremer et al., 1991) and showed that the trypomastigote GIPL structures (to be described elsewhere) were similar. The copy numbers (GIPL/cell) for epimastigotes and trypomastigotes were estimated by GC-MS analysis of the myo-inositol content of the GIPL fractions (Table 1). The mucin-containing fraction was further purified by Octyl-Sepharose chromatography. The mucins eluting within the propan-1-ol gradient (at about 25-35% propan-1-ol) were pooled and the number of molecules per cell was estimated by GC-MS analysis of the myo-inositol content (Table 1). These figures assume quantitative recovery Fig. 1. Epimastigote form of T. cruzi stained by Ruthenium Red. A thin coat is observed on the plasma and flagellar membranes and at the zone of adhesion (arrows) between the cell body (cb) and the flagellum (f). Bar, 0.5 µm. Table 1. Content of GIPLs, mucins and sialic acid in trypomastigote and epimastigote forms of T. cruzi Ratio Trypomastigotes Epimastigotes (trypo:epimastigotes) Parasite surface area (µm 2 ) a GIPLs per cell b,c 0.7± ± GIPLs per µm 2 29, , Mucins per cell c 1.8± ± Mucins per µm 2 75,000 59, Sialic acid per cell d 1.1± ± Sialic acid per µm 2 460, , Sialic acid per mucin 6 11 a The surface area of the parasites was estimated based on lengths and diameter as determined by SEM, and assuming that each parasite is cylindrical. b The number of cells was determined before extraction by counting live parasites in hemocytometer. Values are means ± s.d. of three determinations. c The amount of GIPLs and mucins was determined for a known number of parasites used in the extraction. The values were obtained assuming one myoinositol residue per GIPL or mucin molecule. d The sialic acid content was measured by the TBA-HPLC assays (Powell and Hart, 1986). Fig. 2. Trypomastigotes show a coat after TEM and Ruthenium Red staining that is denser than the epimastigote coat. Cell-derived trypomastigotes were fixed and stained as described in Materials and Methods. A uniform staining can be observed on the cell body and flagellar membranes (arrows), as well as on the membrane lining the flagellar pocket (arrowheads) and in the zone of adhesion between the cell body (cb) and the flagellum (f) (A). No difference is observed in the thickness of Ruthenium Red precipitate in trypomastigotes devoid of sialic acid (B), incubated with SL (C), or grown in the presence of 10% fetal calf serum (D). Bars, 0.5 µm (A); 0.1 µm (B-D). of the GPI-anchored mucins and are therefore, minimum figures. Some mucins appear to elute in the void volume of the column and might represent molecules without a GPI anchor (Acosta et al., 1994).

4 1302 V. L. Pereira-Chioccola and others Fig. 3. Effect of Ch anti-α-gal IgG on the trypomastigote coat detected by the Ruthenium Red method. Trypomastigotes were incubated for 15 minutes at 37 C with 20 µg/ml Ch antiα-gal, fixed and processed for TEM as described in Materials and Methods. The treated parasites present large blebs protruding from the cell body membrane (arrows in A-C), or a fuzzy coat can be seen detaching from the cell surface (D,E) or associated to cell debris (*) (F). Bars, 0.5 µm (A,B,D-F); 0.25 µm (C). As suggested previously by antibody staining (Golgher et al., 1993), the data show that trypomastigotes contain fewer GIPLs than the epimastigotes: about 9% in absolute terms and about 24% in terms of surface density, taking into account the smaller size of the trypomastigote form. However, trypomastigotes contain a slightly higher density of mucins compared with the epimastigotes: 47% in absolute terms but 127% in terms of surface density. Furthermore, trypomastigote mucins appear to be substantially larger than the epimastigote mucin, as judged by SDS-PAGE and gel filtration (Acosta et al., 1994), and therefore may occupy more space than the epimastigote mucin. If we assume, based on the 5 nm cross-sectional diameter of mammalian mucins (Bansil et al., 1995), that each mucin covers about µm 2, then there are sufficient mucin molecules to cover 36 µm 2 of membrane surface. This value is similar to the estimated surface of the trypomastigote form (about 24 µm 2 ) and suggests that the trypomastigote mucins form a dense and continuous cell surface coat. By analogy, the epimastigote forms are also expected to have a mucin surface coat, but one that is slightly less dense and, due to their smaller size, less thick. There are about and sialic acid molecules per trypomastigote and epimastigote, respectively (Table 1). As the density of mucins is slightly high in trypomastigotes, the surface charge of trypomastigotes is almost the same as of the epimastigotes.

