INFECI1ON AND IMMuNrrY, Aug. 1992, p. 3025-3032 0019-9567/92/083025-08$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 60, No. 8

Purification of a Trypanosoma cruzi Trypomastigote 60-Kilodalton Surface Glycoprotein That Primes and Activates Murine Lymphocytes FERNANDO VILLALTA,1* MARIA F. LIMA,2 SYLVIA A. HOWARD,' LIN ZHOU,' AND ANTONIO RUIZ-RUANO' Division of Biomedical Sciences' and Department of Microbiology,2 Meharry Medical College, Nashville, Tennessee 37208 Received 14 November 1991/Accepted 30 April 1992

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host. Trypanosoma

Trypanosoma cruzi, the protozoan which causes Chagas' disease and affects millions of people in South America and Central America (1, 8), must attach to mammalian cells before it can invade them. The disease is acquired by infection with invasive trypomastigotes, which are transmitted by insect vectors, or via blood infected with trypomastigotes during blood transfusion. Recently, cases of blood transfusion-related Chagas' disease were reported in the United States (2, 15). Although the disease is not common, there are estimated to be more than 100,000 infected persons residing in the United States, and it has been suggested that cases will be encountered in increasing numbers (3, 7). Trypomastigotes attach to and penetrate mammalian host cell membranes to multiply intracellularly and disseminate in the body. Knowledge of the molecular basis for immunoprophylaxis against the infection induced by invasive T. cnrzi trypomastigotes is limited. Therefore, the elucidation, characterization, and mapping of T and B cell epitopes of immunological importance on surface polypeptides of invasive forms of T. cnrzi are relevant for molecular and immunological intervention in T. cnrzi infection. Purified T. cnszi trypomastigote surface molecules capable of priming and stimulating the proliferation of lymphocytes have not been reported. In this study, we purified a surface glycoprotein with a relative molecular mass of 60 kDa from T. cnuzi trypomastigotes in native form and to homogeneity and studied its ability to prime and activate lymphocytes as well *

the macrophage response induced by specific antibodies against this parasite glycoprotein.

as

MATERIALS AND METHODS

Animals. Inbred, 5-week-old CBA mice were used as the of spleen cells and were purchased from Jackson Laboratory (Bar Harbor, Maine). CBA mice were also used as the source of nonelicited peritoneal macrophages. New Zealand rabbits were obtained from a local breeder and used to produce antibodies against the purified T. cruzi trypomastigote 60-kDa surface protein. Parasites. Highly infective trypomastigote clone MMC 20A of the Tulahuen strain of T. cnrzi (11) was used in this work. Pure-culture trypomastigotes were obtained from the supernatant of infected rat heart myoblast monolayers (10, 11). To study the interaction between trypomastigotes and macrophages, we adjusted suspensions of parasites to 107 organisms per ml in Dulbecco's minimal essential medium (DMEM) supplemented with 1% bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, Mo.). For other purposes, parasites were washed by centrifugation with Hanks' balanced salt solution and resuspended at the appropriate concentrations as described below. Pure-culture trypomastigotes (1010 organisms) were radioiodinated with 5 mCi of Nal'I (specific activity, 17 Ci/mg; ICN Radiochemicals, Irvine, Calif.) in the presence of Iodobeads (Pierce Chemical Co., Rockford, Ill.) as previously described (10, 11). Trypomastigote surface proteins were also biotinylated with N-hydroxysuccinimide-biotin (Bio-Rad Laboratories, source

Corresponding author. 3025

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VILLALTA ET AL.

