INFECTION AND IMMUNITY, Apr. 1991, p. 1409-1416 0019-9567/91/041409-08$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 59, No. 4
Characterization of a Glycosyl-Phosphatidylinositol-Anchored Membrane Protein from Trypanosoma cruzi CRISTINA HERNANDEZ-MUNAIN, MONICA A. FERNANDEZ, ANTONIO ALCINA, AND
MANUEL FRESNO*
Centro de Biologia Molecular Consejo Superior de Investigaciones CientfficasUniversidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 4 October 1990/Accepted 18 January 1991
Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.
The protozoan flagellate Trypanosoma cruzi is the causative agent of Chagas' disease, which affects several million people in Central America and South America (6, 15). This protozoan has a complex life cycle and exists in at least three morphologically distinct forms: infective (metacyclic or blood trypomastigotes), insect (epimastigotes), and intracellular (amastigotes). Epimastigotes multiply in the insect gut and differentiate into infective metacyclic trypomastigotes as they move through the digestive tract. Once in the vertebrate host, they enter susceptible cells, in which they replicate intracellularly as amastigotes (6, 15). Several alterations of the immune response have been described in this disease (6, 15); these include a severe immunosuppression of the humoral and cellular responses to unrelated antigens during the acute phase of the infection and functional defects in the responding cell population. However, the mechanisms underlying these defects are poorly understood (14, 18, 25). Furthermore, massive lymphocyte polyclonal activation (21) resulting in the generation of autoantibodies cross-reacting with the parasites and host cells and tissues has also been described (8, 17, 33). Analyses of T. cruzi cell surface antigens have been carried out to achieve a better understanding of this complex host-parasite relationship (31). Several cell surface proteins have been identified with the help of monoclonal antibodies (MAbs). Among them are the glycoproteins GP 72 (32), GP 90 (24), and recently GP 57/51 (27). GP 72 has been shown to partially protect the host from infection with metacyclic trypomastigotes (29) and seems to be involved in parasite differentiation (30). GP 90 has been claimed to be trypomastigote specific (24), and GP 25, which recently has been shown to be derived from GP 57/51 (27), is a good serological marker for the disease (26). In addition, some not-so-well characterized MAbs have been reported to be stage specific * Corresponding author. t Present address: Instituto
(4, 34) or strain specific (12). Recently, several MAbs have been used to characterize an amastigote-specific 70- to 84-kDa protein (3). Many eukaryotic proteins are anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) linkage (9, 19). Among them are several proteins from members of the family Trypanosomatidae, such as the variant surface glycoprotein from Trypanosoma brucei and the major surface protease of Leishmania spp. (5, 7, 11). Recently, two proteins from T. cruzi, GP 90 (28) and the amastigote-specific 70- to 84-kDa protein, also have been shown to be GPIanchored membrane proteins (3). We describe here four MAbs against a T. cruzi 50- to 55-kDa surface protein which is present in all differentiation stages and which seems to be anchored to the membrane by GPI. MATERIALS AND METHODS Parasites. The strain of T. cruzi used was originally obtained from a patient with Chagas' disease in the Instituto Nacional de la Salud, Madrid, Spain. It was cloned and named strain G (2). Strains Y and Tulahuen were kindly supplied by John David (Harvard University, Boston, Mass.). The trypanosomes were continuously cultured in liver infusion-tryptose medium supplemented with 10% fetal calf serum as described previously (13). Metacyclic trypomastigotes were prepared by differential centrifugation of parasite cultures at 150 x g for 5 min, and this centrifugation was repeated several times with the resulting supernatant. The final supernatant contained >95% metacyclic trypomastigote forms. Epimastigotes were obtained during exponential growth from parasites cultures which contained more than 95% epimastigotes. The cultures were centrifuged for 15 min at 1,000 x g and washed twice with phosphate-buffered saline (PBS). Amastigotes were obtained from infected cultures of J774 cells as described previously (1) and separated
L6pez-Neyra, Granada, Spain. 1409
1410
INFECT. IMMUN.
