cDNA cloning and partial characterization of amastigote specific surface protein from Trypanosoma cruzi

Infection, Genetics and Evolution 9 (2009) 1083–1091

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cDNA cloning and partial characterization of amastigote specific surface protein from Trypanosoma cruzi Marybell Olivas-Rubio a, Salvador Herna´ndez-Martı´nez b, Patricia Talama´s-Rohana a, Victor Tsutsumi a, Pedro A. Reyes-Lo´pez c, Jose´ Luis Rosales-Encina a,* a b c

Departamento de Infecto´mica y Patoge´nesis Molecular, Centro de Investigacio´n y de Estudios Avanzados del I.P.N., Me´xico D.F. 07360, Mexico Departamento de Gene´tica, Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pu´blica, Cuernavaca, Morelos, 62508, Mexico Direccio´n de Investigacio´n, Instituto Nacional de Cardiologı´a ‘‘Ignacio Cha´vez’’, Me´xico D.F. 14080, Mexico

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 December 2008 Received in revised form 20 May 2009 Accepted 20 May 2009 Available online 27 May 2009

Trypanosoma cruzi amastigote surface proteins are the target of both humoral and cell-mediated immune responses; however, few such molecules have been thoroughly studied. In order to study a T. cruzi amastigote-specific protein (SSP4), we used antibodies against the deglycosylated form of this molecule to clone cDNA. The selected cDNA clone (2070 bp) encodes for a 64 kDa protein product whose sequence analysis revealed no N-glycosylation signal. The DNA sequence showed high homology with a member of a previously reported dispersed repetitive gene family of T. cruzi. Antibodies against the recombinant protein reacted strongly with a 66 kDa protein and weakly with an 84 kDa protein in amastigote extracts. Immunoelectron microscopy studies showed that intracellular amastigotes express the native protein on their surfaces and flagellar pockets. The antibody label was also associated with an amorphous material present in the parasitic cavity and in direct contact with the parasite surface, which suggest that amastigotes are releasing this material. On cell-free amastigotes, the antibody showed strong decoration of the cell surface and labeling of intracellular vesicles. Immunofluorescence analysis showed that the superficial protein is expressed shortly after trypomastigotes begin to transform into amastigotes. Antirecombinant protein antibodies recognized proteins of 100 kDa and 50–60 kDa in protein extracts of rat heart and skeletal muscle, respectively. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Trypanosoma cruzi Amastigotes Surface protein Gene expression Molecular mimetism

1. Introduction Trypanosoma cruzi, the causative agent of Chagas’ disease, undergoes morphological and physiological changes during the course of its life cycle. This cycle is initiated when metacyclic trypomastigotes are eliminated in the feces of the triatomine vector and enter mammalian cells. Inside the cell, trypomastigotes transform into amastigotes in a process that is characterized by changes in major surface glycoproteins (Andrews et al., 1987). Amastigotes are found both inside the host cells and in circulation and, as in trypomastigotes, they can infect cells both in vivo and in vitro (Ley et al., 1988; Noisin and Villalta, 1989; Mortara, 1991). To continue the cycle, amastigotes multiply, transform again into trypomastigotes, and lyse the cell. They are then released into circulation and spread the infection to other tissues.

* Corresponding author at: Departamento de Infecto´mica y Patoge´nesis Molecular, Centro de Investigacio´n y de Estudios Avanzados del I.P.N., Avenida Instituto Polite´cnico Nacional No. 2508, Col. San Pedro Zacatenco, Delegacio´n Gustavo A. Madero, CP 07360, Me´xico D.F., Mexico. Tel.: +52 55 5747 3349; fax: +52 55 5747 3377. E-mail address: [email protected] (J.L. Rosales-Encina). 1567-1348/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.05.016

It has been suggested that intracellular amastigotes play an important role in the persistence of T. cruzi infection (Verbisck et al., 1998). Using monoclonal antibodies, it has been shown that amastigotes from distinct strains and clones express different epitopes in a polymorphic manner, similar to patterns described for epitopes of other parasite stages (Verbisck et al., 1998). Previous work has shown that newly transformed amastigotes – both intracellular and extracellular – express a major surface glycoprotein (SSP4) that is bound to the plasma membrane by a GPI anchor (Andrews et al., 1987, 1988a,b). The cloning of a closely related gene family that is highly expressed in amastigotes (accomplished by differential screening) has also been reported. These genes encode for amastin (Teixeira et al., 1994), surface glycoproteins that show a half-life 7 times longer in amastigotes than in epimastigotes and a level of mRNA 68-fold higher in amastigotes than in epimastigotes (Coughlin et al., 2000). Other amastigote-specific genes (ASP-1 and ASP-2) were shown to be members of the sialidase/trans-sialidase gene superfamily 2 of T. cruzi (Low and Tarleton, 1997; Santos et al., 1997). Here, we report the purification of an 84-kDa protein reactive to the monoclonal antibody 2C2, which defines the SSP4 amastigotespecific surface glycoprotein, and the partial cDNA cloning of an

