A novel cell surface trans-sialidase of trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells

Cell, Vol. 65, 1117-l 125, June 26, 1991, Copyright

0 1991 by Cell Press

A Novel Cell Surface TranslSialidase of Ttypanosoma cruzi Generates a Stage-Specific Epitope Required for Invasion of Mammalian Cells Sergio Schenkman,‘t Man-Shiow Jiang,* Gerald W. Hart,+ and Victor Nussenxweig’ *Department of Pathology and Kaplan Cancer Center NYU Medical Center New York, New York 10016 *Department of Biological Chemistry The Johns Hopkins University Baltimore, Maryland 21218

Summary When trypomastigotes of T. cruri emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-slalidase activity. This enzyme specifically transfers a(2-3)-linked siallc acid from extrinsic hostderived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastlgote-specific epitope. The T. cruri trans-sialidase does not utilize cytidine 5’monophospho-N-acetylneuramlnic acid as a donor substrate, but readily transfers siallc acid from exogenously supplied a(2-3)-sialyllactose. Man* clonal antibodies that recognize sialic acid residues of Ssp3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp3 with structural features required for target cell recognition. Introduction Trypanosoma cruzi, an obligatory intracellular protozoan parasite that causes Chagas’ disease in humans (de Souza, 1984; Marsden, 1988; Kirchhoff, 1990), divides in the cytoplasm of mammalian cells as amastigotes. At the end of the intracellular cycle, the amastigotes transform into trypomastigotes, which enter the circulation. The trypomastigotes can invade a wide variety of mammalian cells. Notwithstanding this broad cellular specificity, the recognition of target cells is receptor mediated (Zingales and Colli, 1985; de AraujoJorge, 1989; Schenkman et al., 1988) and requires parasite energy (Schenkman et al., 1991 b). The nature of the T. cruzi ligand(s) and target cell receptor(s) remains controversial (Ouaissi et al., 1986; de Arrudaet al., 1989; Abuinet al., 1989; Yoshidaet al., 1989; Rimoldi et al., 1989; Boschetti et al., 1987). In an attempt to identify specific receptors and the ligands of T. cruzi, we developed an assay to quantitate the attachment phase of invasion and raised panels of monoclonal antibodies (MAbs) to surface membrane components of infective trypomastigote forms of this parasite. The few MAbs that inhibited attachment recognize in a t Present address: Escola Paulista de Medicina, 04023 Sao Paula, SP, Brazil.

Rua Botucatu 662,

Western blot a group of molecules migrating as a broad band between 60 and 250 kd, showing a peak of intensity around 160 kd (Schenkman et al., 1991 a). The same group of molecules is immunoprecipitated by MAb 3C9 (Schenkman et al., unpublished data), which defines the trypomastigote-specific Ssp3 epitope (Andrews et al., 1987). For over two decades it has been postulated that cell surface glycosyltransferases might specifically mediate intercellular interactions via binding and specific transglycosylation reactions (Roseman, 1970). A major objection to this hypothesis has been the demonstrated lack of sugar nucleotide donors extracellularly. In this report we show that the Ssp3 epitope, involved in T. cruzi adhesion to target cells, contains host-derived sialic acid residues, acquired directly from extrinsic glycoconjugates by means of a unique trans-sialidase activity. Results Presence of Siallc Acid In the Ssp3 Epitope If live trypomastigotes are treated with Vibrio cholerae neuraminidase, or with protease-free Clostridium perfringens neuraminidase, the immunofluorescence staining of the parasites with MAb 3C9 and 46 is abolished (Figure 1). These antibodies recognize the Ssp9related epitopes that have been associated with trypomastigote attachment to host cells (Schenkman et al., 1991a). The same treatment does not affect binding of the control MAb 14 to a different surface antigen of trypomastigotes. Others have obtained indirect but persuasive evidence that T. cruzi does not synthesize sialic acids de novo (Schauer et al., 1983). but rather scavenges it from glycoproteins in the medium, without metabolic energy requirement (Previatoet al., 1985; Zingales et al., 1987). To determine the origin of the sialic acid residues recognized by our MAbs, we examined the reactivity of trypomastigotes from cells maintained in 0.2% BSA-DMEM (“BSA trypomastigotes”), rather than in a medium with FBS, which contains sialyllated glycoproteins. As shown in Figure 2, MAb 3C9 and MAb 46 do not react with the BSA trypomastigotes. However, if these parasites are incubated for 3 hr at 37OC with 1 mM sialyliactose or 0.5 mglml fetuin (but not with 1 mM free N-acetylneuraminic acid), more than 80% of parasite staining by MAb 3C9 is recovered. In Figure 3 we show the kinetics of sialic acid transfer to live trypomastigotes at 4OC and 37OC. At either temperature, maximum reactivity with 3C9 is observed after 15 s of incubation. In parallel with the appearance of the 3C9 epitope, the reactivity of the BSA trypomastigotes with peanut agglutinin lectin (which recognizes terminal 8-galactosyl residues) diminishes (Figure 36). To document the transfer of sialic acid further, we studied the reactivity of MAb 3C9 with antigens transferred to nitrocellulose from various preparations of trypomastigotes. As shown in Figure 4, the Ssp-3 epitope is present in parasites grown in serum (lane a), but not in BSA trypomastigotes incubated with sialic acid (lane b). As shown in

