2Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil, 3Pós-graduação em Imunologia Básica e Aplicada, Universidade de São Paulo, Ribeirão Preto, SP 14049-900, Brazil, 4Centro de Química de Proteínas, Universidade de São Paulo, Ribeirão Preto, SP 14049-900, Brazil, 5Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP 14049-900, Brazil, 6Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG 38400-902, Brazil, and 7Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP 04044-020, SP, Brazil.
Received on November 19, 2000; revised on February 12, 2001; accepted on March 2, 2001.
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Abstract |
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Key words: Toxoplasma gondii/microneme/MIC1/MIC4/lactose-binding lectin.
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Introduction |
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Host cellparasite interaction is a complex stepwise process involving multiple receptors and complementary ligands on both cell types. Toxoplasma gondii is an obligate intracellular parasite and a highly cosmopolitan protozoan capable of invading and replicating within nucleated cells of warm-blooded animals (Jacobs, 1956). During the invasion process the parasite exhibits gliding motility on the surface of the cell, continually probing it with the conoid, an apical organelle. In response to a still unidentified signal during the probing process, the parasite reorients itself with its apical end attached to the host cell surface. Once the parasite has been oriented, invasion is preceded and accompanied by apical protein release from two secretory organelles of the parasite, that is, micronemes (Achbarou et al., 1991
) and rhoptries (Saffer et al., 1992
). The secretion of micronemal proteins is the first event to be visualized, and it is followed by parasite attachment to the host cell. This is rapidly followed by the discharge of rhoptries from the apical end, which decrease the viscosity of the target cell membrane, initiate its invagination, and enhance invasion (Bonhomme et al., 1999
). There is a great interest in the identification of ligand and receptor in the process of attachment and infection by T. gondii. The broad host range of T. gondii suggests that one or more host cell receptors are fairly common across different cell types or that multiple potential receptors are present.
In the present study we report lectin activity in a protein obtained from soluble T. gondii antigen adsorbed to immobilized lactose and eluted with a lactose solution. This preparation, designated Lac+, presented two major proteins identified as the micronemal proteins MIC1 and MIC4. When the nitrocellulose-transferred Lac+ proteins were incubated with erythrocytes or biotinylated fetuin, MIC1, but not MIC4, bound these ligands. These data demonstrate that MIC1 is a lectin.
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Results |
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The Lac+ fraction was characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) under nonreducing and reducing conditions (Figure 1). Under nonreducing conditions (Figure 1, lane 2), the Lac+ fraction contained one protein of 60 kDa, a double band at
70 kDa, and a protein of
120 kDa. Some less abundant smaller proteins were also observed. Under reducing conditions (Figure 1, lane 3), two major proteins of
63 and
78 kDa were observed.
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Discussion |
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Lectins, because of their property of recognition and their ability to discriminate among sugars, are excellent candidates to mediate interactions of parasites with complementary ligands on host cells. In an Apicomplexa species closely related to T. gondii, Sarcocystis muris, a microneme protein, was characterized as a lectin with affinity for N-acetylgalactosamine (Klein et al., 1998). Although several investigators have suggested the participation of lectin molecules in the host cell invasion by the T. gondii, no lectin from T. gondii has been identified. Some clues to the presence of lectin in the T. gondii have been obtained, although the precise involvement of glycoconjugates or lectins in infection has remained elusive. Incubation of tachyzoites in the presence of gold-labeled albumin-N-acetyl-D-glucosamine or albumin-galactose, but not in the presence of albumin-mannose, led to labeling of the rhoptries (Carvalho et al., 1991
). The interaction between SAG1 and host cell surface can be inhibited by the bovine serum albumin (BSA)glucosamide, suggesting that glycan receptors might be important in infection of cells (Mineo et al., 1993
). Gross et al. (1993)
showed that fetuin bound specifically to a 15-kDa antigen of tachyzoites, a phenomenon inhibited by sialic acid and the lectin Sambucus nigra agglutinin. Monteiro et al. (1998)
demonstrated that mutant cells that present few or no surface-exposed sialic acid residues were infected to a lower extent by T. gondii. Similar results were obtained if sialic acid residues were removed by previous neuraminidase treatment. These investigators observed that the addition of sialic acid residues to surface-exposed glycoconjugates using fetuin as a sialic acid donor and the trans-sialidase of Trypanosoma cruzi rendered the cells more easily infected by T. gondii.