5 The role of T. cruzi sialylated coat 1303 Fig. 4. Scanning electron micrographs (SEM) of trypomastigotes treated with Ch anti-α-gal antibodies. Trypomastigotes were incubated for 15 minutes at 37 C with 20 µg/ml Ch anti-α-gal, fixed and processed for SEM as described in Materials and Methods. (A) Trypomastigotes without antibody. (B-F) Some representative images seen in the presence of Ch anti-α- Gal. Bars, 1 µm. Visualization of the surface coat by EM To evaluate the presence of a surface coat in epimastigotes and trypomastigotes, the parasites were fixed, treated with Ruthenium Red, a stain for carbohydrate, and processed for TEM. Fig. 1 shows that epimastigote forms have a thin coat that is more visible in the flagellum attachment region. In contrast, trypomastigotes have a much clearer and thicker staining on the entire parasite surface, including the flagellar pocket, suggesting the presence of a uniform glycocalyx (Fig. 2A), not seen in parasites untreated with Ruthenium Red (de Souza et al., 1978). The continuous and homogeneous deposit of the Ruthenium Red is not affected whether the parasites are sialylated, nonsialylated or grown in the presence of serum (Fig. 2B-D). This result is not surprising, since Ruthenium Red, although used to stain mucopolysaccharides, shows only a slight decrease in the staining of mammalian cells after neuraminidase (Weiss and Zeigel, 1969; Huet and Herzberg, 1973). Evidence for a glycocalix coat enveloping the parasite has also been detected by flip-etching procedures (Pimenta et al., 1989). In both cases trypomastigotes display a thicker coat. Fig. 5. Sialylation protects trypomastigotes from killing by Ch anti-α-gal. Tissue culturederived trypomastigotes ( /ml) grown in a medium without sialic acid donors (circles) or treated with 1 mm SL (squares) were incubated for 30 minutes at 37 C, at the indicated concentrations of Ch anti-α-gal antibodies (A) or Concanavalin- A (Con-A; B). In (C), parasites were treated with Ch anti-α-gal (20 µg/ml) in the presence of the indicated concentrations of MgCl 2. Values are means ± s.d. of triplicate measurements expressed as a percentage of values from controls incubated in absence of antibodies. Motile (live) trypomastigotes were counted in a hemocytometer. MgCl 2 (up to 100 mm) did not affect the parasites in the absence of Ch anti-α-gal. Effect of anti-α-gal antibodies Based on the fact that mucins cover the surface of the parasite and Ruthenium Red stains highly glycosylated molecules on cell surfaces, it is possible that the observed coat corresponds mainly to mucins. To test this hypothesis we studied in detail the effect of antibodies isolated from Chagasic patients that recognize anti-α-gal epitopes also present in the mucins, and induce parasite lysis in the absence of complement (Almeida et al., 1994b). Purified Ch anti-α-gal antibodies produce intense damage of trypomastigotes after a few minutes. The damage is seen by direct microscopic observation, with the immediate cessation of movement and loss of refringency. With high concentrations of parasites, an extensive agglutination is seen before the damage, while damage occurs without agglutination at low parasite concentration. Observation of thin sections of Ruthenium Red-stained parasites reveals different degrees of membrane damage (Fig. 3). In some parasites large blebs are seen protruding from the surface (Fig. 3A-C). In other cells, the continuous coat appears damaged or wrinkled (Fig. 3D,E), or it seems detached from the parasite surface (Fig. 3F). SEM images confirm the damage to the plasma membrane compared with untreated parasites (Fig. 4A). After addition of Ch anti-α-gal, parasites at high density agglutinate immediately (Fig. 4B). The membrane becomes rough (Fig. 4C,D), has blebs that some times seem to