Richmond, Calif.) by use of living parasites as described in detail previously (10, 11). Trypomastigotes (109 organisms) were metabolically labeled with 2 mCi of L-[35S]methionine (Trans[355]-label; specific activity, 1,123 Ci/mmol; ICN) in minimal essential medium deficient in methionine (ICN) for 4 h at 37°C with 5% CO2. Labeled or unlabeled trypomastigotes were washed four times with Hanks' balanced salt solution at 4°C. Live parasites or their cell membranes, obtained as described previously (22, 23), were solubilized with 0.8% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1propanesulfonate (CHAPS) (10, 11) in phosphate-buffered saline (PBS) in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM N-p-tosyl-L-lysine chloromethyl ketone, and 2.8 ,ug of aprotinin per ml) (10, 11). T. cruzi trypomastigote protein separation by preparative isoelectrofocusing. Proteins from trypomastigote membrane preparations were initially separated by preparative isoelectrofocusing with a pH 3 to 10 ampholine range (Bio-Rad) in an isoelectrofocusing cell at 4°C. Labeled or unlabeled trypomastigote membrane fractions (8.0 mg of protein) solubilized with 0.8% CHAPS in Milli-Q-deionized water supplemented with protease inhibitors as described above were mixed with ampholines to yield a final ampholine concentration of 2%. This mixture was focused for 4 to 6 h at 4°C and a constant 12 W (22). Twenty fractions were collected, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9) under nonreducing conditions in 10% polyacrylamide gels, stained with silver stain (BioRad), and autoradiographed when membrane proteins were radioiodinated or when trypomastigotes were metabolically labeled with L-[355]methionine. Fractions were blotted onto nitrocellulose and stained with avidin-alkaline phosphatase (A-AP; Bio-Rad) (12) when biotinylated surface proteins were separated. Fraction 6 of the trypomastigote membrane protein fractions collected from the isoelectrofocusing cell, enriched for the 60-kDa protein at pH 4.2, was further separated by reapplication to the preparative isoelectrofocusing cell. After chromatofocusing, 20 fractions were again collected and analyzed as described above. Fractions obtained after the rechromatofocusing step were dialyzed against water and lyophilized, and their purity was analyzed by SDS-PAGE as described above. Autoradiograms, nitrocellulose membrane blots, or stained gels containing parasite proteins were scanned with an Ultroscan XL laser densitometer (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) as described previously (10, 11). Gel filtration of a chromatofocused fraction of T. cruzi trypomastigotes. Fraction 6, highly enriched for the trypomastigote 60-kDa protein at pH 4.2, was collected from the preparative isoelectrofocusing cell (see Fig. 2, fraction 6) and applied to a Superose gel filtration column attached to a fast protein liquid chromatography (FPLC) system. In brief, 0.5 mg of protein from rechromatofocused T. cruzi fraction 6 resuspended in 0.01 M phosphate-0.15 M NaCl buffer (pH 7.2) supplemented with 0.2% CHAPS was applied at a flow rate of 0.3 ml/min to a Superose 12 HR10/30 column (Pharmacia) equilibrated with the same buffer and attached to an FPLC system (Pharmacia), which was operated with FPLC manager software (Pharmacia). Elution was performed at 4°C, and fractions of 0.3 ml were collected. Fractions were automatically read atA280 and, when radioiodinated samples were used, the radioactivity was determined in a gamma counter. Fractions were dialyzed, lyophilized, and resuspended in 50 ,ul of PBS, and their purity was analyzed by SDS-PAGE under nonreducing conditions, with silver staining of the gels. When radioiodinated or biotinylated samples

INFECT. IMMUN.

were used, chromatographic fractions were also analyzed by autoradiography or by staining of blots with A-AP. Detection of sugars on the purified trypomastigote 60-kDa protein. The presence of sugar residues on the purified 60-kDa protein was investigated by use of a Lectin-Link kit (Genzyme Corp., Boston, Mass.) containing a panel of biotinylated lectins and a mixture of glycoprotein standards. This kit is an established procedure for detecting glycoproteins. In brief, 50-,ul (1-pug) volumes of the purified trypomastigote 60-kDa protein were dot blotted onto nitrocellulose membranes (Genzyme) by use of a dot blot apparatus (Bio-Rad). Samples were probed with the following panel of biotinylated lectins: wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin. A mixture of glycoprotein standards (transferrin, al-acid glycoprotein, and RNase B), denoted GMS, was included as a positive control. The negative control was a nonglycosylated molecule, denoted NGM (cytochrome c; Sigma), probed with biotinylated lectins. Nitrocellulose membranes were incubated with A-AP as described in detail by the manufacturer. Antibodies. Specific immunoglobulin G (IgG) antibodies against the purified trypomastigote 60-kDa protein were prepared in rabbits. The purified 60-kDa glycoprotein was resuspended in Freund's complete adjuvant and injected subcutaneously into two rabbits. Rabbits received two additional subcutaneous injections of the 60-kDa glycoprotein resuspended in Freund's incomplete adjuvant over a period of 4 weeks (20). IgG was purified through a protein A column (Pierce). The IgG concentration was determined by use of the Pierce Micro-BCA assay. The IgG purity was analyzed by SDS-PAGE (9) under reducing and nonreducing conditions, and the gels were stained with silver stain. Specific anti-60-kDa protein IgG was obtained by dissociating the specific IgG that had bound to the 60-kDa protein blotted onto nitrocellulose membranes as described in detail previously (17). Purified specific rabbit anti-60-kDa protein IgG was diluted in PBS. Preimmune IgG was also purified through a protein A column (Pierce). Analysis of parasite surface proteins. Trypomastigotes or epimastigotes at the same concentration (109 organisms) were biotinylated at the same time and under the same conditions as those described above. The same protein concentrations of both biotinylated trypomastigote and epimastigote samples were resolved by SDS-PAGE under nonreducing conditions in 10% acrylamide gels as described by Laemmli (9), electroblotted onto nitrocellulose membranes, and developed with A-AP as described previously (11, 12). Biotinylated molecular mass markers (Bio-Rad) were included in the gels. Similarly, the same concentration of trypomastigotes or epimastigotes (109 organisms) was radioiodinated or metabolically labeled with L-[35S]methionine at the same time and under the same conditions as those described above, and the same concentrations of labeled proteins (105 cpm) were separated by SDS-PAGE and autoradiographed. Binding of specific anti-60-kDa protein IgG to the surface of formaldehyde-fixed or live parasites was evaluated by indirect immunofluorescence (6, 20). Immunoblotting. The ability of antibodies produced during human infection to recognize the purified trypomastigote 60-kDa glycoprotein was analyzed by Western blotting (immunoblotting). The purified trypomastigote 60-kDa glycoprotein (0.5 ,ug) was subjected to SDS-PAGE and blotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked and incubated with a 1:200 dilution of individual or pooled samples of human chagasic serum. Controls consisted of human serum obtained from healthy