HERNANDEZ-MUNAiN ET AL.
from epimastigotes and cells by metrizamide (Nyegaard, Oslo, Norway) discontinuous gradient (8 and 16%) centrifu-
gation. Preparation of MAbs. Female BALB/c mice were infected by injection with cultures which contained a mixture of morphologies of live T. cruzi (106 trypanosomes per mouse) on the first (intraperitoneal), 15th (intraperitoneal), and 27th (intramuscular) days. Five days after the last injection, spleen cells were obtained and fused with P3/X63-Ag 8.653 myeloma cells as described previously (1). Successful hybrids were selected and screened for antibody production by an enzyme-linked immunoabsorbent assay (ELISA) and an indirect immunofluorescence assay. The selected hybrids were cloned three times. Immunoglobulin subclasses of the MAbs were determined by double immunodiffusion with antimouse subclass-specific antibodies (Nordik Laboratories, Tilburg, The Netherlands). MAb C10 (immunoglobulin Gl [IgGl) was purified from the peritoneal fluid of ascitic tumors in BALB/c mice by affinity chromatography in protein A-Sepharose CL-4B (Sigma Chemical Co., St. Louis, Mo.). The other MAbs were of the IgM subclass. Antibody to the cross-reacting determinant (anti-CRD) was a generous gift from M. L. Cardoso de Almeida (Escola Paulista de Medicina, Sao Paulo, Brazil). Localization of the 50- to 55-kDa antigen by immunogold labeling. T. cruzi parasites from a liver infusion-tryptose culture at 27°C were washed three times with PBS and incubated with MAb CIO ascitic fluid diluted 1:50 in PBS for 1 h at room temperature. After the washes, the pellets were incubated with rabbit antimouse immunoglobulin (RMIG) (50 mg/ml in PBS) for 1 h at room temperature. After three washes with PBS, protein A complexed to 5-nm colloidal gold particles (Janssen, Beerse, Belgium) diluted 1:50 in PBS was added and the mixture was incubated for 2 h at room temperature. Parasites were washed with PBS, and the pellets were fixed with 4% formaldehyde-2% glutaraldehyde-2% tannic acid in PBS for 1 h at 4°C. After three PBS rinses, the pellets were treated with 1% osmium tetraoxide in PBS for 30 min at 4°C. After another three rinses of 20 min each with PBS, the pellets were progressively dehydrated by incubation with different dilutions of acetone: 30% for 30 min, 50% for 1 h, and 70% for 1 h. The samples were completely dehydrated by incubation with 70% acetone in PBS for 2 days at 4°C and incubation with 100% acetone for 1 h. The samples were embedded in vegetal resin (Serva, Heidelberg, Federal Republic of Germany), and thin sections of 50 to 80 nm were picked up, stained with 1% aqueous uranyl acetate-2% lead citrate, and examined in an electron microscope. Flow cytometry analysis. T. cruzi parasites were centrifuged twice in PBS with 2% bovine serum albumin (BSA)0.1% sodium azide. For each assay, 1 x 106 to 2 x 106 parasites were resuspended and incubated in 100 p. of MAb hybridoma supernatant for 30 min at 4°C. The parasites were washed and incubated for 30 min at 4°C in the dark with 50 p.1 of fluorescein isothiocyanate-labeled F(ab')2 RMIG (Southern Biotechnology Associates Inc., Birmingham, Ala.). After three rinses, the parasites were resuspended in the same buffer containing 1% paraformaldehyde and the fluorescence was analyzed in an EPICS cytofluorimeter. Treatment of intact parasites. To study the biochemical nature of the antigen recognized by the MAbs, we subjected the parasites to several treatments. (i) Before the blocking solution was added to ELISA wells, the parasites were treated with a 1 M solution of sodium hydroxide for 30 min at 37°C or subjected to nitrous acid deamination by incu-
bation with 0.25 M sodium acetate (pH 3.5) plus 0.2 M fresh sodium nitrite overnight at room temperature. (ii) For the phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, a suspension of 108 inactivated (by heating at 56°C for 15 min) parasites was treated with 20 to 200 U of PI-PLC of Bacillus cereus (Boehringer, Mannheim, Federal Republic of Germany) per ml in 100 p.l of 0.27 M sucrose-25 mM ethanolamine-NaOH (pH 7.5)-l mg of BSA per ml0.002% sodium azide for 1 to 2 h at 37°C. (iii) For the protease (Sigma) treatment, a suspension of live parasites was incubated with 200 p.g of each protease per ml for 16 h at 37°C.