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amastigote-specific surface protein (SSP4). Surface expression of SSP4 was also detected in intermediate forms between trypomastigotes and amastigotes, indicating that the gene starts expressing when trypomastigotes begin the transformation into amastigotes. Anti-fusion protein MBP::SSP4 antibodies recognized proteins of 100 kDa and 50–60 kDa in protein extracts of rat heart and skeletal muscle, respectively. 2. Materials and methods 2.1. Parasites Strain Y of T. cruzi was maintained in monolayers of LLC-MK2 cells in Dulbecco’s minimum essential medium (DMEM) containing 5% fetal calf serum (FCS) at 37 8C, epimastigote forms were grown in LIT medium at 28 8C with gentle shaking (Andrews et al., 1987). One to two weeks after infection, the supernatant containing trypomastigote, intermediate, and amastigote forms was collected. To obtain trypomastigotes, the heterogeneous parasite suspension was centrifugated at 2000  g for 5 min and then incubated at 37 8C. After 2 h, the motile, slender, and highly infective trypomastigotes were collected from the supernatant. Trypomastigotes released from infected cells were incubated in LIT medium at 37 8C for 24–48 h for extracellular transformation into amastigotes (Andrews et al., 1987, 1988a). 2.2. TcSSP4 purification and deglycosylation TcSSP4 purification was carried out as described (Iida and Ley, 1991). Briefly, 8  109 parasites were suspended in 10 mM Tris– HCl, pH 7.7, containing 10 mM NaCl and a cocktail of protease inhibitors (10 mg/ml leupeptin, 0.2 mM phenylmethane sulfonate fluoride, and 0.1 mM EDTA), quickly frozen in liquid nitrogen and thawed in a 37 8C water bath. After three cycles of freezing and thawing procedure, disrupted parasites were centrifugated at 10,000  g for 30 min. The supernatant was passed through a DEAE-5PW column (Pharmacia LKB) and washed with 10 mM NaCl, 10 mM Tris–HCl, pH 7.7 containing 0.02% Tween-20 (Buffer A), bound proteins were eluted with a linear gradient of NaCl (0.1–0.5 M) in Buffer A. In order to check for the presence of SSP4 glycoproteins, fractions were assayed by silver staining of 10% SDS–PAGE and Western blot with the monoclonal antibody (mAb) 2C2 (Andrews et al., 1987). Positive fractions were desalted in a 3.0 ml Sephadex G-25 column equilibrated with 10 mM NaCl, 10 mM acetate buffer pH 4.1 containing 0.02% Tween-20 (Buffer B), then passed through a Mono-S HR 5/5 column (Pharmacia LKB) previously equilibrated with Buffer B. Proteins were eluted with a linear gradient of NaCl (0–0.5 M) in the same buffer. SSP4 purity was assayed by silver staining and Western blot with the mAb 2C2. SSP4 protein (25–100 mg) was deglycosilated under denaturating conditions using N-glycanase (Genzyme). Samples were heated at 100 8C during 3 min in the presence of 0.25 M sodium phosphate buffer, pH 8.6 and 0.5% SDS. o-Phenantroline, NP-40, and N-glycanase were added to a final concentration of 10 mM, 0.6%, and 100 U/ ml respectively. Samples were incubated for 16 h at room temperature. 2.3. DNA and cDNA expression library construction and screening Genomic DNA was prepared from epimastigotes using a standard protocol (Maniatis et al., 1982). DNA was partially digested with HaeIII restriction enzyme, and fragments of 1000– 6000 bp were isolated by a sucrose gradient. EcoRI adapters were ligated to the DNA HeaIII fragments and then ligated into lgt11 expression vector (Stratagene, La Jolla, CA). Also an amastigote