VOL. 60, 1992

T. CRUZI 60-kDa GLYCOPROTEIN STIMULATES

LYMPHOCYTES

3027

volunteers. The presence of specific antibodies against T. cruzi in the human sera was verified by indirect immunofluorescence (6, 20). Some of the sera from infected individuals were a kind donation from Mauro Peralta of the Institute of Microbiology at the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, and the rest of the sera came from our collection. These sera had immunofluorescence titers of 2512. The membranes were incubated with a 1:100 dilution of biotinylated goat anti-human IgG (Cappel, West Chester, Pa.) and developed with A-AP (12). Immunoprecipitation. The ability of purified anti-60-kDa protein IgG obtained from rabbits to immunoprecipitate the labeled 60-kDa surface protein from trypomastigote extracts was analyzed by immunoprecipitation as described previously (18). In brief, 100-p,g extracts of surface biotinylated or radioiodinated trypomastigotes or epimastigotes (106 cpm) were incubated for 2 h at room temperature with 10 ,ul of Sepharose-protein A beads (Sigma). Ten microliters of these treated fractions was then incubated with 10 ,ul of purified anti-60-kDa protein IgG (20 p,g/ml) and 100 p,l of Sepharoseprotein A beads overnight in 0.01 M Tris-HCl (pH 8.0)-0.14 M NaCl-0.1% Triton X-100 (TSA) in the presence of 0.1% BSA as a carrier at 4°C. For controls, preimmune IgG was added in place of anti-60-kDa protein IgG. The beads were then sequentially washed two times with 1 ml of TSA, once with 1 ml of 0.01 M Tris-HCl (pH 8.0)-0.14 M NaCl, and once with 1 ml of 0.05 Tris-HCl (pH 6.8); 50 1LI of SDS sample buffer (9) was added to the beads, which were then boiled at 100°C for 10 min. The beads were microcentrifuged, and the supernatant was analyzed by SDS-PAGE under nonreducing conditions. Gels were either autoradiographed when parasite proteins were radioiodinated or transferred to nitrocellulose when proteins were biotinylated, and the membranes were developed with A-AP. Priming of mice with the T. cruzi 60-kDa protein. The lyophilized trypomastigote 60-kDa protein was resuspended in PBS at 200 p,g/ml. The protein concentration was determined using by use of the Pierce Micro-BCA assay. Four CBA mice were primed with the purified trypomastigote 60-kDa protein resuspended in PBS. Each mouse received 25 ,ug of the 60-kDa glycoprotein subcutaneously and 25 p,g of the 60-kDa glycoprotein intramuscularly once a week for three consecutive weeks. Control animals received only PBS instead of the trypomastigote 60-kDa glycoprotein. Seven days after the last injection, spleens from primed and unprimed CBA mice were obtained and used for the experiments detailed below. Lymphocyte proliferation assays. The ability of the purified trypomastigote 60-kDa glycoprotein to induce the lymphoproliferation of CBA mouse spleen cells primed with the 60-kDa glycoprotein was investigated. Suspensions of pooled murine spleen cells from inbred CBA mice primed with the purified trypomastigote 60-kDa glycoprotein or from control mice receiving only PBS were prepared as described previously (14) and resuspended at a final concentration of 107 cells per ml in RPMI 1640 medium supple-

presence or absence of the trypomastigote 60-kDa glycoprotein. When added to cultures, this protein replaced an equivalent volume of medium. The cultures were pulsed with 1 RCi of [3H]thymidine (specific activity, 2 Ci/mmol; ICN) on the sixth day of incubation and harvested 24 h later with an automated PHD cell harvester (Cambridge Technology, Inc., Boston, Mass.). The ability of spleen cells from 60-kDa glycoprotein-primed or unprimed CBA mice to respond to concanavalin A (ConA) was investigated by incubating 2.5 x 105 spleen cells from unprimed mice with 2.5 ,ug of ConA per ml in the presence of various concentrations of the purified trypomastigote 60-kDa protein at 37°C in a 5% C02-in-air atmosphere saturated with water vapor. This concentration of ConA was selected because in previous experiments performed with spleen cells obtained from unprimed CBA mice, it was found to be the optimal mitogenic concentration. Spleen cells were pulsed after 2 days of incubation with 1 RCi of [3H]thymidine and harvested 24 h later with an automated PHD cell harvester. Incorporated radioactivity in all cultures was determined with a liquid scintillation counter. All determinations were performed in triplicate, and the results were expressed as counts per minute + 1 standard deviation (SD). Mouse peritoneal macrophages. The methods for collecting and processing peritoneal macrophage monolayers from CBA mice have been described previously (19, 21). Macrophages consisted of 298% nonspecific esterase-positive cells with typical macrophage morphology. Adherent mouse peritoneal macrophages were incubated overnight in DMEMfetal bovine serum at 37°C in an atmosphere of 5% CO2 before the parasites were added (16). Effect of specific IgG on trypomastigote uptake and killing by mouse peritoneal macrophages. Mouse peritoneal macrophage monolayers were washed with DMEM prior to the addition of anti-60-kDa protein IgG-treated trypomastigotes or preimmune IgG-treated trypomastigotes (107 organisms) resuspended in DMEM-BSA. Trypomastigotes (107 parasites) were incubated with the same nonagglutinating concentration (20 ,ug) of either purified rabbit anti-60-kDa protein IgG or purified rabbit preimmune IgG diluted in DMEMBSA at 4°C for 1 h. Treatment of trypomastigotes under these conditions did not affect trypomastigote motility or numbers of parasites. Treated trypomastigotes were then added to macrophage cultures in triplicate at a 10:1 ratio. The cocultures were incubated at 37°C for 2 h, and nonbound trypomastigotes were removed by washing of cultures with DMEM. Some cultures were fixed with absolute methanol and others, for studying parasite killing, received fresh DMEM-fetal bovine serum and were incubated for 24, 48, or 72 h. After fixation as described above and staining with Giemsa stain, the number of parasites per 200 macrophages was microscopically determined (16, 21). Independent experiments were performed in triplicate. Presentation of results and statistical analysis. Each set of results (see Table 1 and figures) is typically representative of