Cell 1118

mAb

SC9

mAb

46

a

Fluorescence

intensity

Figure 1. Effect of Bacterial Sialidase on the Binding of MAbs 3C9, 46, and 14 to Live Trypomastigotes T. cruzi trypomastigotes were isolated from culture supernatants of LLC-MK, cells grown in serum, washed, resuspended to 1 x 108/ml in0.15M NaCI, IOmM HEPES(pH7.0), 4 mMCaCI,, and5mglml BSA. and treated for 2 hr at room temperature with 50 mu/ml V. cholerae sialidase. Control samples were incubated at identical conditions, but with heat-inactivated sialidase. At the end of incubation the trypomastigotes remained fully motile. The parasites were washed twice with 0.2% BSA-DMEM and stained by immunofluorescence with MAb 3C9 at 5 uglml (a and b), MAb 46 at 1 uglml (c and d), and MAb 14 at 5 Kg/ ml (e and f). The figure shows the FACS scan of trypomastigotes incubated in the absence (a, c, and e) or in the presence (b, d, and 9 of sialidase.

lane a, Ssp-3 is expressed in molecules that migrate as a smear between 60 and 200 kd, with a few defined bands, the strongest at about 160 kd. If the BSA parasites are incubated with sialyllactose for 15 min at O°C (lane c), or for 1 and 5 min at 37% (lanes d and e), the reactivity with MAb 3C9 is regained. When the nitrocellulose strips with antigens from parasites grown in serum are treated with protease-free C. perfringensor V. cholerae neuraminidase, reactivity with 3C9 is lost (Figure 4, lane 9. The total amount of sialic acid in the parasites was also measured by the thiobarbituric acid-HPLC assay. Samples of BSA trypomastigotes, before and after incubation with sialyllactose, contain 3.2 and 19.4 pmol of sialic acid per 1 O6parasites, respectively. Trypomastigotes grown in the presence of serum also contain relatively high amounts of sialic acid (12.2 pmolll O6 parasites). To demonstrate directly that the sialic acid detected in the Ssp-3 epitope originated from sialyllactose, we incubated BSA trypomastigotes at 37% for 3 hr with sialyllactose containing tritium-labeled sialic acid. The trypomastigotes were lysed with detergent, and one sample of the lysate was analyzed directly by SDS-PAGE (Figure 4, lane

Fluorescence

tntenslty

Figure 2. Acquisition by BSA Trypomastigotes of Epitopes Recognized by MAb 3C9 and 46, Following Incubation with Conjugated Sialic Acid Purified slender T. cruzi trypomastigotes (5 x lO’/ml), obtained from culture fluids of LLC-MK, cells grown in 0.2% BSA-DMEM (BSA trypomastigotes), were incubated for 3 hr at 37OC in 0.2% BSA-DMEM with 1 m M sialic acid (a and b), 1 m M a(2-3)sialyllactose (c and d), and 0.5 mglml fetuin (e and 9. At the end of the incubation, the parasites were washed by centrifugation, stained by immunofluorescence using MAb 46 and 3C9 as primary antibodies, and analyzed by FACS.

h). The remaining lysate was immunoprecipitated with the 3C9 MAb before SDS-PAGE (lane i). Autoradiographs of both samples show diffuse bands, similar to the appearance of the Ssp3-bearing molecules on Western blots. The MAb 3C9, however, only immunoprecipitates about 10% of the sialylated, radiolabeled molecules. The remaining 90% could express sialic acid-containing epitopes distinct from that recognized by MAb 3C9, or the sialic acid could have been enzymatically released from the Ssp-3 epitope during the experimental manipulations. In Table 1, we show some of the requirements for the transfer of sialic acid to live trypomastigotes. The parasites acquire sialic from a(2-3)sialyllactose at concentrations as low as 10 PM. In contrast, a(2-6)-sialyllactose, CMPsialic acid, or colominic acid (a(2-8)-polysialic acid) are not sialyl donors, even at concentrations as high as 1 mM. Fetuin is also a sialic acid donor. The transfer is not prevented by azide. The presence of 10 mM EDTA in the incubation medium is also without effect. Fixation of the trypomastigotes with paraformaldehyde only partially blocks the reaction. On the other hand, lactose inhibits the sialic acid transfer at concentrations much lower than those of galactose or melibiose. Other monosaccharides, such as N-acetylglucosamine, glucose, and N-acetylgalactosamine, are poor inhibitors (data not shown).

Surface Trans-Sialidase 1119

of T. cruzi

abcdefg

h

i

j

Figure 4. The Ssp-3 Epitope Is Restored after Incubation of BSA Trypomastigotes with a(2-3)Sialyllactose

0

5

10

15

120

180

Time (min)

600

,

0

60

Time (min) Figure 3. Kinetics of Sialic Acid Transfer to Live Trypomastigotes BSA trypomastigotes (5 x 10’lml) were incubated in 0.2% BSADMEM at 4OC with 0.1 m M a(2-3)sialyllactose (A), or at 37OC with 1 m M a(2-3)-sialyllactose (B). At the indicated time the parasites were diluted 10 times with cold 0.2% BSA, centrifuged, and washed with 0.2% BSA-DMEM at 4OC. The staining with MAb 3C9 by indirect immunofluorescence or with FITC-labeled peanut agglutinin (PNA) was analyzed by FACS. In (A), the values shown in the ordinate represent the relative increase in 3C9 mean fluorescence, while in (B) they represent the fluorescence measurements.