Reactivity with antibodies was determined by ELISA or flow cytometry analysis. ELISA. In brief, parasites were washed three times with PBS, heated at 56°C for 15 min, and resuspended to 106/ml in PBS. They were added to a 96-well flat-bottom polyvinyl chloride plate (Titertek; Flow Laboratories, Irvine, Scotland) and incubated overnight at 4°C. The wells were saturated by incubation with 5% BSA-0.05% Tween 20 (Sigma) in PBS for 1 to 2 h at room temperature. The wells were incubated with 100 p.l of serial dilutions of hybridoma supernatants or serum in PBS containing 0.1% BSA and 0.05% Tween 20 for 1 h at room temperature, and ELISA was carried out as described previously (2). Immunoblotting. Parasites were washed with PBS and disrupted with lysis buffer (1% Nonidet P-40 [Fluka Chemie, Buchs, Switzerland]), 150 mM NaCl, 20 mM Tris-HCI [pH 8], and a cocktail of protease inhibitors [Boehringer] containing 1 mM phenylmethylsulfonyl fluoride, 1 p.g of aprotinin per ml, 1 p.g of pepstatin per ml, 1 p.g of leupeptin per ml, and 2 mM EDTA) for 30 min at 4°C. They were electrophoresed on 10 to 12% acrylamide gels and blotted to nitrocellulose paper (BioRad, Richmond, Calif.) as described previously (2). The paper was saturated with PBS containing 0.05% Tween 20, 5% skim milk, and 0.1% sodium azide, and the strips were treated with the hybridoma supernatants of the different MAbs and 1% skim milk for 4 h at room temperature. The strips were washed with PBS0.05% Tween 20 and incubated with 125I-RMIG (specific activity, S x 106 cpm/p.g; 106 cpm/ml diluted in PBS-0.05% Tween 20) for 1 h at room temperature. Phase separation of glycolipid-anchored membrane proteins in TX-114 solution. After treatment with PI-PLC, the parasites were washed two times with 1 ml of Tris-HCI (pH 7.4)-140 mM NaCl (TBS) containing the cocktail of protease inhibitors described above. The supernatants obtained were filtered through 0.2-p.m-pore filters to eliminate possible contaminating parasites and were kept at 4°C to be mixed together with the aqueous phase resulting from Triton X-114 (TX-114) partition of the parasites. The parasites in the pellets were lysed by 30 min of incubation at 4°C with 2% precondensed TX-114 (Serva) in TBS with protease inhibitors. The lysates were centrifuged at 100,000 x g for 30 min at 4°C to remove the insoluble material, incubated at 37°C for 5 min, and centrifuged at 17,000 x g in a minicentrifuge at room temperature for 1 min. The resulting aqueous (detergent-depleted) and detergent-enriched phases were separated, and a volume of TBS containing protease inhibitors or precondensed TX-114 was added to the opposite phase to restore the initial detergent/H20 ratio to start a new phase partitioning. The two samples were incubated at 4°C for 10 min with occasional stirring, warmed in a 37°C bath, and centrifuged at 17,000 x g in a minicentrifuge at room temperature for 1 min. New phases were separated. The corresponding aqueous and detergent-enriched phases were
VOL. 59, 1991
NEW GPI-LINKED T. CRUZI MEMBRANE PROTEIN
Epimastigotes 84.79%
Amastigotes 96.84%
Tripomastigotes 97.54%
92.04%
89.64%
95.39%
93.99%
90.94%
96.09 %
94.94%
91.54%
96.64%
95.74%/*
86.39%
94.54%
1411
oKTcG
MAb C2
MAb C4
MAb C7
MAb C10 _
m~ _t
Fluorescence Intensity FIG. 1. Flow cytometry analysis of T. cruzi with specific MAbs. The reactivities of MAb C10, C2, C4, and C7 culture supernatants with purified epimastigotes, amastigotes, or trypomastigotes were determined. Shown is the number of cells versus the logarithm of fluorescence intensity. The percentage of positive cells in each case is also indicated. The blank profiles represent staining with an irrelevant antibody. aTcG, Antisera from immune mice.