mented with 2% fetal bovine serum. Cultures of 60-kDa glycoprotein-primed or unprimed mouse spleen cells were incubated in the presence or absence of the trypomastigote 60-kDa glycoprotein at 37°C for 7 days in a 5% C02-in-air atmosphere saturated with water vapor. Triplicate cultures, set up in flat-bottom wells of 96-well microculture plates (Linbro, New Haven, Conn.), consisted of 0.1 ml of RPMI 1640 medium supplemented with 2% (vol/vol) heat-inactivated fetal bovine serum, 100 U of penicillin per ml, and 100 p,g of streptomycin per ml and 2.5 x 105 spleen cells in the

represent the mean of triplicate determinations + 1 SD. Differences between mean values were considered statistically significant when P was c0.05, as determined by Student's t test.

three or four experiments with similar protocols. The results

RESULTS A 60-kDa surface protein is present on trypomastigotes but not epimastigotes. Comparative surface labeling of trypomastigote and epimastigote forms of T. cruzi with biotin

3028

VILLALTA ET AL.

kDa

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IN-FEcr. IMMUN.

pH

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FIG. 1. Trypomastigote but not epimastigote forms of T. cnuzi have a 60-kDa surface protein. Biotinylated trypomastigote (lane T) and epimastigote (lane E) extracts (3 p,g) were separated by SDSPAGE, blotted onto nitrocellulose membranes, incubated with A-AP, and developed as described in Materials and Methods. Biotinylated molecular mass standards (Bio-Rad) are indicated. The arrowhead points to the trypomastigote 60-kDa surface protein.

kD

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showed that a protein with a relative molecular mass of 60 kDa was present on the surface of invasive trypomastigotes (Fig. 1, lane T). This protein was not found on the surface of noninvasive epimastigote forms of the parasite when the same concentration (3 ,ug) of labeled solubilized parasites was used (Fig. 1, lane E) or when higher protein concentrations were used, indicating that the expression of the 60-kDa surface protein is developmentally regulated in T. cnuzi. Similar results were obtained when the same concentrations of trypomastigotes or epimastigotes were radioiodinated or metabolically labeled with L-[3 S]methionine and the same amounts of radiolabeled lysed parasites (105 cpm) were resolved by SDS-PAGE and autoradiographed (results not shown). Purification and characterization of the trypomastigote 60kDa glycoprotein. Preparative isoelectrofocusing of T. cnuzi trypomastigote membranes yielded a fraction, obtained at pH 4.2, that was enriched for a protein with a relative molecular mass of 60 kDa (Fig. 2, lane 6). Laser densitometric analysis of this band with respect to its counterpart in the crude membranes applied to the isoelectrofocusing cell indicated that this protein was enriched approximately five times by this one-step chromatofocusing separation. Fraction 6 (Fig. 2), enriched for the 60-kDa protein, was rechromatofocused, and a fraction highly enriched for the 60-kDa protein and containing very few proteins as contaminants at very low concentrations was obtained. The trypomastigote 60-kDa protein from this fraction was further purified by gel filtration chromatography on a Superose column attached to an FPLC system. Figure 3A shows the chromatographic profile of the separation of fraction 6 on a Superose column. A major peak containing the 60-kDa protein was eluted at 58 min. Fractions contained in that peak were dialyzed against water, lyophilized, analyzed by SDS-PAGE, and stained

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FIG. 2. Separation of T. cnmzi trypomastigote membrane proteins by preparative isoelectrofocusing. A solubilized membrane fraction (8.0 mg of protein) obtained from trypomastigotes was applied to a preparative isoelectrofocusing cell as described in Materials and Methods. Twenty fractions were harvested, and their pHs were determined. The fractions (5 p.1) were analyzed by SDS-PAGE and stained with silver stain. Fraction 6 was highly enriched for the 60-kDa protein. Lane A shows SDS-PAGE of 0.5 pLg of the crude trypomastigote membrane extract applied to the isoelectrofocusing cell. Molecular mass standards (Bio-Rad) are indicated.