Purification and Characterization of the T. cruzi Tram-Sialidase To isolate the T. cruzi tram+sialidase, we developed a quantitative assay based on the incorporation of sialic acid into radiolabeled N-acetyllactosamine or lactose, and retention of the product by an anion exchange column. Using this assay, the enzyme was purified from n-octyl glucoside lysates of trypomastigotes by affinity chromatography on concanavalin A-Sepharose, followed by FPLC separation on Mono Q (Figure 5). Enzymatic activity was recovered in a single peak. The purification achieved was about 100 times, and the recovery was 10% of the initial activity in the crude extracts. The enzyme does not express the Ssp-3 epitope, since MAb 3C9 linked to Sepharose beads was unable to deplete trans-sialidase from total trypomastigote extracts (data not shown).

(Experiment 1) T. cruzi trypomastigotes (2.5 x 10’ in 0.5 ml) cultured in medium containing serum (lane a) or BSA (lanes b-e) were incubated for 15 min at 37OC in 0.2% BSA-DMEM (lane a), for 15 min at 37OC in 0.2% BSA-DMEM with 1 m M sialic acid (lane b), for 15 min at 4OC in the same medium with 1 m M a(2-3)-sialyllactose (lane c), or for 1 and 5 min at 37OC in the same medium with 1 m M sialyllactose (lanes d and e). At the end of the incubations, the parasites were washed by centrifugation in Hanks’solution and boiled in SDS sample buffer, and the binding of MAb 3C9 was assayed by Western blotting. (Experiment 2) T. cruzi trypomastigotes obtained from cells grown in media containing serum were centrifuged, washed in Hanks’ solution, and boiled in SDS sample buffer. Samples were subjected to SDSPAGE and transferred to nitrocellulose. One of the nitrocellulose strips was treated for 3 hr at room temperature with 10 mu/ml C. perfringens sialidase (lane f), whereas the other strip was treated with boiled sialidase (lane g). The strips were then washed and parasite antigens revealed with MAb 3C9. (Experiment 3) BSA trypomastigotes were incubated with [~Hjsialyllactose and extracted as described in Experimental Procedures. Twenty percent of the lysate was mixed with concentrated sample buffer and analyzed directly by SDS-PAGE (lane h). The remaining lysate was precleared with Sepharose 48 beads. The supernatant was divided in two aliquots and immunoprecipitated with MAb 3C9 (lane i) or with MAb 27 (lane j). The immunoprecipitated materials were analyzed by SDS-PAGE and fluorography. The standards correspond to myoglobin (200 kd), f3-galactosidase (116 kd), phosphorylase b (93 kd), BSA (66 kd), and ovalbumin (43 kd).

The specificity of the enzyme in the total extracts, or in the partially purified preparation, is identical to that observed in living parasites. Both in vitro and in vivo, the enzyme utilizes as sialic acid donors a(2-3)-sialyl-Nacetyllactosamine or a(2-3)-sialyllactose at 10 PM, but not a(2-6)-sialyllactose, CMP-sialic acid, or free sialic acid at 1 mM. The purified enzyme is active at neutral or alkaline pH, but the activity is substantially reduced below pH 6.0 (Table 2). Almost 100% of the trans-sialidase activity is displayed on the surface membrane of the trypomastigotes. If live parasites are first treated with trypsin, the lysate is enzymatically inactive. We also studied the stage specificity of the enzyme. Metacyclic trypomastigotes, the infective insect stage of the parasite, contain substantial levels of the trans-sialidase. Enzymatic activity is much reduced in extracts of amastigotes, which correspond to the intracellular, dividing form of the parasite (Table 2). In vitro grown amastigotes (Andrews et al., 1987) contain a small number of intermediate forms between trypomastigotes and amastigotes. which could account for the low levels of transsialidase activity.

Cell 1120

Table 1. Specificity and Characteristics of T. cruzi Trans-Sialidase Reaction in Live Parasites

Experimental

Conditions”

Control Sialic acid 1 m M a(2-3)sialyllactose 1 mM a(2-3)sialyllactose 0.1 m M a(2-3)sialyllactose 0.01 m M r a(2-3)sialyllactose 0.001 m M a(2-3)sialyllactose 1 mM CMP-sialic acid 1 m M Colominic acid 0.3 mglml Fetuin 1 mglml Asialofetuin 1 mg/ml a(2-3)sialyllactose 1 m M + 0.05% NaN3 a(2-3)sialyllactose 1 m M + 10 m M EDTA a(2-3)sialyllactose 1 mM/fixed T. cruziC Sialic acid 1 mM/fixed T. cruziC a(2-3)sialyllactose 20 nM + 10 m M lactose a(2-3)sialyllactose 20 nM + 1 m M lactose a(2-3)sialyllactose 20 uM + 0.1 m M lactose a(2-3)sialyllactose 20 uM + 10 m M galactose u(2-3)sialyllactose 20 uM + 1 m M galactose a(2-3)sialyllactose 20 uM + 10 m M melibiose