6 1304 V. L. Pereira-Chioccola and others Fig. 6. Anti-TS antibodies that prevent sialylation increase the killing mediated by Ch anti-α-gal antibodies. (A) The amount of surviving parasites after incubation with mouse anti-ts antiserum diluted (1:2) in NMS (circles) or NMS (squares), followed by the indicated amounts of Ch anti-α-gal. (B) The survival after incubation with a fixed amount of Ch anti-α-gal (20 µg/ml) (closed bars), or medium alone (open bars) in the presence of increased dilutions of anti-ts in NMS. Each point represents the mean ± s.d. of triplicates. Controls include incubations in the presence of NMS only, or medium only ( ). Fig. 7. Anti-TS antibodies have no effect in sialylated trypomastigotes, which are more resistant to the Ch anti-α-gal antibodies. Trypomastigotes grown in BSA-containing medium (hatched bars) or parasites resialylated with 1 mm SL (open bars) were incubated for 10 minutes either with anti-ts antiserum diluted in NMS, NMS alone or medium alone. Parasites were then incubated for 30 minutes at 37 C, with Ch anti-α-gal (20 µg/ml), normal human serum or medium, as indicated. The results are means± s.d. of triplicate measurements. detach from the surface (Fig. 4E), or exhibits even more severe damage (Fig. 4F). We then tested whether the presence of sialic acid changes parasite susceptibility to lysis induced by Ch anti-α-gal antibodies. We found that sialylation conferred significant resistance to killing. The antibody concentration required to induce 50% killing increased from 5 µg/ml for unsialylated parasites to 100 µg/ml for those treated with SL (Fig. 5A). This effect is not due to a reduced binding of the antibody to the mucin α-galactosylated oligosaccharides. Sialylation does not affect the binding of Ch anti-α-gal, as assessed by enzyme immunoassays with purified mucin (Almeida et al., 1994a). Moreover, sialylated and non-sialylated trypomastigotes bind the same amount of anti-α-gal, as judged by indirect immunofluorescence and FACS analysis (not shown). In addition, the effect is specific for mucin ligands. Agglutination induced by Concanavalin A, which does not bind to mucins (Schenkman et al., 1991), is the same with sialylated and nonsialylated parasites (Fig. 5B). To better understand the mechanism by which sialylation prevents the killing by the Ch anti-α-gal antibodies, sialylated and non-sialylated parasites were treated with increasing concentrations of MgCl 2.. As shown in Fig. 5C, addition of Mg 2+ ions reverses the protection induced by sialylation, whereas it has practically no effect in the killing of non-sialylated parasites. Magnesium ions do not interact specifically with the sialic acid but can inhibit electrostatic repulsion between sialic acids. Antibodies from T. cruzi infected animals and Chagasic patients display high levels of TS inhibitory activity (Pereira- Chioccola et al., 1994). These antibodies prevent in vitro (Schenkman et al., 1994a) and in the infected animal (unpublished results) parasite sialylation, as seen by immunofluorescence using antibodies specific for sialylated epitopes. Sialylation is quite a rapid event, taking place in less than few minutes in the presence of serum (Schenkman et al., Fig. 8. Proposed model for how sialylation protects the trypomastigotes against killing mediated by Ch anti-α-gal. See text for details. 1991). Therefore, by preventing sialylation, anti-ts antibodies could improve the killing effect of Ch anti-α-gal antibodies. To test this possibility, non-sialylated parasites were incubated with a pool of sera from mice immunized with a recombinant TS, or with normal mouse serum (NMS). In both cases, the serum was the source of sialic acid donors. As shown in Fig. 6A, trypomastigotes incubated with anti-ts sera become much more susceptible to killing by Ch anti-α-gal as compared to parasites sialylated in the presence NMS (Fig. 6A). The inhibitory anti-ts sera could be used up to a 2080-fold dilution in normal mouse serum to increase the lytic effect of Ch antiα-gal (Fig. 6B). Similar results were obtained when we used antibodies isolated from Chagasic serum (not shown). Specific anti-ts antibodies were purified by affinity chromatography containing the recombinant TS immobilized on Tresyl- Sepharose. Therefore, individuals with inhibitory anti-ts antibodies must have a decreased parasite sialylation, which in turn could dramatically improve the killing mediated by Ch anti-α-gal. Anti-TS antibodies do not promote parasite desialylation and do not increase the lysis induced by Ch anti-α-gal. When