mixed, and the supernatant obtained in the first centrifugation was added to the aqueous phase obtained. The final volume of each phase was adjusted to 2.5 ml with TBS containing protease inhibitors. These samples were used in ELISAs or immunoprecipitations. Immunoprecipitation. Parasites (2 x 108 epimastigotes) were washed with PBS, resuspended in 150 ,ul of the same buffer, and incubated for 20 min at room temperature with 1 mCi of 125I-Na (16.4 mCi/,ug; Amersham, Buckinghamshire, United Kingdom)-30 p.l of lactoperoxidase (140 U/ml; Sigma)-10 ,ul of H202 (0.06%). Every 5 min, 10 ,ul of H202 was added. The cells were washed once with 10 ml of PBS containing 20 mM Nal and 0.5% BSA. 125I-labeled T. cruzi epimastigotes (108) were either treated or not treated with PI-PLC and extracted with a TX-114 solution. The phases obtained after the TX-114 partitioning were separated as described above. The immunoprecipitations were done with preformed protein A-Sepharose CL-4B-antibody complexes. These complexes were formed by incubating 10 p.g of RMIG and 10 of 50% protein A-Sepharose CL-4B suspension for 2 h at 4°C. After a wash with PBS, ternary complexes were formed by further incubation with 10 p.g of purified MAb C10 or an irrelevant mouse IgGl MAb. The complexed beads were washed with PBS before use, and 10 p.l of a 50% suspension of the preformed complexes was used in each incubation. The samples were precleared three consecutive times by incubation for 1 h at 4°C with RMIG-protein A-Sepharose CL-4B complexes. The supernatants were incubated for 4 h at 4°C with specific or control antibodies in ternary complexes with RMIG and protein A-Sepharose CL-4B. The beads were washed five times with the lysis buffer used for the immunoblotting. The proteins were eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) buffer and subjected to SDS-PAGE amide gels as described previously (2).
on
12% acryl-
RESULTS MAbs C10 (IgGl), C4 (IgM), C7 (IgM), and C2 (IgM) were selected by ELISA and indirect immunofluorescence assay from a pool of hybridomas obtained from T. cruzi-infected mice. These MAbs reacted very strongly with epimastigotes and trypomastigotes and somewhat more weakly with amastigotes, as determined by flow cytometry analysis (Fig. 1). To characterize the antigens recognized by the different T. cruzi-specific MAbs, we used the immunoblotting technique. For this purpose, epimastigote extracts were subjected to SDS-PAGE, blotted, and incubated with the four MAbs. The sera from infected mice but not from normal mice reacted with 72-kDa, 50-kDa, and other, lower-molecular-mass antigens, as well as with the lipopeptidophosphoglycan (LPPG) antigen (Fig. 2). However, the four MAbs detected the same broad band in the 50- to 55-kDa region of the gel. The relationship among all of the MAbs was studied by MAb C10 binding competition experiments with an ELISA. C4 and C7 partially inhibited MAb C10 binding, whereas C2 was ineffective (Fig. 3). The four MAbs were specific for T. cruzi, since they did not react with other members of the family Trypanosomatidae, such as Leishmania mexicana, L. donovani, L. infantum, or T. brucei. However, no significant differences were observed in their reactivities with several strains of T. cruzi (data not shown). MAb C10 recognized in the indirect immunofluorescence assay a determinant that was present in the membranes of both living and fixed parasites. The staining was still visible after 24 h of incubation of live parasites in serum-free
1412
INFECT. IMMUN.