MAb 3C9 Stain Relative Fluorescenceb 1.0 1.2 30.3 17.1 7.2 1.8 1.9 3.4 3.0 23.0 2.0 31.0 21.8 5.1 0.8 1.2 2.7 14.0 5.7 21.9 22.3

a Purified slender T. cruzi trypomastigotes (5 x lO’/ml), obtained from culture supernatant of LLC-MKI cells grown in 0.2% BSA-DMEM. were incubated for 30 min at 37OC in 0.2% BSA-DMEM with the indicated reagents. At the end of incubation the parasites were washed, and the immunofluorescence staining by MAb 3C9 was assayed by FACs. ’ Values shown are the increase in the mean fluorescence relative to the staining of parasites incubated in 0.2% BSA-DMEM alone (control). ’ Trypomastigotes were washed in Hanks’ solution and incubated with 4% paraformaldehyde in PBS at 4OC for 30 min. The trypomastigotes were washed with 0.2% BSA-DMEM before incubation with a(2-3)-sialyllactose or sialic acid.

Figure 5. Partial Purification

of T. cruzi Trans-Sialidase

A total detergent extract obtained from 4 x 1 Ogfrozen trypomastigotes was subjected to affinity chromatography on concanavalin A-Sepharose. The eluate was dialyzed against Tris-HCI (pH 8.0) and loaded into a Mono Q FPLC column equilibrated with the same buffer. After extensive washing, the trans.-sialidase activity (circles) was eluted with a NaCl gradient. The solid line represents the optical density at 280 nm and the dotted line the NaCl concentration.

invasion of target cells. In the experiment illustrated in Figure 7, BSA trypomastigotes, or trypomastigotes grown in medium with serum, were incubated for 30 min at 37% with 3T3fibroblasts. After removal of the extracellular parasites, the infected cells were stained by immunofluorescence with MAb 3C9. As shown, both types of intracellular parasites react with the antibody. As a control, the two preparations of parasites were incubated on slides coated with poly-L-lysine. The BSA trypomastigotes attached to the glass are not recognized by 3C9. Discussion

T. cruzi Trans-Sialidase Reaction Product To identify the reaction product of T. cruzi trans-sialidase, we incubated parasite extracts with cold a(2-3)sialyllactose and [‘Cllactose, with the radiolabel in the glucose portion. The mixture was subjected to anion exchange chromatography to retain charged oligosaccharides. These were eluted with 1 M ammonium formate and analyzed by thin-layer silica gel chromatography, followed by autoradiography. Radioactive a(2-3)-sialyllactose is the only product detected (Figure 6). If tritiated N-acetyllactosamine was used as the sialic acceptor (instead of lactose), only a(2-3)-sialyllactosamine was detected on the thinlayer chromatography plate (data not shown). T. cruzi Trans-Sialidase Transfers Sialic Acid from Mammalian Cells to the Ssp-3 Epitope The trypomastigote trans-sialidase can also utilize cell surface glycoproteins as donors of sialic acid. For example, by FACS analysis, there is a significant increase in 3C9 reactivity in the BSA trypomastigotes, following incubation of the parasites with a suspension of human erythrocytes (data not shown). Perhaps of greater biological significance, the BSA trypomastigotes acquire sialic acid during

Here we identify a unique transglycosidase on the membrane of T. cruzi trypomastigotes, which specifically transfers sialic acid from extrinsic and host glycoconjugates to form a developmentally regulated surface epitope involved in the invasion of host cells. Our findings demonstrate directly that T. cruzi utilizes host carbohydrates for the biosynthesis of its glycoconjugates and highlight the importance of stage-specific carbohydrates in host-parasite interactions. Several independent lines of evidence suggesting the presence of a sialyltransferase-like enzyme in T. cruzi were previously reported by Previato and collaborators for epimastigotes, but the enzyme, its substrate, or the parasite acceptor molecules were either not characterized or were characterized incompletely (Zingales and Colli, 1985; Previato et al., 1985). Sialylation of molecules on the trypomastigote surfaces by the trans-sialidase leads to the formation of the Ssp-3 epitope. This epitope was originally defined by MAb 3C9 (Andrews et al., 1987) and later by MAb 46, 50, and 87 (Schenkman et al., 1991a). It is found exclusively on the plasma membrane of the infective, trypomastigote stage of T. cruzi, but not on amastigotes or insect forms of the parasite. Reactivity of the MAbs with the Ssp-3 epitope is strong in parasites grown in serum, but very weak in

$$ce

Trans-Sialidase

Table 2. Specificity

of T. cruzi

of T. cruzi Trans-Sialidase

In Vitro Sialyllactosamine

Produced (cpm)

Enzyme Source

Substrate

Total Lysate

Purified Enzyme

Trypomastogotes

No substrate a(2-3)eialyllactose 1 mM a(2-3)eialyllactose 0.1 m M a(2-3)-sialyllactose 0.01 m M a(2-6)eialyllactose 1 mM a(2-3~sialyllactosamine 1 mM a(2-6)~sialyllactosamine 1 mfvl CMP-sialic acid Sialic acid a(2-3)eialyllactose 1 mM