cDNA expression library was constructed in the same vector (Gonzalez et al., 1990). A rabbit polyclonal antibody (see below) developed against purified and deglycosilated SSP4 molecule was used to screen 120,000 phages from the genomic expression library. After three rounds of screening one clone (C2-1) out of 10 highly positives was selected for further characterization. DNA inserts of lgt11 clones (genomic and cDNA) were subcloned in the EcoRI site of the plasmid pBluescript SK. A 50 -end 450 bp PstI fragment from clone C2-1 was used to screen the cDNA expression library (1.5  104 pfu) by standard procedures. A cDNA clone (TcSSP4) was isolated after three rounds of screening. 2.4. DNA sequencing DNA inserts of selected clones were subcloned into the pBluescript SK-vector and subjected to automated doublestranded sequencing using Taq Fluorescence-Based Dye Terminator Cycle Sequencing on a PerkinElmer/Applied Biosystems 37718Eln DNA sequencer, and also by the dideoxynucleotide chain termination method using Sequenase (United States Biochemicals, Cleveland, OH). 2.5. Fusion protein expression The cDNA insert (GeneBankTM, EMBL, and DDJ databases accession number AF480943) was cloned in the EcoRI site of the expression vector pMAL-C2 (New England Biolabs), and expressed in Escherichia coli DH-5a as fusion protein with Maltose Binding Protein (MBP). The fusion protein MBP::SSP4 and MBP were affinity purified by using amylose resin according to the manufacturer. Purity of proteins was assayed by SDS–PAGE and Western blot. The TcHsp70 cDNA insert (GeneBankTM EMBL and DDJ databases accession number AY576621) was cloned in the same vector and the MBP::Hsp70 was affinity purified. 2.6. Antibodies Amastigote-specific 2C2 mAb has been previously described (Andrews et al., 1987). Polyclonal antibodies (R188) against the deglycosilated protein were obtained by immunization of rabbits with 50 mg of antigen in the presence of complete Freund’s adjuvant. After 4 weeks rabbits were bled and the antibody titer was measured by Western blot against pure protein. Antibodies directed against MBP::SSP4 and MBP were elicited by immunization of mice 4 times at 2-week intervals with 500 ng of protein per dose, and bled 2 weeks after the last booster injection. Complete Freund’s adjuvant was used in the first immunization and incomplete Freund’s adjuvant in the following. 2.7. Immunoblotting Parasites (amastigotes, epimastigotes, and trypomastigotes) were pelleted by centrifugation and washed twice with cold phosphate-buffered saline (0.02 M sodium phosphate pH 7.4). Pelleted parasites were boiled in sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 2% sodium dodecylsulfate, and 0.001% bromophenol blue), run in a 10% SDS–PAGE, and transferred to nitrocellulose paper. Blots were incubated with primary antibodies (anti-MBP::SSP4 at a 1:1000), followed by incubation with a 1:3000 of the alkaline phosphatase conjugated secondary antibody. 2.8. Indirect immunofluorescence Infected LLC-MK2 cell monolayers were washed twice with PBS and fixed in absolute methanol for 15 min at 4 8C. Collected free

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parasites were washed with PBS, fixed with 2% formaldehyde in PBS, and air-dried on glass slides for 45 min at 37 8C. These preparations were incubated with anti-MBP::SSP4 (1:100) and anti-MBP (1:100) antisera in PBS containing 5% fat free milk for 1 h at room temperature; a FITC-labelled affinity purified goat antimouse IgG antibody was used as secondary antibody. After 1 h at room temperature, slides were rinsed in PBS, mounted with fluoprep (bioMe´rieux), and observed with a fluorescence microscope (Nikon E600). 2.9. Transmission electron microscopy Free parasites released to the culture medium of infected LLCMK2 cells were collected and fixed by 2 h at 37 8C with 2% formaldehyde, 0.1% glutaraldehyde in PBS. Parasites were pelleted and dehydrated in a graded ethanol series, and then embedded in LR-white resin 48 h at 37 8C. T. cruzi infected LLCMK2 cells were fixed as above, collected by scraping, and pelleted. Thin sections mounted in nickel grids were incubated with anti-MBP::SSP4 or anti-MBP antibodies (1:50 in PBS/1% BSA) for 1 h at room temperature; bound antibody was detected with pig anti-mouse gold particles (20 nm) conjugated antibody. Sections were stained with 5% aqueous uranyl acetate and 2% lead citrate and examined in a Zeiss (EM-10) transmission electron microscope. 2.10. Rat’s heart and skeletal muscle (Tibialis anterioris) sample preparation Male Wistar rats (130 g) were from the animal facility at Cinvestav (Me´xico, D.F., Me´xico). The experiments were conducted in accordance with National Research Guidelines (NOM-062-ZOO1999). Frozen heart and skeletal muscle were pulverized in a mortar containing liquid nitrogen. For protein extraction, pulverized tissues were homogenized at 4 8C in 20 mM Tris–HCl buffer (pH 7.4) containing protease inhibitors (10 mg/ml leupeptin, 0.2 mM phenylmethane sulfonate fluoride, and 0.1 mM EDTA). Each sample was homogenized using a polytron at 4 8C and then sonicated on ice. The homogenate was centrifugated at 3000  g for 15 min at 4 8C, and supernatant fractions were used for 10% SDS–PAGE and immunoblotting.