7 The role of T. cruzi sialylated coat 1305 parasites were sialylated before the incubation with anti-ts, there was less killing by addition of the Ch anti-α-gal (Fig. 7). This experiment also shows that anti-ts does not promote lysis in both sialylated and non-sialylated parasites, and that addition of NMS as a source of sialic acid protects the parasites as does pre-sialylation with SL. DISCUSSION We have provided evidence that mucin molecules form a dense and continuous cell surface coat in trypomastigote T. cruzi cells. This coat seems to have a primarily protective role and the addition of sialic acid into the mucin molecules, through the unique TS reaction, seems to confer to this parasite an important and efficient mechanism for surviving in different environments. The notion suggesting that the coat, as seen by TEM, corresponds to the parasite mucins is based on the fact that these molecules are highly abundant, and being hydrated as mammalian mucins, their surface area is large enough to cover most of, or the entire, parasite surface. Our calculations are based on the assumption that the cross-sectional area of a mammalian mucin is similar to the parasite mucins. This assumption is valid, considering that the cross-sectional area depends on the type of polypeptide and glycosylation, which is similar in both cases. The presence of the surface coat is also in agreement with electron microscopy and fluorescence staining using Ch anti-α-gal antibodies and anti-mucin monoclonal antibodies, which reveal a dense labeling (Schenkman et al., 1991; Souto-Padron et al., 1994). Another abundant surface glycoprotein, such as the members of the 85 kda family, are present at only about 10 5 molecules per parasite (Cross and Takle, 1993), and cover a smaller fraction of the surface area. Secondly, addition of Ch anti-α-gal antibodies induced extensive coat damage and parasite lysis, in a manner compatible with the idea that mucins cover the entire parasite surface. Homologous coats are found in other protozoan parasites such as T. brucei variable surface glycoprotein coat (Cross, 1975), and Leishmania lipophosphoglycan coat (Saraiva et al., 1995), and similar types of membrane damage have been observed for lectins that recognize abundant surface antigens in other microorganisms (Alderete and Kasmala, 1986). The finding that trypomastigote coat is thicker than the coat found in epimastigotes could be due to (1) smaller density of the epimastigote mucins, (2) the presence of large amounts of GIPLs, which are small molecules in epimastigotes, (3) on the fact that trypomastigote mucins show a more complex glycosylation pattern, and (4) the larger size of trypomastigote mucin. Interestingly, epimastigotes can acquire more sialic acid per mucin in agreement with the idea that other types of glycosylation are found in the trypomastigote forms, such as the α-galactosylation. Nevertheless, this difference is less important taking in consideration the density of sialic acid per cell, which is quite similar in both cases. We also provide an important evidence for the role of T. cruzi sialylation. Since large amounts of Ch anti-α-gal antibodies are required to induce the same lysis when the mucin coat is sialylated, sialylation of the mucin coat seems to be an efficient escape mechanism. The fact that large amounts of lytic anti-α- Gal IgM are produced in the acute phase of Chagas disease (Gazzinelli et al., 1991), argue that, at least in the human case, sialylation must favor parasite survival, and the eventual inhibition of TS by antibodies must be an important defense mechanism against the parasite. The protective effect of sialylation is most likely due to a charge effect since it is counterbalanced by the presence of Mg 2+ ions, although we cannot exclude the possibility that Mg 2+ ions interfere with the structure of sialylated mucin layer. Ch anti-α-gal antibodies are elicited during infection and recognize additional epitopes to natural anti-α-gal, including structures present in the O-linked oligossacharide of the mucins, with high affinity. Moreover, a