Lac+ has an electrophoretic profile (two main bands of 70 and 60 kDa) similar to the pattern obtained by Ortega-Barria and Boothroyd (1999), who examined electrophoresed T. gondii tachyzoite proteins following transfer to nitrocellulose and their interactions with glutaraldehyde-fixed rabbit erythrocytes. These investigators reported hemagglutination activity in the cytoskeleton, membrane, and total antigen from T. gondii. Although this hemagglutination was inhibited by three sulfated carbohydrates (fucoidan, dextran sulfate, and heparin) or by glycoproteins (fetuin and asialofetuin), no inhibition was observed with lactose or D-galactose, the inhibitory sugars for the hemagglutination reaction triggered by Lac+ in the present study. They reported that sulfated carbohydrates, depending on the concentration used, could also facilitate or block parasite attachment to host cells. In addition, T. gondii shows a decreased ability to bind to cells that are deficient in proteoglycan synthesis. In contrast, we found that heparin did not interfere with Lac+ lectin activity, even when used at concentrations as high as 0.2 mg/ml. It is known that D-galactose in aqueous equilibrium preferentially occurs as the ß-galactopyranoside. Thus, the inhibition of the hemagglutinating activity of Lac+ by lactose and D-galactose, but not by other carbohydrates, including melibiose, is indicative that the lectin contained in the Lac+ preparation preferentially recognizes ß-galactoside. Interestingly, when the nitrocellulose-transferred proteins were incubated with erythrocytes or biotinylated fetuin, MIC1, but not MIC4, bound ligands even when Lac+ was electrophoresed under native conditions. This lectin activity of MIC1 was inhibited by 100 mM lactose, but not by 100 mM mannose (data not shown). Although a variety of glycosaminoglycans may function as receptors for Toxoplasma in the adhesion to the substratum during gliding and for attachment to host cells during invasion (Carruthers et al., 2000a
), glycosaminoglycan-binding proteins from T. gondii were not identified. Our results demonstrate that Lac+ from T. gondii has a lectin activity different from that described by Ortega-Barria and Boothroyd (1999)
and that probably is not a glycosaminoglycan-binding protein.
Our N-terminal data for MIC4 do not correspond to those reported by Brecht et al. (2001). Their amino terminal sequence [X(G/S)E(P/N)(D/A)(K/P)LDLA(P/L)V] starts at residue 14 of the MIC4 sequence obtained in the present study (ITPAGDDVSANVTSSEP). This suggests a difference in the posttranslational processing of the protein. Posttranslational processing of MIC2 has also been reported (Carruthers et al., 2000b
).
We propose that MIC1 is a lectin and that MIC4, complexed to this lectin, may copurify with MIC1 on the lactose-agarose column. Brecht et al. (2001) suggested that MIC4, which lacks a transmembrane or lipid anchor domain, could act as a bridge between an unknown receptor in the parasite and a receptor on the host cell, and that MIC4 forms a complex with MIC1 and MIC6. In this complex MIC1 may be directly and stably associated with MIC4, because the two proteins were coimmunoprecipitated, even in the absence of MIC6. The present demonstration of lectin activity in MIC1, but not MIC4, can partly explain the results obtained by Brecht et al. (2001)
, who observed that increasing concentrations of free galactose had a biphasic effect on MIC4 binding to host cells. Lower concentrations of the carbohydrate enhanced the MIC4 binding to host cell, whereas minimal binding inhibition was observed at higher doses. The role played by MIC1, through its lectin activity, in T. gondii infection is not known.
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Materials and methods |
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Preparation of STAg
Parasites resuspended in PBS containing 0.8 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., St. Louis, MO) were submitted to sonication (Vibra-cell, Sonics & Materials Inc., Danbury, CT) with three pulses of 10 kHz, 50 W, for 4 min each. The soluble proteins recovered by centrifugation at 3000 x g, for 30 min at 4°C, constituted the STAg preparation.
Isolation of lactose-binding proteins
Twenty-milligram portions of STAg were submitted to affinity chromatography on a 5.0 ml -lactose-agarose column (Sigma) previously equilibrated at 4°C with PBS containing 0.5 M NaCl. After washing with equilibrating buffer, the adsorbed material (Lac+) was eluted with 10 ml of 0.1 M lactose in equilibrating buffer, concentrated, and dialyzed against water in an ultradiafiltration system using YM10 membrane (Amicon® Division, W.R. Grace & Co., Beverly, MA).
Assay for hemagglutinating activity
Human blood samples groups A, B, and O were collected into heparinized tubes. Erythrocytes were separated from platelet-rich plasma and the buffy coat by centrifugation at 150 x g for 15 min. They were then pelleted by centrifugation at 350 x g for 15 min, washed three times in PBS, and resuspended in the same buffer at a concentration of 2% (v/v). Hemagglutination assays were performed as previously described (Rüdiger, 1993) using 96-conical well microtiter plates (Nalge Nunc International Co., Naperville, IL). Twenty-five microliters of Lac+ protein (40 µg/ml) were serially diluted (twofold) in PBS, and 25 µl of one of the 2% suspensions of human erythrocytes were added to each diluted sample. After 1 h at room temperature, each well was examined for agglutination. Lectin titer is reported as the reciprocal of the highest dilution of Lac+ protein able to cause detectable agglutination of erythrocytes. One hemagglutinating unit (HU) is defined as the minimum lectin concentration required to provide complete agglutination. Specific hemagglutination activity corresponds to HU per microgram lectin (HU/µg). For inhibition of hemagglutinating activity, 25 µl of a serial dilution of a potential carbohydrate inhibitor (D-glucose, D-mannose, D-galactose, L-fucose, lactose, melibiose, or heparin) was mixed with an equal volume containing 2 HU of Lac+ preparation, for 1 h at room temperature. At the end of this incubation, 25 µl of a 2% suspension of human erythrocytes were added, and the minimum concentration of carbohydrate able to inhibit hemagglutination was recorded after 1 h at room temperature.