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3. Results 3.1. SSP4 purification and polyclonal anti-SSP4 antibodies characterization SSP4 was described as the major stage specific antigen of T. cruzi amastigotes, that is expressed on the surface of newly developed amastigotes (Andrews et al., 1987). This protein is recognized by the 2C2 mAb. This antibody immunoprecipitates two 125I-surface labelled glycoproteins (70 kDa and 84 kDa) that are GPI-anchored and can be released from the plasma membrane by a parasite phospholipase C (Andrews et al., 1988b). To further characterize the amastigote surface proteins reactive to 2C2 mAb, parasite total extract was subjected to fast protein liquid chromatography and the purification of an 84 kDa protein was achieved by two step ion exchange chromatography. Carbohydrate residues were removed from this molecule and after treatment with N-glycanase the electrophoretic mobility increased from 84 kDa to 75 kDa (Fig. 1A), which represents about 12% of its molecular weight. When the deglycosylated molecule was assayed with the 2C2 mAb, no reactivity was detected (Fig. 1B), indicating that the epitope recognized by the antibody is a polysaccharide Asn-linked to the protein. A rabbit antiserum (R188) obtained against the 84 kDa deglycosylated protein was able to recognize both native and deglycosylated molecules (Fig. 1C). 3.2. cDNA cloning and recombinant protein purification Early attempts to isolate amastigote cDNA clones reactive to antibodies against the glycosylated and deglycosylated SSP4 molecules have failed. The R188 polyclonal antibody, able to immunoprecipitate a protein from in vitro translated mRNA from amastigotes (data not shown) was used to screen a genomic expression library, and a highly reactive clone (C2-1) was selected for further characterization. This genomic clone has a 2 kbp DNA insert, and the fusion protein detected by Western blot with an anti-b-galactosidase antibody had a molecular weight of 125 kDa. By using a 450 bp (1–450) from the C2-1 DNA insert, a cDNA clone (TcSSP4) was selected from an amastigote cDNA library. The DNA

2.11. Human chagasic sera Normal and chagasic human sera were kindly supplied by Dr. Carmen Guzme´n Bracho at the ‘‘Instituto de Diagno´stico y Referencia Epidemiolo´gica (InDRE), Me´xico. 2.12. ELISA assays ELISA plates were coated overnight at 4 8C with 2 mg/ml of MBP::SSP4, MBP or MBP::Hsp70, in carbonate buffer (pH 9.6). Plates were washed 6 times with PBS containing 0.05% Tween (PBST), and incubated for 2 h at 37 8C with PBS–BSA (PBS containing 1% bovine serum albumin). Plates were then washed 3 times with PBST and 3 times with PBS, and incubated with 50 ml of human chagasic sera in PBS–BSA (1:100). Plates were washed with PBST and bound antibodies were detected with affinity-purified peroxidase-conjugated goat anti-human IgG antibodies (Zymed Laboratories), at 1:1000 dilution in PBS–BSA, and incubated for 1 h at 37 8C. Plates were washed with PBST and then developed with 2,2-azino-bis[3-ethylbenzthiazoline]6-sulfonic acid (ABTS) (Zymed Laboratories), the reaction was allowed to proceed for 20 min at room temperature. Absorbance was read at 405 nm in an ELISA reader (Labsystem Multiskan MS).

Fig. 1. N-glycanase treatment of the 84 kDa glycoprotein recognized by mAb 2C2. The 84 kDa glycoprotein was treated with N-glycanase and analyzed either by silver staining (A) or by Western blot with the monoclonal antibody 2C2 (B) and with the antiserum R188 (C). Non-treated (1) and treated (2).

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Fig. 2. Amino acid sequence of TcSSP4. The predicted amino acid sequence of TcSSP4 showed a Cys-rich region (187–205, underline), an ARG-rich region (222–390, italic), and a cell attachment sequence RGD (489–491, bold).

insert (2070 bp) was fully sequenced (Fig. 2), and a GenBankTM (BLAST) search showed high homology (87%) with a member of a dispersed repetitive gene family reported previously (Wincker et al., 1990, 1992). The predicted amino acid sequence revealed a long arginine-rich region, a short cysteine-rich region, and a cell

attachment sequence (RGD). No potential N-glycosylation sites were detected. To express the SSP4 protein in E. coli, the TcSSP4 DNA insert was cloned in the pMal plasmid. The affinity purified MBP::SSP4 recombinant protein was obtained as a band of approximately 116 kDa (Fig. 3), accounted by the fusion of MBP (43 kDa) with SSP4 (64 kDa). 3.3. Expression of TcSSP4 in the T. cruzi life cycle In order to study TcSSP4 protein expression in the different developmental stages of the parasite, anti-MBP::SSP4 recombinant protein antibodies were assayed by immunoblot against amastigote, epimastigote, and trypomastigote lysates (Fig. 4). The polyclonal anti-MBP::SSP4 antibody (panel 1) recognized mainly a 66 kDa protein and in less proportion a 84 kDa protein in amastigote lysate (A). The antibody did not recognize any protein in epimastigote lysate (E), and showed a weak recognition of a 66 kDa protein in trypomastigote (T). 3.4. Subcellular distribution of TcSSP4