much higher concentration of anti-α-gal antibodies isolated from non- Chagasic patients is required to induce trypomastigote lysis (Almeida et al., 1994a,b). Anti-α-Gal antibodies are known to be restricted to humans and Old World monkeys. For this reason, sialylation cannot be considered to be a general mechanism to prevent killing induced by these antibodies. It is possible that other mammalian species might also produce specific, or crossreacting antibodies that recognize non-sialylated mucins and damage the parasite. Immunization of mice with a recombinant TS generates inhibitory anti-ts antibodies that protect animals against a parasite challenge (Pereira-Chioccola et al., 1999), perhaps by preventing sialylation and damage by such kind of antibody. Sialylation of β-gal residues could be important to prevent clearance, binding to opsonins and, eventually, to other antibodies produced by mammalian species. In addition, the sialylated coat could prevent damage induced by potentially hazardous agents, such as enzymes and oxidants used as a host defense mechanism (Pereira-Chioccola and Schenkman, 1999). In the parasite stages growing in the insect gut the presence of a surface coat could help them resist the harsh conditions, such as desiccation and digestive enzymes. In the mammalian host, the coat could improve parasite survival during the initial steps of the nonspecific immune response. Although sialic acid does not seem to improve complement resistance (Tomlinson et al., 1992), it could affect complement opsonization, which may influence the distribution of parasites in the different tissues (Pereira and Hoff, 1986; Pereira, 1990). We are currently evaluating whether this coat can protect the parasite against lysosomal hydrolases and eventually against oxidative molecules. It should be mentioned that definitive proof for the role of sialylation may come from genetic ablation, which is not currently possible. In Fig. 8 we propose a mechanism by which Ch anti-α-gal induce lysis and why sialylation reduces killing. In the absence of sialic acid substitution, mucins are aggregated on the parasite surface by the bivalent anti-α-gal antibodies. Killing is not observed with Fabs (not shown), in support of this explanation. Aggregation of mucins, which are abundant on the surface, results in irreversible surface damage and lysis. The exact reason why local aggregation induces such a damage is unknown. One possibility is that clustering of two mucin chains results in a mechanical distortion of the molecule leading to membrane destabilization. When parasites are concentrated (above /ml) anti-α-gal antibodies also induce an extensive parasite agglutination followed by killing. Interestingly, parasite agglutination is not enough to promote membrane destabilization since antibodies against the sialylated mucins, although highly agglutinating, are unable to

8 1306 V. L. Pereira-Chioccola and others induce lysis (Franchin et al., 1997). This could be explained by the fact that sialylation, which is required for antibody binding, also prevents mucin clustering, possibly by electrostatic repulsion of negatively charged sialic acid molecules. Alternatively, α-galactosyl epitopes could be localized in a special position in the mucin molecules which, upon local aggregation, could induce alterations in the glycoprotein topology, resulting in membrane destabilization. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Ministério da Ciência e Tecnologia-PADCT, from Brazil. A.A.S. was a fellow from CONICIT (Venezuela). I.C.A. was recipient of a FAPESP postdoctoral fellowship (no. 96/4260-0). We thank Dr Paul Englund for reading the manuscript and Edna Haapalainen for the use of the SEM facilities. The authors are grateful to Luiz S. Silva (UNIFESP) for the purification of Chagasic anti-α-gal antibodies. REFERENCES Acosta, A., Schenkman, R. P. F. and Schenkman, S. (1994). Sialic acid acceptors of different stages of Trypanosoma cruzi are mucin-like glycoproteins linked to the parasite membrane by GPI anchors. Braz. J. Med. Biol. Res. 27, Alderete, J. F. and Kasmala, L. (1986). Monoclonal antibody to a major glycoprotein immunogene mediates differential complement-independent lysis of Trichomonas vaginalis. Infect. Immun. 