20 6340 4320 1120 32 nt nt 320 26 640

30 4560 np nt 300 6600 1740 260 nt nt

a(2-3)-sialyllactose

1 mf.4

8340

nt

a(2-3)-sialyllactose a(2-3)-sialyllactose a(2-3)-sialyllactose pH 5.5 pH 6.5 pH 6.7 pH 7.0 pH 7.5 pH 6.5

1 mM 1 mM 1 mM

3750 1330

nt nt

nt nt rlt nt nt nt

1350 2400 3260 3500 2550 2500

Trypsin trypomastigotes+ Control trypomastigotesb Meta Amastigotes Trypomastigotes

Pellets of 1 x lb T. cruzi trypomastigotes, metacyclic trypomastigotes, or amastigotes were extracted by treatment with 100 ul of 3% n-octyl glucoside, 50 m M Tris-HCI (pH 7.4) 0.1 m M EDTA, 1 m M PMSF, 5 nglml leupeptin, antipain, and pepstatin. Five microliters of the lysates, or of the purified enzyme from bypomastigotes (fraction 64 of the Mono Cl column), was incubated 30 min at 37% in 50 ul of 50 m M PIPES (pH 7.0) containing 25,000 cpm of [SH]N-acetyllactosamine and the various substrates. Where indicated, the following Sigma buffers at 50 m M were used: pH 5.5 and 6.0, MES; pH 6.7 and 7.0, PIPES; pH 7.5, HEPES; and pH 8.5, Tris-HCI. ant = not tested. b Live bypomastigotes were washed twice with Hanks’ solution, resuspended to 5 x lO’/ml, and incubated for 30 min with 0.1 mglml trypsin. At the end of the incubation, 0.2 mglml soybean trypsin inhibitor was added, and the parasites were washed and extracted as described above.

a

b

c

d

Figure 6. Chromatography on Silica Gel Thin-Layer action Products of T. cruzi Trans-Sialidase

e

Plates of the Re-

NP-40 extracts of trypomastigotes were incubated with 1 m M a(23)-sialytlactose and [YJlactose, and the product of the reaction was isolated by elution from a QAE-Sephadex A50 column, as described in Experimental Procedures. The eluate was lyophilized and analyzed by chromatography in silica gel 60 plates using ethanol-n-butanolpyridine-water-acetic acid (100:10:10:30:3 [v/v]). The standards, a(23)-sialyllactose (a), a(2-6)~sialytlactose (b), a(2-3)eialyllactosamine (c), and a(2-6)~sialyllactosamine (d), were visualized with the orcinol ferric chloride spray reagent (Veh et al., 1961). The reaction product of T. cruzi trans-sialidase (e) wasvisualized by spraying with En3Hance followed by autoradiography.

trypomastigotes developing inside the host’s cells, or on the BSA trypomastigotes. Within seconds of contact with medium containing serum, fetuin, orsialyllactose, the BSA trypomastigotes acquire macromolecular-bound sialic acid and express the Ssp-3 epitope. The exact structure of Ssp-3 epitope is unknown. Several lines of evidence, however, indicate that it contains a sialyl-a(2-3)+galactose structure. First, recognition of Ssp-3 is strictly sialic acid dependent, as shown by its sensitivity to sialidase both in vivo and following transfer of Ssp9bearing molecules to nitrocellulose. Second, the T. cruzi trans-sialidase transfers sialic acid to galactosebearing oligosaccharides in vitro, forming a(2-3)-linked and not a(2-6)-linked sialic acid. Third, the sialic acid transfer is inhibited by galactose and lactose. Fourth, peanut agglutinin reacts strongly with BSA trypomastigotes, but the reactivity diminishes progressively following incubation of the parasites with sialyllactose and incorporation of sialic acid into Ssp-3. Analogous observations were reported elsewhere (Previato et al., 1965). We emphasize, however, that although antibodies to Ssp-3 probably recognize the sialyl-a(2-3)+galatose structure, the epitope is not sialyl-a(2-3)N-acetyllactosamine; this structure is commonly found in glycoproteins and glycolipids, and our MAbs do not react with fetuin, other serum glycoproteins, and human red cells (data not shown). In addition, the epitopes for the different MAbs

Cell 1122

Figure 7. Mammalian

Cells Are Sialic Acid Donors for T. cruzi Trans-Sialidase

lmmunofluorescence staining with MAb 3C9, observed under the fluorescence microscope, of trypomastigotes attached to glass coverslips pretreated with polylysine (a and c) or to 3T3 fibroblasts after incubation for 30 min at 37°C (b and d). BSA trypomastigotes were used in the coverslips shown in (a) and (b), and trypomastigotes from cells cultivated in 10% FBS-DMEM were used in coverslips shown in (c) and (d).