Fig. 3. Purification of MBP::SSP4. Coomassie blue-stained SDS gel of proteins from pMal-TcSSP4 transformed E. coli DH-5a extracts before induction with IPTG (1), after induction (2) and affinity purified MBP::SSP4 (3). Molecular weight markers are shown.

The subcellular distribution of TcSSP4 protein was studied by immunoelectron microscopy of LLC-MK2 parasite-infected cells and axenic culture amastigotes by using the anti-MBP::SSP4 antisera. Ultrastructural analysis of parasite-infected cells showed that the TcSSP4 protein is present on the surface membrane and in the flagellar pocket of amastigotes. The antibody label was also associated with an amorphous material in the parasitic cavity and in direct contact with the parasite surface (Fig. 5A), which suggest that amastigotes could be releasing this material. Anti-MBP antisera were used as a negative control (Fig. 5B). On free cell

Fig. 4. Reactivity of anti-MBP::SSP4 antibodies with the developmental forms of T. cruzi. Extracts from amastigotes (A), epimastigotes (E), and trypomastigotes (T), were assayed by Western blot with the anti-MBP::SSP4 fusion protein antibodies (1), with the anti-MBP protein antibodies (2), and with preimmune sera (3).

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amastigotes, the antibody heavily decorated the parasite surface as well as intracellular vesicles (Fig. 6). Recently released parasites from LLC-MK2 infected cells were incubated in LIT medium to follow the expression of TcSSP4. As soon as the parasites were released (Fig. 7A) they were stained with the anti-MBP::SSP4 antibody and the fluorescent label was found only in amastigotes (Fig. 7B). This parasite population was centrifugated and the parasitic forms that migrated from the pellet to the supernatant were collected (Fig. 7E) and stained with the same antibody. In this case, only intermediate forms were detected (Fig. 7F). Furthermore, this population was incubated for another 4 h period (Fig. 7C) to induce the transformation of motile trypomastigotes into amastigotes. Also in this condition the anti-MBP::SSP4 antibody recognized the intermediate forms as well as fully transformed amastigotes (Fig. 7D). On the other hand, the reactivity of anti-MBP::SSP4 antibodies with trypomastigote lysate detected by immunobloting (see Fig. 4) could be explained by the copurification of these intermediate forms. 3.5. Human immune response to MBP::SSP4

Fig. 5. Cellular localization of the TcSSP4 antigen. Ultrathin sections of LR Whiteembedded amastigote-infected cells were incubated with: (A) anti-MBP::SSP4 antisera and (B) anti-MBP antisera. Bound antibodies were detected with pig antimouse antibodies conjugated to gold particles. Arrow, released material; Arrow head: amorphous material in the parasitic cavity; N, host cell nucleus; k, kinetoplast; n, parasite nucleus; *, flagellar pocket. Bars; A = 1.0 mm, B = 0.5 mm.

It has been shown that TcSSP4 is shed during the intracellular development of the parasite (Fig. 5). To investigate whether humans develop a humoral immune response during the course of Chagas’ disease against TcSSP4, ELISA assays using MBP::SSP4 as antigen showed that human chagasic sera contain low or negligible levels of anti-MBP::SSP4 antibodies (Fig. 8), compared with antibody levels to MBP::Hsp70 that was used as a control. These results suggest that the polypeptide chain of TcSSP4 is a poor immunogen for human beings. These sera did not show reactivity against the carrier protein MBP used to produce the fusion proteins. In addition, the crossreactivity between the antiMBP::SSP4 antibodies and host’s proteins was investigated. To this purpose, rat’s heart and skeletal muscle proteins were assayed by Western blot with mouse anti-MBP::SSP4 antiserum and human chagasic sera (Fig. 9). Anti-MBP::SSP4 antibodies recognized a 100 kDa and 50–60 kDa proteins from heart and skeletal muscle respectively. On the other hand, human chagasic sera did not recognize any protein in heart extract, but they showed a faint recognition of the 50–60 kDa proteins in skeletal muscle compared with the anti-MBP::SSP4 antibodies reactivity.