with silver stain. The insert in Fig. 3A shows the purified 60-kDa protein obtained as a single band by gel filtration on a Superose column. Lane a indicates that the lyophilized Superose column peak (indicated by the arrowhead) contained only a single protein with a relative molecular mass of 60 kDa when analyzed by SDS-PAGE and stained with silver

T. CRUZI 60-kDa GLYCOPROTEIN STIMULATES LYMPHOCYTES

VOL. 60, 1992

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FIG. 3. Purification and characterization of the trypomastigote 60-kDa membrane glycoprotein. (A) Gel filtration of rechromatofocused fraction 6 of trypomastigote membranes on a Superose column. Fraction 6 at pH 4.2 (Fig. 2) and harvested from a preparative isoelectrofocusing cell was rechromatofocused as described in Materials and Methods and applied to a Superose 12 HR10/30 column attached to an FPLC system. Fraction 6 (0.5 mg of protein) resuspended in 0.01 M phosphate-0.15 M NaCI buffer (pH 7.2) supplemented with 0.2% CHAPS was applied at a flow rate of 0.3 m/min to the Superose column equilibrated with the same buffer. Fractions of 0.3 ml were automatically read at A2so. The purity of lyophilized fractions was analyzed by SDS-PAGE, and gels were stained with silver stain. The second peak (indicated by the arrowhead) shows the purified 60-kDa protein. The insert shows SDS-PAGE analysis of the purified T. cnrzi trypomastigote 60-kDa protein obtained by gel filtration. Lanes: a, SDS-PAGE analysis of 0.4 p.g of the lyophilized second peak obtained by gel filtration on the Superose column and stained with silver stain as described in Materials and Methods; b, autoradiogram of the lyophilized second peak (5 x 104 cpm) obtained by gel filtration on the Superose column and separated by SDS-PAGE under conditions similar to those used for lane a, except that trypomastigotes were radioiodinated. (B) Detection of sugar residues on the purified trypomastigote 60-kDa protein. Fifty microliters (1 p.g) of the purified trypomastigote 60-kDa protein was dot blotted onto nitrocellulose membranes and probed with the following biotinylated lectins: wheat germ agglutinin, R. communis agglutinin, and D. stramonium agglutinin. Nitrocellulose membranes were then stained with A-AP.

stain. Lane b shows an autoradiogram of the lyophilized Superose column peak (indicated by the arrowhead) obtained when trypomastigote surface proteins were radioiodinated and subjected to the same purification process involving preparative isoelectrofocusing and gel filtration. The autoradiogram of this lyophilized second peak eluted from the Superose column (Fig. 3A, lane b) revealed only a 60-kDa band, indicating that the purified 60-kDa protein is a trypomastigote membrane protein. Similar results were obtained when the surface proteins of trypomastigotes were biotinylated or when parasites were metabolically labeled with L-[35S]methionine and subjected to the same purification process involving chromatofocusing and gel filtration. In this case, blots or autoradiograms after SDS-PAGE of the lyophilized second peak eluted from the Superose column showed a single 60-kDa band (results not shown). SDSPAGE analysis of the purified 60-kDa protein stained with silver-Coomassie blue stain and Schiff-periodate stain indicated that the 60-kDa molecule is a glycoprotein. Biotinylated wheat germ agglutinin, R communis agglutinin, and D. stramonium agglutinin bound to dot blots of the purified trypomastigote 60-kDa protein, also indicating that the 60-

kDa protein is glycosylated, as shown in Fig. 3B. GMS yielded similar positive reactions with the biotinylated lectins (Fig. 3B). NGM probed with biotinylated lectins yielded negative or background reactions (Fig. 3B). Antibodies against the purified trypomastigote 60-kDa glycoprotein immunoprecipitate a biotinylated 60-kDa surface protein in trypomastigotes, and chagasic sera recognize the purified trypomastigote 60-kDa glycoprotein. Figure 4, lanes A to D, shows that specific anti-60-kDa protein IgG immunoprecipitates a single band of 60 kDa from surface biotinlabeled trypomastigotes (lane A) but does not recognize any protein on surface biotin-labeled epimastigotes (lane C), indicating that a 60-kDa protein that is able to be recognized by specific IgG against the purified trypomastigote 60-kDa glycoprotein is present on the surface of trypomastigote but not epimastigote forms of T. cruzi. This result also confirms the findings described in Fig. 1, which showed that the 60-kDa protein is a developmentally regulated surface molecule in T. cnuzi. Figure 4, lanes A to D, also shows that preimmune IgG does not immunoprecipitate any protein from biotin-labeled trypomastigotes (lane B) or epimastigotes (lane D). Positive fluorescence on the surface of

3030

INFECT. IMMUN.