HERNANDEZ-MUNAIN ET AL.
I-
CD
a
L)4
c:
O
Vol. 59, No. 4
Characterization of a Glycosyl-Phosphatidylinositol-Anchored Membrane Protein from Trypanosoma cruzi CRISTINA HERNANDEZ-MUNAIN, MONICA A. FERNANDEZ, ANTONIO ALCINA, AND
MANUEL FRESNO*
Centro de Biologia Molecular Consejo Superior de Investigaciones CientfficasUniversidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 4 October 1990/Accepted 18 January 1991
Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.
The protozoan flagellate Trypanosoma cruzi is the causative agent of Chagas' disease, which affects several million people in Central America and South America (6, 15). This protozoan has a complex life cycle and exists in at least three morphologically distinct forms: infective (metacyclic or blood trypomastigotes), insect (epimastigotes), and intracellular (amastigotes). Epimastigotes multiply in the insect gut and differentiate into infective metacyclic trypomastigotes as they move through the digestive tract. Once in the vertebrate host, they enter susceptible cells, in which they replicate intracellularly as amastigotes (6, 15). Several alterations of the immune response have been described in this disease (6, 15); these include a severe immunosuppression of the humoral and cellular responses to unrelated antigens during the acute phase of the infection and functional defects in the responding cell population. However, the mechanisms underlying these defects are poorly understood (14, 18, 25). Furthermore, massive lymphocyte polyclonal activation (21) resulting in the generation of autoantibodies cross-reacting with the parasites and host cells and tissues has also been described (8, 17, 33). Analyses of T. cruzi cell surface antigens have been carried out to achieve a better understanding of this complex host-parasite relationship (31). Several cell surface proteins have been identified with the help of monoclonal antibodies (MAbs). Among them are the glycoproteins GP 72 (32), GP 90 (24), and recently GP 57/51 (27). GP 72 has been shown to partially protect the host from infection with metacyclic trypomastigotes (29) and seems to be involved in parasite differentiation (30). GP 90 has been claimed to be trypomastigote specific (24), and GP 25, which recently has been shown to be derived from GP 57/51 (27), is a good serological marker for the disease (26). In addition, some not-so-well characterized MAbs have been reported to be stage specific * Corresponding author. t Present address: Instituto
(4, 34) or strain specific (12). Recently, several MAbs have been used to characterize an amastigote-specific 70- to 84-kDa protein (3). Many eukaryotic proteins are anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) linkage (9, 19). Among them are several proteins from members of the family Trypanosomatidae, such as the variant surface glycoprotein from Trypanosoma brucei and the major surface protease of Leishmania spp. (5, 7, 11). Recently, two proteins from T. cruzi, GP 90 (28) and the amastigote-specific 70- to 84-kDa protein, also have been shown to be GPIanchored membrane proteins (3). We describe here four MAbs against a T. cruzi 50- to 55-kDa surface protein which is present in all differentiation stages and which seems to be anchored to the membrane by GPI. MATERIALS AND METHODS Parasites. The strain of T. cruzi used was originally obtained from a patient with Chagas' disease in the Instituto Nacional de la Salud, Madrid, Spain. It was cloned and named strain G (2). Strains Y and Tulahuen were kindly supplied by John David (Harvard University, Boston, Mass.). The trypanosomes were continuously cultured in liver infusion-tryptose medium supplemented with 10% fetal calf serum as described previously (13). Metacyclic trypomastigotes were prepared by differential centrifugation of parasite cultures at 150 x g for 5 min, and this centrifugation was repeated several times with the resulting supernatant. The final supernatant contained >95% metacyclic trypomastigote forms. Epimastigotes were obtained during exponential growth from parasites cultures which contained more than 95% epimastigotes. The cultures were centrifuged for 15 min at 1,000 x g and washed twice with phosphate-buffered saline (PBS). Amastigotes were obtained from infected cultures of J774 cells as described previously (1) and separated
L6pez-Neyra, Granada, Spain. 1409
1410
INFECT. IMMUN.