Fig. 6. Subcellular localization of TcSSP4 antigen. LR white-embedded extracellularly developed amastigotes were strongly labeled in the membrane with anti-MBP::TcSSP4 antibodies. Label was also detected in cytoplasmic vesicles. Bars, left = 0.5 mm, magnifications = 0.2 mm.

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Fig. 7. TcSSP4 surface membrane expression. (A and B) Recently released parasites, (C and D) released parasites incubated 4 h in LIT medium, and (E and F) purified trypomastigote sample, were fixed with paraformaldehyde and processed for indirect immunoflurescence (B, D, and F) with the anti-MBP::TcSSP4 antibodies. Bound antibodies were detected by incubation with FITC-labeled affinity purified goat antibody to mouse IgG. (A, C, and E) Nomarski differential interference contrast of respective samples.

4. Discussion Transformation of T. cruzi from one stage to another is associated with morphological changes and expression of stagespecific surface molecules. Although several surface proteins of trypomastigotes have been described (Harth et al., 1989; Schenkman et al., 1991; Hall et al., 1992; Ramirez et al., 1993; Fresno et al.,

Fig. 8. ELISA assay of human chagasic sera to MBP::SSP4. Plates were covered with MBP::HSP70, MBP::SSP4 and MBP proteins, and 1:100 diluted human chagasic sera were assayed by the ELISA as described.

1994; Herrera et al., 1994; Giordano et al., 1999) and their role the in host–parasite relationship has been studied (Schenkman et al., 1994; Chuenkova and Pereira, 1995; Gao and Pereira, 2001; Magdesian et al., 2001; Malaga and Yoshida, 2001), relatively few amastigote surface proteins have been investigated (Andrews et al., 1988b; Pan and McMahon-Pratt, 1989; Iida and Ley, 1991; Teixeira et al., 1994; Verbisck et al., 1998; Silva et al., 1998). Amastigote surface proteins are important targets of the host immune response, including lytic antibodies (Lima-Martins et al., 1985; Iida and Ley, 1991), as well as cytotoxic T lymphocyte recognition of parasite-infected cells (Nickell et al., 1993; Garg et al., 1997; Low et al., 1998). To further characterize the role that SSP4 molecules play in the pathogenic mechanisms of T. cruzi infection, we isolated an 84-kDa glycoprotein recognized by the mAb 2C2, which defines stagespecific proteins on the amastigote surface (Andrews et al., 1987). The reactivity of mAb 2C2 toward the 84-kDa glycoprotein was abolished by N-glycanase treatment, indicating that the antibody recognized the Asn-linked carbohydrate moiety. Previous studies have shown that different mAbs recognizing the 84-kDa glycoprotein lose their reactivity upon periodate oxidation (Barros et al., 1997), which suggests that the main antibody immune response against the 84-kDa glycoprotein is directed to its carbohydrate constituent. This could explain why early attempts to clone the gene from expression libraries using antibodies against the glycosylated protein were unsuccessful.

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Fig. 9. Reactivity of anti-MBP::SSP4 antibodies with of rat’s heart and skeletal muscle proteins. Total protein extracts from rat’s heart (A) and muscle (B) were assayed by Western blot with 1:100 dilution of: (1) pool of romal human sera, (2) pool of human chagasic sera, (3) mouse anti-MBP::SSP4 antibodies, (4) mouse antiMBP antibodies, and (5) mouse preimmnune sera.

As a first approach to clone the gene coding for the 84-kDa protein, antibodies against the deglycosylated protein were used to screen a genomic expression library. A highly reactive clone (C2-1) was selected, and Southern blot analysis of digested genomic DNA with the C2-1 DNA insert revealed that this clone is a member of a family of related genes (data not shown). The TcSSP4 cDNA sequence showed high similarity (87%) with a representative gene (6C) of a dispersed repetitive gene family (DRGF) reported previously (Wincker et al., 1990, 1992), and showed two open reading frames. A T. cruzi blast search at The J. Craig Venter Institute (http://blast.jcvi.org/er-blast/index.cgi?project=tca1) with the amino acid sequence derived from frame 2 showed identities from 83% to 89% with probable cell-surface proteins (DGF-1). When the amino acid sequence was derived from frame 1 (this work), identities of 84% and 88% were found in a small stretch of the protein sequence of members of the DGF-1 family and identities of 80–84% were found with small amastigote surface proteins. Experiments to clarify whether the two open reading frames are expressed in the parasite should be conducted. It was shown by Northern blot assay that a single RNA band (10.5 kb) hybridized when a DNA fragment of clone 6C was used as a probe. However, only epimastigote RNA was analyzed; RNA from trypomastigotes and amastigotes was not included in this analysis. The fact that the TcSSP4 cDNA clone was selected from an amastigote cDNA library and the DNA fragment of clone 6C hybridized with RNA from epimastigotes suggests that members of the DRGF are expressed in both amastigotes and epimastigotes. A member of the dispersed gene family may be expressed in amastigotes and another in epimastigotes, or the expression of members of this gene family may be regulated by a posttranscriptional mechanism, as has been shown determined for the amastin mRNA levels in amastigotes (Teixeira et al., 1994, 1995), for the expression of the trans-sialidase and 85-kDa glycoprotein genes in trypomastigotes (Abuin et al., 1999) and for the gp72 and gp85 genes in epimastigotes (Nozaki and Cross, 1995). Thus, the TcSSP4 gene is a member of a dispersed repetitive gene family and it appears that cell surface proteins in T. cruzi are encoded by complex multigene families, such as those coding for an 83-kDa amastigote protein (Low and Tarleton, 1997), SA85-1.1 (Kahn et al., 1990), mucin-like glycoproteins (Salazar et al., 1996; Di Noia et al., 1998), trans-sialidase (Uemura et al., 1992), gp85 (Takle and Cross, 1991), and adenylyl cyclase (Taylor et al., 1999).