VILLALTA ET AL. 8000 60 kDa-

60 kDa

7000 -

T

6000 5000 A

B

c

D

FIG. 4. Antibodies against the purified trypomastigote 60-kDa glycoprotein immunoprecipitate a biotinylated 60-kDa surface molecule in trypomastigotes but not epimastigotes, and chagasic sera recognize the purified trypomastigote glycoprotein in Western blots. Lanes A to D, immunoprecipitation of biotinylated surface polypeptides of trypomastigotes (100 pLg) with anti-60-kDa protein IgG (A) or with preimmune IgG (B) and immunoprecipitation of biotinylated surface polypeptides of epimastigotes (100 ,ug) with anti-60-kDa protein IgG (C) or with preimmune IgG (D). Lanes E and F, Western blots of the purified trypomastigote 60-kDa glycoprotein (0.2 p.g) probed with pooled serum from patients with Chagas' disease (E) or probed with pooled serum from healthy volunteers (F).

TI

4000 3000 2000 1000 -

0

1. 0

trypomastigotes was observed in indirect immunofluorescence studies when formaldehyde-fixed or live trypomastigotes were incubated with specific IgG against the purified 60-kDa glycoprotein. Negative fluorescence was observed when parasites were incubated with preimmune IgG (results not shown). Figure 4, lanes E and F, shows that the purified trypomastigote 60-kDa glycoprotein is recognized by pooled chagasic serum in Western blots (lane E), whereas pooled healthy volunteer serum is unable to recognize the purified trypomastigote 60-kDa glycoprotein (lane F), indicating that this trypomastigote 60-kDa glycoprotein is immunogenic for humans. Ninety-three percent of 54 serum samples from chagasic individuals with positive serological results in indirect immunofluorescence recognized the trypomastigote 60kDa molecule in Western blots. Ability of the trypomastigote 60-kDa protein to induce the proliferation of primed spleen cells. We found that the T. cruzi trypomastigote 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in the levels of [3H]thymidine incorporation by cultures of spleen cells obtained from CBA mice primed with the purified 60-kDa protein and incubated with 0.2 and 0.8 ,ug of the purified 60-kDa protein per ml, with respect to control values (Fig. 5). The T. cruzi trypomastigote 60-kDa glycoprotein did not stimulate unprimed spleen cells (Fig. 5), showing that the effect induced by the 60-kDa protein is not polyclonal activation. The purified trypomastigote glycoprotein was unable to suppress the response of spleen cells obtained from mice to a known mitogen in vitro. Several concentrations of the purified trypomastigote 60-kDa glycoprotein were unable to suppress the response of mouse spleen cells to ConA in vitro, since the levels of [3Hlthymidine incorporation by unprimed spleen cells exposed to an optimal mitogenic concentration of ConA (2.5 ,ug/ml) were similar in the presence or absence of different concentrations of the purified trypomastigote 60-kDa glycoprotein (Table 1). Effect of specific antibodies against the trypomastigote 60kDa glycoprotein on the ability of macrophages to take up and kill T. cruzi. The addition of purified specific anti-60-kDa protein IgG (20 ,ug) to T. cruzi trypomastigotes enhanced the uptake of the parasites by nonelicited macrophages, com-

0.2

0.8

60 kDa (ug/mi) FIG. 5. The trypomastigote 60-kDa glycoprotein induces the lymphoproliferation of spleen cells obtained from CBA mice primed with the purified glycoprotein. Spleen cells were incubated in the presence of different concentrations of the 60-kDa protein or in its absence as described in Materials and Methods. Symbols: 1, proliferation of 60-kDa protein-primed spleen cells; *, proliferation of unprimed spleen cells. This is a representative experiment of three independent experiments performed with the same protocol, with similar results. Each column represents the mean of triplicate determinations, and each bar represents 1 SD. Differences between 60-kDa protein-primed and unprimed spleen cell responses to the purified 60-kDa protein were statistically significant (P c 0.05).

pared with that of parasites incubated with the same concentration of preimmune IgG (Fig. 6A). The addition of anti-60-kDa protein IgG also induced the destruction of trypomastigotes by these phagocytic cells (Fig. 6B). There was a considerable decrease in the number of parasites per 200 macrophages (from 414 to 88) during the first 24 h of the TABLE 1. The purified T. cruzi trypomastigote 60-kDa glycoprotein does not suppress the response of CBA mouse spleen cells to ConAa Purified 60-kDa glycoprotein concn

(big/mI)

0.0 0.0 0.1 0.2 0.4 0.8 1.6

ConA concn

(Lg/nml) 0.0 2.5 2.5 2.5 2.5 2.5 2.5

cpm (mean ± SD)

576 105,867 99,241 98,603 115,495 98,102 115,410

283

t t t

7,582c 5,190d

± ± ± ±

29,517d 13,860d 13,309d 4,861d

a Spleen cells were incubated with different concentrations of the purified trypomastigote 60-kDa glycoprotein in the presence or absence of 2.5 ,g of ConA per ml as described in Materials and Methods. This is a representative experiment of three independent experiments performed in triplicate, with similar results. Differences between values marked by superscripts b and c were statistically significant (P c 0.05). Differences between values marked by superscripts c and d were not statistically significant (P < 0.05).