may not be identical, but cross-reactive. Complex oligosaccharide chains or neighboring groups most likely contribute to differences in specificity. Other related issues are also left unresolved. We do not know if all the surface-associated sialic acid molecules in trypomastigotes are included in Ssp-3 epitopes. The fact that the SDS-PAGE autoradiographic patterns of lysates of SSA trypomastigotes labeled with tritiated sialic acid, before and after immunoprecipitation with MAb 3C9, are similar (Figure 4) suggests that Ssp3 may contain most of the transferred sialic acid. Another question is whether the very heterogeneous pattern of migration of Ssp-3 in SDSPAGE is a reflection of the diversity in the primary structure of the carrier molecules, or of the posttranslational modifications of the same carrier molecule. Nevertheless, all Ssp-3-bearing molecules share common properties, that is, they are all glycoproteins that cannot be labeled on the parasite surface with jZ51and are synthesized while the parasite is still inside the host cells (Andrews et al., 1987; Schenkman et al., 1991a; unpublished data). The unique trypomastigote trans-sialidase has not been fully characterized, but the fact that following purification from concanavalin A-Sepharose, it migrates as a sharp

peak in Mono Cl suggests that it is a single molecule. The purified enzyme retains the properties displayed in vivo; that is, its substrates must contain an a(2-3)-linked sialic acid end unit, different from other known enzymes that transfer sialic acid moieties (Paulson and Colley, 1989) all of which require CMP-sialic acids, and its activity can be inhibited by b-galactosides. In addition, the enzyme is active at low temperatures, is divalent cation independent, and the optimum pH of activity is in the physiologic range. Most important, the trans-sialidase is on the parasite surface membrane (or in the flagellar pocket), as shown by its sensitivity to trypsin in live parasites. The speed of sialic acid transfer at 4%, and in the presence of metabolic inhibitors, raised the possibility that the Ssp-3-bearing molecules were sialylating themselves. This was rendered unlikely by the observation that the trans-sialidase does not express the Ssp-3 epitope. Perhaps the enzyme is closely associated with the substrate on the trypomastigote surface membrane, or the enzyme is secreted and acts from the outside. In any case, by altering the sialic acid content of the host glycoproteins or glycolipids, the trans-sialidase may contribute to the pathology of Chagas’ disease. In vitro, the T. cruzi trans-sialidase reaction product (sia-

Surface Trans-Sialidaseof T. cruzi 1123

lyllactose) can be identical to the substrate (Figure 6). This finding raises the question of the possible relationship between the trans-sialidase and the previously described T. cruzi sialidases (Pereira, 1963; Harth et al., 1967). It is not known whether the invasive blood stages of T. cruzi bear two separate sialic acid-targeted enzymes, a sialidase and a trans-sialidase, or a single regulated enzyme, which can release or transfer sialic acid to modulate specific parasite functions. As for the function of the transferred sialic acid, several findings suggest that the Ssp-3 epitope participates in target cell recognition. Fab fragments of antibodies reacting with Ssp3containing molecules inhibit attachment of the parasite to mammalian cells (Schenkman et al., 1991a). Others have reported that the infectivity of trypomastigotes increases substantially following incubation with macromolecules containing sialic acid (Piras et al., 1967), and by inhibition of the parasite’s own membrane-associated sialidase with monoclonal antibodies (Prioli et al., 1990). On the other hand, treatment of target cells with bacterial sialidases decreases trypomastigote infectivity (de Titto and Araujo, 1967; de AraujoJorge, 1969). Sialic acid is known to play a role in cell-cell and cellsubstrate interactions. Several microorganisms, such as influenza virus (Crowell and Lonberg-Holm, 1966), Mycoplasma (Roberts et al., 1969), and Plasmodium falciparum (Hadley et al., 1967), recognize sialic acid during attachment and/or invasion of target cells. Moreover, the glycosyltransferases themselves may function as recognition molecules, as originally proposed by Roseman et al. (Roseman, 1970; Roth et al., 1971). Migrating embryonic cells have high levels of cell surface glycosyltransferases, and in some instances their migration seems to be mediated by the successive enzymatic recognition of the appropriate sugar residues from the extracellular glycoconjugate matrices (Runyan et al., 1966). Also, the mouse sperm surface galactosyltransferase is the principal receptor for sperm binding to the zona pellucida during fertilization (Wassarman, 1967). It could be similarly argued that the T. cruzi trans-sialidase recognizes its substrate on the surface of some target cells. As indicated by the experiment depicted in Figure 7, the trypomastigotes can utilize host cell sialic acids during invasion and assemble the Ssp-3 epitope. T. cruzi may have to traverse vascular endothelial cells to invade muscle and nervous system cells. Endothelial cells transiently bear on their surface membrane members of the LECCAM family of receptors (Osborn, 1990), whose N-terminal lectin domain specifically recognizes ligands containing sialyl-a(2-3)+galactose (Phillips et al., 1990; Walz et al., 1990; for review see Brandley et al., 1990). One intriguing possibility is that the Ssp9-bearing molecules may interact with members of this family during their migration within the host. Others have shown that sialidase treatment enhances the sensitivity of blood stream T. cruzi trypomastigotes to lysis by complement (Kipnis et al., 1961). Sialic acids inhibit the assembly of the C3-convertase C3bBb (Kazatchkine et al., 1979) and control complement activity on the surface of some microorganisms, such as Neisseria (Jar-

vis and Vedros, 1967) and type Ill group B Streptococcus (Edwards et al., 1962). Interestingly, N. gonorrhoeae acquires sialic acid from the host and becomes serum resistant (Nairn et al., 1966). The proposed functions for the T. cruzi transsialidase are still speculative, but seem now amenable to experimental approaches. Furthermore, it is possible that similar, yet to be discovered transglycosylation reactions may play a key role in cellular recognition reactions in other biological systems. Experimental Procedures Parasites