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In the present work, immunoblot and immunofluorescence experiments with anti-MBP::SSP4 antibodies showed that a main 66-kDa and a minor 84-kDa protein are expressed only on the amastigote’s surface and that the TcSSP4 gene begins to express early in the transformation process from trypomastigote to amastigote. The R188 antiserum was able to immunoprecipitate only a 66-kDa protein from the in vitro translated products from amastigote mRNA (data not shown). The 66-kDa protein could represent the primary product with no post-transductional modifications, such as glycosylation, and the 84-kDa protein could represent one of the final products. Experiments are being conducted to obtain the complete cDNA for TcSSP4 and to determine the possible glycosylation site(s) within the molecule. Immunoelectron microscopy studies showed that the TcSSP4 protein is present on the surface membrane, in the flagellar pocket, and in internal vesicles of amastigotes (Figs. 5 and 6). The same pattern was observed when mAb 2C2 was assayed for immunocytochemical localization of the SSP4 antigen in both intra- and extracellular amastigotes (Silva et al., 1998). The anti-MBP::SSP4 antibody label was also associated with an amorphous material in the parasitic cavity in direct contact with the parasite surface, which suggests that amastigotes are releasing this material (Fig. 6). The labeling of an amorphous material in the parasitic cavity was also detected with anti-Ssp-3 (trans-sialidase) antibodies, when trypomastigotes shed the antigen shortly after cell invasion (2 h) (Frevert et al., 1992) and started transforming into amastigotes (Tomlinson et al., 1995). This indicates that, in the transformation process of trypomastigote to amastigote, shedding of transsialidase and synthesis of TcSSP4 occurs at the same time. Further studies are required to investigate the signaling pathway involved in the TcSSP4 gene expression and its possible post-transcriptional regulation. The fact that TcSSP4 is shed during extra- or intracellular development allows investigation of the presence of antibodies against this glycoprotein in sera from patients with Chagas’ disease (Andrews et al., 1989). All tested sera recognized the TcSSP4 glycoprotein and human antibodies inhibited the binding of mAb 2C2 in immunoradiometric assays, indicating that human chagasic antibodies also recognize the epitope for mAb 2C2. In this work, we showed that N-glycanase treatment abolished the reactivity of mAb 2C2 toward the 84-kDa glycoprotein, so we investigated whether sera from chagasic patients recognize the recombinant protein MBP::SSP4. These sera contain low or negligible levels of anti-MBP::SSP4 antibodies, relative to antibody levels in response to MBP::Hsp70 which was used as a control (Krautz et al., 1998). This suggests that the main humoral immune response against the shaded TcSSP4 glycoprotein is directed toward the Asn-linked carbohydrate moiety. It has been reported that antibodies against a number of T. cruzi proteins cross-react with host’s proteins (for review see Kierszenbaum, 1999), such as those for the flagellar protein FL-160 (Van Voorhis and Eisen, 1989), the JL5 protein (P ribosomal protein, TcP2b) (Levin et al., 1989), the MAP-like protein (microtubuleassociated protein) (Kerner et al., 1991), and the 140–116 kDa proteins (Cunha-Neto et al., 1995). However, to date, it has been demonstrated that anti-P ribosomal protein antibodies are arrhythmogenic in the setting of a normal heart, thus suggesting a pathogenic role in the development of chagasic disease (Lopez Bergami et al., 2001). Our results show that the sera of chagasic patients did not recognize by immunotransference assay any protein in rat heart extract. Similarly, it has been shown that sera from Argentinean chagasic patients do not contain detectable levels of antibodies capable of binding to cardiac tissue as assayed by immunofluorescence (Baig et al., 1997). In addition, both antiMBP::SSP4 antibodies and human chagasic sera recognize skeletal muscle proteins in the range of 50–60 kDa, although human