HERNANDEZ-MUNAiN ET AL.
from epimastigotes and cells by metrizamide (Nyegaard, Oslo, Norway) discontinuous gradient (8 and 16%) centrifu-
gation. Preparation of MAbs. Female BALB/c mice were infected by injection with cultures which contained a mixture of morphologies of live T. cruzi (106 trypanosomes per mouse) on the first (intraperitoneal), 15th (intraperitoneal), and 27th (intramuscular) days. Five days after the last injection, spleen cells were obtained and fused with P3/X63-Ag 8.653 myeloma cells as described previously (1). Successful hybrids were selected and screened for antibody production by an enzyme-linked immunoabsorbent assay (ELISA) and an indirect immunofluorescence assay. The selected hybrids were cloned three times. Immunoglobulin subclasses of the MAbs were determined by double immunodiffusion with antimouse subclass-specific antibodies (Nordik Laboratories, Tilburg, The Netherlands). MAb C10 (immunoglobulin Gl [IgGl) was purified from the peritoneal fluid of ascitic tumors in BALB/c mice by affinity chromatography in protein A-Sepharose CL-4B (Sigma Chemical Co., St. Louis, Mo.). The other MAbs were of the IgM subclass. Antibody to the cross-reacting determinant (anti-CRD) was a generous gift from M. L. Cardoso de Almeida (Escola Paulista de Medicina, Sao Paulo, Brazil). Localization of the 50- to 55-kDa antigen by immunogold labeling. T. cruzi parasites from a liver infusion-tryptose culture at 27°C were washed three times with PBS and incubated with MAb CIO ascitic fluid diluted 1:50 in PBS for 1 h at room temperature. After the washes, the pellets were incubated with rabbit antimouse immunoglobulin (RMIG) (50 mg/ml in PBS) for 1 h at room temperature. After three washes with PBS, protein A complexed to 5-nm colloidal gold particles (Janssen, Beerse, Belgium) diluted 1:50 in PBS was added and the mixture was incubated for 2 h at room temperature. Parasites were washed with PBS, and the pellets were fixed with 4% formaldehyde-2% glutaraldehyde-2% tannic acid in PBS for 1 h at 4°C. After three PBS rinses, the pellets were treated with 1% osmium tetraoxide in PBS for 30 min at 4°C. After another three rinses of 20 min each with PBS, the pellets were progressively dehydrated by incubation with different dilutions of acetone: 30% for 30 min, 50% for 1 h, and 70% for 1 h. The samples were completely dehydrated by incubation with 70% acetone in PBS for 2 days at 4°C and incubation with 100% acetone for 1 h. The samples were embedded in vegetal resin (Serva, Heidelberg, Federal Republic of Germany), and thin sections of 50 to 80 nm were picked up, stained with 1% aqueous uranyl acetate-2% lead citrate, and examined in an electron microscope. Flow cytometry analysis. T. cruzi parasites were centrifuged twice in PBS with 2% bovine serum albumin (BSA)0.1% sodium azide. For each assay, 1 x 106 to 2 x 106 parasites were resuspended and incubated in 100 p. of MAb hybridoma supernatant for 30 min at 4°C. The parasites were washed and incubated for 30 min at 4°C in the dark with 50 p.1 of fluorescein isothiocyanate-labeled F(ab')2 RMIG (Southern Biotechnology Associates Inc., Birmingham, Ala.). After three rinses, the parasites were resuspended in the same buffer containing 1% paraformaldehyde and the fluorescence was analyzed in an EPICS cytofluorimeter. Treatment of intact parasites. To study the biochemical nature of the antigen recognized by the MAbs, we subjected the parasites to several treatments. (i) Before the blocking solution was added to ELISA wells, the parasites were treated with a 1 M solution of sodium hydroxide for 30 min at 37°C or subjected to nitrous acid deamination by incu-
bation with 0.25 M sodium acetate (pH 3.5) plus 0.2 M fresh sodium nitrite overnight at room temperature. (ii) For the phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, a suspension of 108 inactivated (by heating at 56°C for 15 min) parasites was treated with 20 to 200 U of PI-PLC of Bacillus cereus (Boehringer, Mannheim, Federal Republic of Germany) per ml in 100 p.l of 0.27 M sucrose-25 mM ethanolamine-NaOH (pH 7.5)-l mg of BSA per ml0.002% sodium azide for 1 to 2 h at 37°C. (iii) For the protease (Sigma) treatment, a suspension of live parasites was incubated with 200 p.g of each protease per ml for 16 h at 37°C.