T. cruzi trypomastigotes,Y strain

(Silvaand Nussenzweig, 1953), were grown in cultures of LLC-MK, cells (American Type Culture Collection CCL-7). Usually 75 cm2 flasks, with subconfluent cultures of LLC-MIG cells, were infected with 5 x lb bypomastigotes. The LLC-MK, cells were grown in low glucose D M E M with penicillin and streptomycin (GIBCO), containing 10% FBS at 37OC, 5% C02. Free parasites were removed 24 hr later, and the cultures were maintained in 10% FBSDMEM. When indicated, the FBS-DMEM was removed during the third day following infection, the monolayers were washed twice with Hanks’solution, and the medium was replaced with D M E M containing 0.2% BSA (Ultrapure, Boehringer Mannheim) and 20 m M HEPES (pH 7.4) (0.2% BSA-DMEM). There was no difference in numbers or morphologyof parasites obtained from cultures in 0.2% BSA-DMEM (BSA trypomastigotes) or in FBS-DMEM. After the fifth day following the initial infection, the culture supernatants contained trypomastigotes, intermediate forms, and amastigotes. To separate the trypomastigates, the heterogeneous parasite suspensions were centrifuged at 2000 x g for 5 min and then incubated at 37OC. After 2 hr, the motile, slender, and highly infective trypomastigotes were collected from the supernatant. The contamination of this fraction with amastigotes and intermediate forms was less than 1%. Amastigotes were prepared by incubating trypomastigotes for 24-48 hr at 37OC in liver infusion tryptose (Ley 81 al., 1988) containing 10% FBS. The metacyclic stage of T. cruzi was obtained from aged cultures of epimastigotes in the same medium at 28OC. The epimastigotes were removed by passage through a DE-52 (Whatman, UK) column (Teixeira and Yoshida, 1988).

Antlbodles and Reagents MAbXg(IgG1) wasgenerated by Andrewset al. (Andrewset al., 1987) and purified from ascitic fluids by DEAE-cellulose chromatography. MAbs 48 and 14 (IgG2a) and MAbs 87,50, and 27 (IgGl) were generatedasdescribed inschenkmanetal. (1991a)and purifiedfromascitic fluids by elution from protein A-Sepharose columns. V. cholerae sialidase was obtained from GIBCO, proteasefree V. cholerae sialidase was from Boehringer Mannheim, and protease-freeC. perfringenssialidase was obtained from Sigma. Labeled a(2-3)~sialyllactose was prepared by incubating 25 pCi of [sialic-9-JH]CMP-sialic acid (20 Cilmmol; New England Nuclear) with 0.15 M lactose in the presence of mammalian a23Galpl-3GalNAc sialyltransferase (Passaniti and Hart, 1988).

Siallc Acid Transfer In Vlvo BSA trypomastigotes were washed in 0.2% BSA-DMEM, resuspended to 5 x lo7 parasites per ml, and incubated at the indicated temperature with various concentrations of a(2-3~sialyllactose, a(28)-sialyllactose, a(2+-sialyllactosamine, a(2-8)-sialyllactosamine (Oxford Glycosystems), CMP-sialic acid, sialyllactose (Boehringer Mannheim), fetuin, asialofetuin, and synthetic sialic acid (Sigma). Inhibitors were added to the parasites before addition of the sialic acid donor. Labeled sialic acid was transferred to the parasites by incubating 4 x 108 ttypomastigotes in 800 pl of 0.2% BSA-DMEM for 3 hr at 37’C with 0.5 pCi of [sialic-9-3H]a(2-3)-sialyllactose. At the end of incubation the parasites were centrifuged and washed twice with Hanks’solution, lysed with 3% NP-40,50 m M Tris-HCI (pH 7.4), 1 m M PMSF, 0.1 m M EDTA, and 5 pglml antipain, pepstatin, and leupetin, and centrifuged 10 min at 10,000 x g.