53, Almeida, I. C., Ferguson, M. A. J., Schenkman, S. and Travassos, L. R. (1994a). Lytic anti-α-galactosyl antibodies from patients with chronic Chagas disease recognize novel O-linked oligosaccharides on mucin-like glycosylphosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. Biochem. J. 304, Almeida, I. C., Ferguson, M. A., Schenkman, S. and Travassos, L. R. (1994b). GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas disease. Braz. J. Med. Biol. Res. 27, Almeida, I. C., Krautz, G. M., Krettli, A. U. and Travassos, L. R. (1993). Glycoconjugates of Trypanosoma cruzi: A 74 kd antigen of trypomastigotes specifically reacts with lytic anti-α-galactosyl antibodies from patients with chronic Chagas disease. J. Clin. Lab. Anal. 7, Almeida, I. C., Milani, S. R., Gorin, P. A. J. and Travassos, L. R. (1991). Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-α-galactosyl antibodies. J. Immunol. 146, Avila, J. L., Rojas, M. and Galili, U. (1989). Immunogenic Gal α 1 3 Gal carbohydrate epitopes are present on pathogenic American Trypanosoma and Leishmania. J. Immunol. 142, Bansil, R., Stanley, E. and LaMont, J. T. (1995). Mucin biophysics. Ann. Rev. Physiol. 57, Camargo, M. M., Almeida, I. C., Pereira, M. E., Ferguson, M. A., Travassos, L. R. and Gazzinelli, R. T. (1997). Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages. J. Immunol. 158, Cross, G. A. M. (1975). Identification, purification and properties of clonespecific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitol. 71, Cross, G. A. M. and Takle, G. B. (1993). The surface trans-sialidase family of Trypanosoma cruzi. Annu. Rev. Microbiol. 46, de Souza, W., Martinez-Palomo, A. and Gonzalez-Robles, A. (1978). The cell surface of Trypanosoma cruzi: cytochemistry and freeze-fracture. J. Cell Sci. 33, Ferguson, M. A. J. (1993). GPI membrane anchors: isolation and analysis. In Glycobiology. A Practical Approach (ed. M. Fukuda and A. Kobata), pp IRL Press. Oxford. Franchin, G., Pereira-Chioccola, V. L., Schenkman, S. and Rodrigues, M. M. (1997). Passive transfer of a monoclonal antibody specific for a sialic acid-dependent epitope on the surface of Trypanosoma cruzi. Infect. Immun. 65, Galili, U., Anaraki, F., Thall, A., Hill-Black, C. and Radic, M. (1993). One percent of human circulating B lymphocytes are capable of producing the natural anti-gal antibody. Blood 82, Galili, U., Mandrell, R. E., Hamadeh, R. M., Shohet, S. B. and Griffiss, J. M. (1988a). Interaction between human natural anti-α-galactosyl immunoglobulin G and bacteria of the human flora. Infect. Immun. 56, Galili, U., Shohet, S. B., Kobrin, E., Stults, C. L. and Macher, B. A. (1988b). Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J. Biol. Chem. 263, Gazzinelli, R. T., Camargo, M. M., Almeida, I. C., Morita, Y. S., Giraldo, M., Acosta-Serrano, A., Hieny, S., Englund, P. T., Ferguson, M. A., Travassos, L. R. and Sher, A. (1997). Identification and characterization of protozoan products that trigger the synthesis of IL-12 by inflammatory macrophages. Chem. Immunol. 68, Gazzinelli, R. T., Pereira, M. E., Romanha, A. J., Gazzinelli, G. and Brener, Z. (1991). Direct lysis of Trypanosoma cruzi: a novel effector mechanism of protection mediated by human anti-gal antibodies. Parasite Immunol. 13, Golgher, D. B., Colli, W., Souto-Padrón, T. and Zingales, B. (1993). Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi. Mol. Biochem. Parasitol. 60, Huet, C. and Herzberg, M. (1973). Effects on enzyme and EDTA on ruthenium red and Concanavalin A labeling of cell surface. J. Ultrastruct. Res. 42, Lederkremer, R. M., Lima, C., Ramirez, M. I., Ferguson, M. A. J., Homans, S. W. and Thomas-Oates, J. (1991). Complete structure of the glycan of lipopeptidophosphoglycan from Trypanosoma cruzi epimastigotes. J. Biol. Chem. 266, Pereira-Chioccola, V. L., Costa, F., Ribeirão, M., Soares, I. S., Arena, F., Schenkman, S. and Rodrigues, M. M. (1999). Comparison of antibody and protective immune responses against Trypanosoma cruzi infection elicited by immunization with a parasite gene delivered as naked DNA or recombinant protein. Parasite Immunol. 