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antibodies react weakly with these proteins. It has also been shown that antisera from C57BL/6 mice infected with T. cruzi react with skeletal muscle proteins of 53 kDa (Tibbetts et al., 1994) and 43– 45 kDa (McCormick and Rowland, 1993), respectively, even though the sera also recognize proteins with the same molecular mass in heart extracts. The only antibodies that reacted with a protein in rat heart extract were the anti-MBP::SSP4 antibodies. This reactivity, as well as that shown against skeletal muscle proteins, could be due either to molecular mimicry between the TcSSP4 protein and that of the rat (Davies, 1997) or to the presence of heterophile antibodies (Kaplan and Levinson, 1999) in the anti-MBP::SSP4 serum and chagasic sera. To clarify this reactivity, it will be important to identify the reacted host proteins and to compare their amino acid sequence to that for the TcSSP4 glycoprotein. Acknowledgements We thank Dr. Victor Nussenzweig in whose laboratory at NYU Medical Center we obtained the initial results of this paper. Our thanks also go to Lidia Baylo´n Pacheco and Enrique Martı´nez de Luna for their technical help, Marı´a Guadalupe Aguilar Gonza´lez for the DNA sequencing, and Amelia Rios for manuscript revision. MOL was recipient of a Ph.D. fellowship from Conacyt, Me´xico. This work was supported by funds from the John D. and Catherine T. MacArthur Foundation and Conacyt, Me´xico (grant 211085-53536PM). References Abuin, G., Freitas-Junior, L.H., Colli, W., Alves, M.J., Schenkman, S., 1999. Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors. J. Biol. Chem. 274, 13041–13047. Andrews, N.W., Hong, K.S., Robbins, E.S., Nussenzweig, V., 1987. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp. Parasitol. 64, 474–484. Andrews, N.W., Robbins, E., Ley, V., Nussenzweig, V., 1988a. Stage-specific surface antigens during the morphogenesis of Trypanosoma cruzi: developmentally regulated expression of a glycosyl-phosphatidylinositol anchored glycoprotein of amastigotes. Mem. Inst. Oswaldo Cruz Suppl. 1, 561–562. Andrews, N.W., Robbins, E.S., Ley, V., Hong, K.S., Nussenzweig, V., 1988b. Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi. J. Exp. Med. 167, 300–314. Andrews, N.W., Einstein, M., Nussenzweig, V., 1989. Presence of antibodies to the major surface glycoprotein of Trypanosoma cruzi amastigotes in sera from chagasic patients. Am. J. Trop. Med. Hyg. 40, 46–49. Baig, M.K., Salomone, O., Cafioro, A.L., Caforio, P., Goldman, J.H., Amuchastegui, M., Caiero, T., McKenna, W.J., 1997. Human chagasic disease is not associated with an antiheart humoral response. Am. J. Cardiol. 79, 1135–1137. Barros, H.C., Verbisck, N.V., Da Silva, S., Araguth, M.F., Mortara, R.A., 1997. Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells. J. Euk. Microbiol. 44, 332–344. Chuenkova, M., Pereira, M.E., 1995. Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas’ disease. J. Exp. Med. 181, 1693– 1703. Coughlin, B.C., Teixeira, S.M., Kirchhoff, L.V., Donelson, J.E., 2000. Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 30 -untranslated region position-dependent cis-element and an untranslated region-binding protein. J. Biol. Chem. 275, 12051–12060. Cunha-Neto, E., Duranti, M., Gruber, B., Zingales, B., de Messias, I., Stolf, N., Bellotti, B., Patarroyo, M.E., Pilleggi, F., Kalil, J., 1995. Autoimmunity in Chagas’ disease cardiomyopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen. Proc. Natl. Acad. Sci. U.S.A. 92, 3541–3545. Davies, J.M., 1997. Molecular mimicry: can epitope mimicry induce autoimmune disease? Immunol. Cell. Biol. 75, 113–126. Di Noia, J.M., D’Orso, I., Aslund, L., Sanchez, D.O., Frasch, A.C., 1998. The Trypanosoma cruzi mucin family is transcribed from hundreds of genes having hypervariable regions. J. Biol. Chem. 273, 10843–10850. Fresno, M., Hernandez-Munain, C., de-Diego, J., Rivas, L., Scharfstein, J., Bonay, P., 1994. Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a GPI anchor. Braz. J. Med. Biol. Res. 27, 431–437. Frevert, U., Schenkman, S., Nussenzweig, V., 1992. Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi. Infect. Immun. 60, 2349–2360.

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