Immunofluorescence Parasites (2.5 x 10’) were resuspended

in 250 pl of 0.2% BSA-DMEM

Cdl 1124

at 4%. An equal volume of antibody, diluted in 0.2% BSA-DMEM containing 0.05% NaN3, was added and the incubation proceeded for 30 min on ice. The suspension was then centrifuged for 2 min at 6000 rpm in a Beckman minifuge using a horizontal rotor. The supernatant was removed and the remaining pellet carefully resuspended in 100 nl of 0.2% BSA-DMEM, followed by addition of 900 ul of 4% paraformaldehyde in PBS. After at least 30 min at 4”C, the fixative was removed, and the parasites were resuspended and washed twice with 1 ml of cold 0.2% BSA-DMEM. The parasites were then incubated for 30 min with anti-mouse IgG conjugated with FITC. The suspensions were centrifuged, washed with 0.2% BSA-DMEM, resupended In 50 ul of PBS, and postfixed with 450 ul of 4% paraformaldehyde. The mixtures were analyzed on a Becton Dickinson FACScan. To study the antigenic properties of intracellular parasites, we infected BALB13T3 fibroblasts, clone A31 (American Type Culture Collection CCL-163) plated in 12 m m glass coverslips placed in 24-well plates. As a control, the parasites were attached to glass coverslips coated with 0.1% poly-L-lysine in PBS. Trypomastigotes were placed 30 min at 37OC in contact with the coverslips. At the end of incubation unbound parasites were removed by aspiration and the coverslips fixed for 1 hr with 4% paraformaldehyde in PBS. The preparations were washed with PBS, treated 1 min with 0.1% Triton X-100, washed again with PBS, and incubated for 30 min with 0.2% BSA-DMEM at room temperature. Preparations were incubated with 50 pglml MAb 3C9 and treated with an anti-mouse IgG antibody labeled with FITC. The stained parasites were examined under a fluorescence mrcroscope. Sialic Acid Measurements Trypomastigotes were washed three times in Hanks’ solution and stored frozen at -70°C until analysis. Total cellular sialic acid was determined after hydrolysis of the cell pellets with 0.1 M HCI. using the thiobarbituric acid method and HPLC analysis (Powell and Hart, 1986). Western Blots and lmmunopreclpltation For Western blots, the trypomastigote suspensions were pelleted for 2 min at 6000 rpm in a microfuge equipped with a horizontal rotor. The trypomastigotes were washed with Hanks’ solution, resuspended in SDS sample buffer, and boiled for 3 min. Samples containing the equivalent of 2 x 10’ trypomastigotes were loaded onto 7.5% SDSPAGE gels. After electrophoresis, the gels were blotted onto nitrocellulose paper. The paper was blocked with 1% BSA in PBS and incubated with 20 pglml MAb 3C9 in 10 m M Tris-HCI, 0.15 M NaCI. and 0.05% Tween 20. Bound antibodies weredetected with anti-mouse IgG conjugated to alkaline phosphatase (Sigma), followed by incubation with 0.3 mglml nitroblue tetrazolium and 0.15 mg/ml5-bromo-4-chloro-3-indolyl phosphate, in 0.1 M Tris-HCI (pH 9.5) 0.1 M NaCI, and 0.005 M MgCI,. In some cases, the nitrocellulose strips were pretreated with the neuraminidases in 0.05 M sodium acetate (pH 5.5) before incubation with MAb 3C9. For immunoprecipitation, extracts were incubated with MAbs coupled to CNBr-Sepharose 48 beads for 3 hr at 4%. The beads were washed and processed as described in Andrews et al. (1987), and the precipitated material was loaded onto 7.5% SDS-PAGE gels. Trans-Sialidase Activity Trans.-sialidase activity was determined by incubating trypomastigote lysates in 50 m M PIPES buffer (pH 7.0) (Sigma) in the presence of a sialic acid donor and [N-acetyl-D-l-3H-glucosamine]N-acetyllactosamine (10 Cilmmol) (Passaniti and Hart, 1988), or [o-glucose-l-‘4C]lactose (60 mCi/mmol) (Amersham). The standard assay contained 1 m M sialyllactose and 25,000 to 40,000 cpm of the radioactive substrate in a final volume of 50 ul. This mixture was incubated 30 min at 37OC, and the reaction was terminated by addition of 1 ml of water followed by passage through a 1 ml QAE-Sephadex A50 column, also equilibrated in water. The radioactive oligosaccharides were eluted with 1 ml of 1 M ammonium formate. Activity is expressed as eluted cpm. Purification of Trans-Sialidase To purify the trans-sialidase, 5 x 1 O9 trypomastigotes were lysed at 4°C in 5 ml of 3% n-octyl glucopyranoside (Sigma), 50 m M Tris-HCI (pH 7.4) 0.1 m M EDTA, 0.1 m M PMSF, and 5 ug/ml leupeptin, pep. statin. and antipain. The insoluble material was removed bycentrifuga-

tion (10 m m at 10,000 x g). and the supernatant was adjusted to 0.5 M NaCI, 1 m M of CaCI,, MgCI,, and MnCI,. The supernatant was incubated with 2 ml of concanavalin A-Sepharose equilibrated with 0.3% n-octyl glucopyranoside, 0.5 M NaCI. and 50 m M Tris-HCI (pH 7.4). After washing with 25 ml of the equilibration buffer, the enzyme was eluted with 0.5 M a-methylmannoside in the same buffer. The eluate was dialyzed with 10 m M Tris-HCI (pH 8.0) and applied mto Mono Q FPLC column HR5/5 (Pharmacia-LKB) preequilibrated in the same buffer. After the absorbance had decreased below 0.002, the enzyme was eluted with a gradient of NaCI. Acknowledgments This work was supported by grants from the MacArthur Foundation, the UNDPMlorld BankMIHO Special Program for Research and Training in Tropical Diseases, American Heart Association (Grant-in-Aid #901216), NIH CA-42486, and Conselho National de Desenvolvimento Cientifico e Tecnolgico (Proc. 50041 l-90.5). We thank Rocilda Schenkman and Antonio Ruiz for technical assistance and fruitful discussions. We thank Dr. L. Travassos for reading the manuscript and making many useful suggestions. The costs of publication of this article were defrayed rn part by the payment of page charges. This article must therefore be hereby marked “advertisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact. Received March 27, 1991

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