T. CRUZI 60-kDa GLYCOPROTEIN STIMULATES LYMPHOCYTES

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experiment. This number remained low throughout the 72-h duration of the experiment when trypomastigotes had been pretreated with anti-60-kDa protein IgG and then allowed to interact with macrophages. When parasites were pretreated with preimmune IgG and then exposed to macrophages, the number of parasites per 200 macrophages remained constant during the first 24 h of the experiment and then increased considerably after 72 h, denoting parasite multiplication within macrophages.

0

o

m E a a

0

2

Time (hr)

,

400

0

X

N

U

E

300

200

i_;

100

0

20

40

60

80

Time (hr)

FIG. 6. Specific anti-T. cruzi 60-kDa protein IgG increases trypomastigote uptake and intracellular killing by macrophages. Trypomastigotes were incubated with the same concentration of either specific anti-60-kDa protein IgG or preimmune IgG at 4°C for 1 h and then exposed to triplicate cultures of macrophages. (A) Trypomastigote uptake by macrophages. Symbols: *, trypomastigote trypomastiuptake in the presence of anti-60-kDa protein IgG; gote uptake in the presence of preimmune IgG. (B) Fate of trypomastigote forms of T. cnuzi taken up by macrophages in the presence of anti-60-kDa protein IgG (N) or preimmune IgG (0). After 2 h of macrophage-trypomastigote interaction, unbound trypomastigotes were washed away and fresh medium was added to the cultures as described in Materials and Methods. Intracellular parasite killing by macrophages was microscopically evaluated as described in Materials and Methods. This is a representative experiment of three independent experiments performed with the same protocol, with similar results. Each column or point represents the mean of triplicate determinations, and each bar represents 1 SD. Differences between trypomastigote uptake and killing by macrophages in the presence of anti-60-kDa protein IgG and preimmune IgG were statistically significant (P < 0.05). 5,

DISCUSSION The results of this study show that the T. cruzi trypomastigote 60-kDa surface glycoprotein can be readily purified in its native state to homogeneity from membranes of T. cnrzi trypomastigotes by two steps involving preparative isoelectrofocusing and gel filtration. The purified trypomastigote 60-kDa acidic membrane surface glycoprotein is able to stimulate in vitro lymphocytes obtained from mice primed in vivo with the purified glycoprotein. Antibodies against this glycoprotein facilitate the uptake and destruction of trypomastigotes by nonelicited macrophages, presumably by binding to the macrophage Fc receptor. This finding is in agreement with previous observations that antibodies against the parasites facilitate their uptake and destruction by macrophages (reviewed in reference 4). The fact that antibodies against the purified trypomastigote 60-kDa glycoprotein immunoprecipitate from extracts of biotinylated parasites a single biotinylated 60-kDa surface protein in trypomastigotes but not epimastigotes indicates that the 60-kDa surface molecule is developmentally regulated in the cell cycle of the parasite. In agreement with this observation is the fact that a biotinylated 60-kDa protein could be seen on the surface of trypomastigotes but not epimastigotes when the same number of trypomastigotes or epimastigotes was biotinylated and the same amount of protein was separated by SDS-PAGE and blotted onto nitrocellulose membranes. This trypomastigote 60-kDa glycoprotein is recognized by antibodies present in the sera of infected individuals, since sera from our collection (up to 50 immunofluorescencepositive serum samples from patients with Chagas' disease) were able to recognize the purified 60-kDa glycoprotein. These results indicate that infected patients can mount an immune response against this parasite surface glycoprotein in the course of a chagasic infection. The fact that antibodies produced against this parasite surface glycoprotein increased trypomastigote uptake and killing by nonelicited macrophages suggests that the host may mount an immune response against this 60-kDa glycoprotein of invasive forms of the parasite. This response may be a beneficial immunological mechanism against a developmentally regulated glycoprotein of invasive forms of the parasite during T. cruzi infection. It is well documented that T. cnzi trypomastigotes or their fractions induce immunosuppression, decreasing the response of lymphocytes to polyclonal activators (reviewed in reference 5). In this paper, we report that the trypomastigote 60-kDa glycoprotein is unable to suppress the response of murine spleen cells to ConA in vitro, since the responses of unprimed splenocytes to ConA in the presence or absence of several concentrations of the purified 60-kDa glycoprotein were similar. In addition, our results show that this molecule is not a polyclonal activator. In contrast, lymphocytes obtained from 60-kDa protein-primed mice were able to be stimulated in vitro with the purified 60-kDa glycoprotein,