Reactivity with antibodies was determined by ELISA or flow cytometry analysis. ELISA. In brief, parasites were washed three times with PBS, heated at 56°C for 15 min, and resuspended to 106/ml in PBS. They were added to a 96-well flat-bottom polyvinyl chloride plate (Titertek; Flow Laboratories, Irvine, Scotland) and incubated overnight at 4°C. The wells were saturated by incubation with 5% BSA-0.05% Tween 20 (Sigma) in PBS for 1 to 2 h at room temperature. The wells were incubated with 100 p.l of serial dilutions of hybridoma supernatants or serum in PBS containing 0.1% BSA and 0.05% Tween 20 for 1 h at room temperature, and ELISA was carried out as described previously (2). Immunoblotting. Parasites were washed with PBS and disrupted with lysis buffer (1% Nonidet P-40 [Fluka Chemie, Buchs, Switzerland]), 150 mM NaCl, 20 mM Tris-HCI [pH 8], and a cocktail of protease inhibitors [Boehringer] containing 1 mM phenylmethylsulfonyl fluoride, 1 p.g of aprotinin per ml, 1 p.g of pepstatin per ml, 1 p.g of leupeptin per ml, and 2 mM EDTA) for 30 min at 4°C. They were electrophoresed on 10 to 12% acrylamide gels and blotted to nitrocellulose paper (BioRad, Richmond, Calif.) as described previously (2). The paper was saturated with PBS containing 0.05% Tween 20, 5% skim milk, and 0.1% sodium azide, and the strips were treated with the hybridoma supernatants of the different MAbs and 1% skim milk for 4 h at room temperature. The strips were washed with PBS0.05% Tween 20 and incubated with 125I-RMIG (specific activity, S x 106 cpm/p.g; 106 cpm/ml diluted in PBS-0.05% Tween 20) for 1 h at room temperature. Phase separation of glycolipid-anchored membrane proteins in TX-114 solution. After treatment with PI-PLC, the parasites were washed two times with 1 ml of Tris-HCI (pH 7.4)-140 mM NaCl (TBS) containing the cocktail of protease inhibitors described above. The supernatants obtained were filtered through 0.2-p.m-pore filters to eliminate possible contaminating parasites and were kept at 4°C to be mixed together with the aqueous phase resulting from Triton X-114 (TX-114) partition of the parasites. The parasites in the pellets were lysed by 30 min of incubation at 4°C with 2% precondensed TX-114 (Serva) in TBS with protease inhibitors. The lysates were centrifuged at 100,000 x g for 30 min at 4°C to remove the insoluble material, incubated at 37°C for 5 min, and centrifuged at 17,000 x g in a minicentrifuge at room temperature for 1 min. The resulting aqueous (detergent-depleted) and detergent-enriched phases were separated, and a volume of TBS containing protease inhibitors or precondensed TX-114 was added to the opposite phase to restore the initial detergent/H20 ratio to start a new phase partitioning. The two samples were incubated at 4°C for 10 min with occasional stirring, warmed in a 37°C bath, and centrifuged at 17,000 x g in a minicentrifuge at room temperature for 1 min. New phases were separated. The corresponding aqueous and detergent-enriched phases were
VOL. 59, 1991
NEW GPI-LINKED T. CRUZI MEMBRANE PROTEIN
Epimastigotes 84.79%
Amastigotes 96.84%
Tripomastigotes 97.54%
92.04%
89.64%
95.39%
93.99%
90.94%
96.09 %
94.94%
91.54%
96.64%
95.74%/*
86.39%
94.54%
1411
oKTcG
MAb C2
MAb C4
MAb C7
MAb C10 _
m~ _t
Fluorescence Intensity FIG. 1. Flow cytometry analysis of T. cruzi with specific MAbs. The reactivities of MAb C10, C2, C4, and C7 culture supernatants with purified epimastigotes, amastigotes, or trypomastigotes were determined. Shown is the number of cells versus the logarithm of fluorescence intensity. The percentage of positive cells in each case is also indicated. The blank profiles represent staining with an irrelevant antibody. aTcG, Antisera from immune mice.