21, Pereira-Chioccola, V. L. and Schenkman, S. (1999). Biological role of Trypanosoma cruzi trans-sialidase. Biochem. Soc. Trans. 27, Pereira-Chioccola, V. L., Schenkman, S. and Kloetzel, J. (1994). Sera from chronic Chagasic patients and animals infected with Trypanosoma cruzi inhibit trans-sialidase by recognizing its catalytic domain. Infect. Immun. 62, Pereira, M. E. A. (1990). Cell biology of Trypanosoma cruzi. In Modern Parasite Biology (ed. D. Wyler) pp W. H. Freeman and Co., New York. Pereira, M. E. A. and Hoff, R. (1986). Heterogeneous distribution of neuraminidase activity in strains and clones of Trypanosoma cruzi and its possible association with parasite myotropism. Mol. Biochem. Parasitol. 20, Pimenta, P. F., de Souza, W., Souto-Padron, T. and Pinto, d. S. (1989). The cell surface of Trypanosoma cruzi: a fracture-flip, replica-staining labelfracture survey. Eur. J. Cell Biol. 50, Piras, M. M., Henriquez, D. and Piras, R. (1987). The effect of fetuin and other sialoglycoproteins on the in vitro penetration of Trypanosoma cruzi trypomastigotes into fibroblastic cells. Mol. Biochem. Parasitol. 22, Powell, L. D. and Hart, G. W. (1986). Quantitation of picomole levels of N- acetyl and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay. Anal. Biochem. 157, Previato, J. O., Gorin, P. A., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M. and Mendonça-Previato, L. (1990). Primary structure of the oligosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi. J. Biol. Chem. 265, Reyes, M. B., Pollevick, G. D. and Frasch, A. C. C. (1994). An unusually small gene encoding a putative mucin-like glycoprotein in Trypanosoma cruzi. Gene 140, Ribeirão, M., Pereira-Chioccola, V. L., Eichinger, D., Rodrigues, M. M. and Schenkman, S. (1997). Temperature differences for transglycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Glycobiol. 7, Saraiva, E. M., Pimenta, P. F., Brodin, T. N., Rowton, E., Modi, G. B. and Sacks, D. L. (1995). Changes in lipophosphoglycan and gene expression

9 The role of T. cruzi sialylated coat 1307 associated with the development of Leishmania major in Phlebotomus papatasi. Parasitol. 111, Schenkman, S., Andrews, N. W., Nussenzweig, V. and Robbins, E. S. (1988). Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner. Cell 55, Schenkman, S., Chaves, L. B., Pontes de Carvalho, L. and Eichinger, D. (1994a). A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxy-terminal domain is active, monomeric and generates antibodies that inhibit enzymatic activity. J. Biol. Chem. 269, Schenkman, S., Eichinger, D., Pereira, M. E. A. and Nussenzweig, V. (1994b). Structural and functional properties of Trypanosoma cruzi transsialidase. Annu. Rev. Microbiol. 48, Schenkman, S., Ferguson, M. A. J., Heise, N., Cardoso de Almeida, M. L., Mortara, R. A. and Yoshida, N. (1993). Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi. Mol. Biochem. Parasitol. 59, Schenkman, S., Man-Shiow, J., Hart, G. W. and Nussenzweig, V. (1991). A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stagespecific epitope required for invasion of mammalian cells. Cell 65, Silva, L. H. P. and Nussenzweig, V. (1953). Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clin. Biol. 20, Souto-Padron, T., Almeida, I. C., de Souza, W. and Travassos, L. R. (1994). Distribution of α-galactosyl-containing epitopes on Trypanosoma cruzi trypomastigote and amastigote forms from infected Vero cells detected by Chagasic antibodies. J. Euk. Microbiol. 41, Tomlinson, S., Pontes de Carvalho, L., Vandekerckhove, F. and Nussenzweig, V. (1992). Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes. Glycobiol. 2, Towbin, H., Rosenfelder, G., Wieslander, J., Avila, J. L., Rojas, M., Szarfman, A., Esser, K., Nowack, H. and Timpl, R. (1987). Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl (alpha 1-3)-galactose epitopes. J. Exp. Med. 166, Weiss, L. and Zeigel, R. (1969). Cell surface negativity and the binding of positively charged particles. J. Cell Physiol. 73, Yoshida, N. (1983). Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi. Infect. Immun. 40,