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suggesting that this trypomastigote surface glycoprotein, which is developmentally regulated in the cell cycle of the parasite and is recognized by antibodies produced during human infection with T. cnrzi, may be a candidate for vaccination studies. A limited amount of information concerning immunoprotective molecules and their lymphocyte epitopes in mammalian forms of T. cruzi is available (13). Therefore, the identification and characterization of surface molecules of mammalian forms of T. cruzi that are developmentally regulated in the cell cycle of the parasite and that could potentially be targets of immunological attack by the host may facilitate the definition of T and B cell epitopes relevant to molecular and immunological intervention in T. cruzi infection. The 60-kDa molecule discussed in this paper is a good candidate, since it does not induce immunosuppression and appears to induce a beneficial response in vitro. Current studies in our laboratory are devoted to defining the molecular structure of this glycoprotein and to searching for potential immunoprotective epitopes for this molecule. ACKNOWLEDGMENTS This work was supported by grants DAN-5053-G-SS-9111-00 from the United States Agency for International Development, AI-25637 from the National Institutes of Health, and RII-9005826 from the National Science Foundation. A.R.-R. is a postdoctoral trainee supported by grant 5G12RR03032 from the National Institutes of Health. S.A.H. is a fellow trainee supported by training grant AI-07181 in molecular parasitology from the National Institutes of Health. REFERENCES 1. Brener, Z. 1973. Biology of Trypanosoma cruzi. Annu. Rev. Microbiol. 27:347-382. 2. Grant, I. H., J. W. M. Gold, M. Wittner, H. B. Tanowitz, C. Nathan, K. Mayer, L. Reich, N. Wollner, L. Steinherz, F. Ghavimi, R. J. O'Reilly, and D. Armstrong. 1989. Transfusion associated acute Chagas' disease acquired in the United States. Ann. Intern. Med. 111:849-851. 3. Hagar, J. M., H. Hahbudin, and M. B. Rahimtoola. 1991. Chagas' heart disease in the United States. N. Engl. J. Med. 325:763-768. 4. Hudson, L., and V. Britten. 1985. Immune responses to South American trypanosomiasis and its relationship to Chagas' disease. Br. Med. Bull. 41:175-180. 5. Kierszenbaum, F., and M. B. Sztein. 1990. Mechanisms underlying immunosuppression induced by Trypanosoma cruzi. Parasitol. Today 6:201-204. 6. Kierszenbaum, F., F. Villalta, and P. C. Tai. 1986. Kinetics of human eosinophil activation upon interaction with intracellular (amastigote) forms of Trypanosoma cruzi. J. Immunol. 136:662666. 7. Kirchhoff, L. V., A. A. Gam, and F. C. Gilliam. 1987. American trypanosomiasis (Chagas' disease) in Central American immi-

INFECr. IMMUN.

grants. Am. J. Med. 82:915-920. 8. Kirchhoff, L. V., and M. E. Wilson. 1991. Chagas' disease, African trypanosomiasis, and leishmaniasis. Curr. Opin. Infect. Dis. 4:273-281. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Lima, M. F., and F. Villalta. 1988. Host-cell attachment by Trypanosoma cruzi: identification of an adhesion molecule. Biochem. Biophys. Res. Commun. 155:256-262. 11. Lima, M. F., and F. Villalta. 1989. Trypanosoma cruzi trypomastigote clones differentially express a parasite adhesion molecule. Mol. Biochem. Parasitol. 33:159-170. 12. Lima, M. F., and F. Villalta. 1990. Trypanosoma cnrzi receptors for human transferrin and their role. Mol. Biochem. Parasitol. 38:245-252. 13. Miles, M. A., G. R. Wallace, and J. L. Clarke. 1989. Multiple peptide synthesis (Pepscan Method) for the systematic analysis of B- and T-cell epitopes: application to parasite proteins. Parasitol. Today 5:397-400. 14. Mishell, B. B., S. M. Shiigi, C. Henry, E. L. Chan, J. North, R. Gallily, M. Slomich, K. Miller, J. Marbrook, D. Parks, and A. H. Good. 1980. Preparation of mouse cell suspensions, p. 1-166. In B. B. Mishell and S. M. Shigii (ed.), Selected methods in cellular immunology, 1st ed. W. H. Freeman & Co., New York. 15. Nickerson, P., P. Orr, M. L. Shroeder, L. Sekla, and J. B. Johnston. 1989. Transfusion associated Trypanosoma cruzi infection in non-endemic area. Ann. Intern. Med. 111:851-853. 16. Noisin, E. L., and F. Villalta. 1989. Fibronectin increases Trypanosoma cruzi amastigote binding and uptake by murine macrophages and human monocytes. Infect. Immun. 57:10301034. 17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Sringer, T. A. 1991. Immunoprecipitation, p. 8.3.1-8.3.11. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology, vol. 1. John Wiley & Sons, Inc., New York. 19. Villalta, F., and F. Kierszenbaum. 1983. Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi. Biochim. Biophys. Acta 736:39-44. 20. Villalta, F., and F. Kierszenbaum. 1983. Cross-reactivity of vector-borne metacyclic forms of Trypanosoma cruzi with mammalian cells and culture stages. J. Protozool. 3:329-331. 21. Villalta, F., and F. Kierszenbaum. 1984. Role of inflammatory cells in Chagas' disease. II. Interactions of mouse macrophages and human monocytes with intracellular forms of Trypanosoma cruzi: uptake and mechanisms of destruction. J. Immunol. 133:3338-3343. 22. Villalta, F., M. F. Lima, and L. Zhou. 1990. Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells. Biochem. Biophys. Res. Commun. 172:925-931. 23. Wilson, M. E., and K. K. Hardin. 1988. The major concanavalin A-binding surface glycoprotein of Leishmania donovani chagasi promastigotes is involved in attachment to human macrophages. J. Immunol. 141:265-272.

Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied i...
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