mixed, and the supernatant obtained in the first centrifugation was added to the aqueous phase obtained. The final volume of each phase was adjusted to 2.5 ml with TBS containing protease inhibitors. These samples were used in ELISAs or immunoprecipitations. Immunoprecipitation. Parasites (2 x 108 epimastigotes) were washed with PBS, resuspended in 150 ,ul of the same buffer, and incubated for 20 min at room temperature with 1 mCi of 125I-Na (16.4 mCi/,ug; Amersham, Buckinghamshire, United Kingdom)-30 p.l of lactoperoxidase (140 U/ml; Sigma)-10 ,ul of H202 (0.06%). Every 5 min, 10 ,ul of H202 was added. The cells were washed once with 10 ml of PBS containing 20 mM Nal and 0.5% BSA. 125I-labeled T. cruzi epimastigotes (108) were either treated or not treated with PI-PLC and extracted with a TX-114 solution. The phases obtained after the TX-114 partitioning were separated as described above. The immunoprecipitations were done with preformed protein A-Sepharose CL-4B-antibody complexes. These complexes were formed by incubating 10 p.g of RMIG and 10 of 50% protein A-Sepharose CL-4B suspension for 2 h at 4°C. After a wash with PBS, ternary complexes were formed by further incubation with 10 p.g of purified MAb C10 or an irrelevant mouse IgGl MAb. The complexed beads were washed with PBS before use, and 10 p.l of a 50% suspension of the preformed complexes was used in each incubation. The samples were precleared three consecutive times by incubation for 1 h at 4°C with RMIG-protein A-Sepharose CL-4B complexes. The supernatants were incubated for 4 h at 4°C with specific or control antibodies in ternary complexes with RMIG and protein A-Sepharose CL-4B. The beads were washed five times with the lysis buffer used for the immunoblotting. The proteins were eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) buffer and subjected to SDS-PAGE amide gels as described previously (2).
on
12% acryl-
RESULTS MAbs C10 (IgGl), C4 (IgM), C7 (IgM), and C2 (IgM) were selected by ELISA and indirect immunofluorescence assay from a pool of hybridomas obtained from T. cruzi-infected mice. These MAbs reacted very strongly with epimastigotes and trypomastigotes and somewhat more weakly with amastigotes, as determined by flow cytometry analysis (Fig. 1). To characterize the antigens recognized by the different T. cruzi-specific MAbs, we used the immunoblotting technique. For this purpose, epimastigote extracts were subjected to SDS-PAGE, blotted, and incubated with the four MAbs. The sera from infected mice but not from normal mice reacted with 72-kDa, 50-kDa, and other, lower-molecular-mass antigens, as well as with the lipopeptidophosphoglycan (LPPG) antigen (Fig. 2). However, the four MAbs detected the same broad band in the 50- to 55-kDa region of the gel. The relationship among all of the MAbs was studied by MAb C10 binding competition experiments with an ELISA. C4 and C7 partially inhibited MAb C10 binding, whereas C2 was ineffective (Fig. 3). The four MAbs were specific for T. cruzi, since they did not react with other members of the family Trypanosomatidae, such as Leishmania mexicana, L. donovani, L. infantum, or T. brucei. However, no significant differences were observed in their reactivities with several strains of T. cruzi (data not shown). MAb C10 recognized in the indirect immunofluorescence assay a determinant that was present in the membranes of both living and fixed parasites. The staining was still visible after 24 h of incubation of live parasites in serum-free
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HERNANDEZ-MUNAIN ET AL.
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