PAGE and electroblotting
SDSPAGE was performed with either a 1015% PhastGel gradient using a Phast System (Amersham-Pharmacia Biotech, Uppsala, Sweden) (Figure 1) or a 10% polyacrylamide gel using a Mini V-8.10 Vertical Gel Electrophoresis System (Gibco BRL, Rockville, MD) (Figures 2 and 5). For the PhastGel System, dried samples were suspended in 10 mM TrisHCl buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, with 2.5% (w/v) SDS, and, in some cases, 1% (w/v) dithiothreitol was also added to reduce the samples. After running (250 V, 10 mA), gels were stained with Coomassie blue. Low molecular mass standards from Amersham Pharmacia Biotech were used.
For the 10% gel, samples were suspended in 0.5 M TrisHCl buffer, pH 8.8, with 12% (v/v) glycerol, 2% (w/v) SDS (Laemmli, 1970), and running at 200 V, 80120 mA. Gels were stained as described above.
Native PAGE was performed in a 10% polyacrylamide gel without SDS or reducing agent using a Mini V-8.10 Vertical Gel Electrophoresis System (Gibco BRL) (Figure 6). Samples were suspended in 0.5 M TrisHCl buffer, pH 8.8, with 12% (v/v) glycerol. Running and staining were performed as described above.
In some experiments, after electrophoresis proteins were blotted to nitrocellulose or polyvinylidene difluoride (PVDF) membranes using the Mini V-8.10 Blot System, according to the manufacturers instructions. The PVDF membrane was stained with Coomassie blue, and the bands were cut out for sequencing by Edman degradation. The nitrocellulose membranes were blocked with 3% (w/v) gelatin in PBS for 2 h at 37°C and immunostained with mouse polyclonal antiserum or used to assay binding of erythrocytes or fetuin to electrophoretically separated proteins.
Lectin-binding assays
Erythrocyte binding.
The nitrocellulose membrane containing electrophoresed Lac+ proteins was incubated for 1 h at room temperature in PBS containing 1% (v/v) Triton X-100, followed by 1 h in PBS containing 1% (w/v) BSA. The membrane was incubated with a 1% suspension of human erythrocytes (A, B, or O) in 1% BSA and the suspension allowed to settle for 1 h at room temperature. After gentle washing, the sheet was fixed for 10 min in 3% buffered formalin and rinsed with PBS.
Glycoprotein binding.
The nitrocellulose membrane containing electrophoresed Lac+ proteins was incubated with biotinylated fetuin (14 µg/ml) for 1 h at room temperature, washed three times with PBS, and incubated with streptavidin-peroxidase (Pierce Chemical Co., Rockford, IL) for 30 min at room temperature. The reaction was visualized with diaminobenzidine (DAB) (Sigma) in the presence of 0.03% hydrogen peroxide (Sigma).
Production of mouse polyclonal antiserum
Bands of 70 and 60 kDa from the Lac+ preparation obtained by preparative SDSPAGE under nonreducing conditions were emulsified in Freunds complete adjuvant (Sigma). Mice were injected IP with 10 µg protein. On days 21, 28, and 35 postinoculation, they were reinjected IP with the same amount of antigen emulsified in Freunds incomplete adjuvant (Sigma). After 8 days, the animals were bled, and the titer of specific serum IgG was tested by enzyme-linked immunosorbent assay.
Immunoblotting assay
Nitrocellulose membranes containing electrophoresed and blotted T. gondii proteins were incubated with 1:3000 mouse polyclonal anti-STAg serum, 1:3000 mouse polyclonal anti-60/70-kDa serum, or 1:500 nonimmunized mouse serum for 1 h at room temperature, washed three times with PBS, and developed with 1:1500 peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h at room temperature. The reactions were visualized with DAB (Sigma) in the presence of 0.03% hydrogen peroxide (Sigma).
Screening of a T. gondii cDNA library
A cDNA library from ME49-PDS T. gondii tachyzoites was prepared in ZAP II (Stratagene, La Jolla, CA) and kindly provided by Dr. Ian Manger, Department of Microbiology and Immunology, Stanford University School of Medicine (Stanford, CA). The cDNA library (amplified titer, 4 x 108 pfu/ml) was screened with polyclonal mouse anti-Lac+ preparation antiserum. Clone protein expression was induced using nitrocellulose filters soaked in 10 mM isopropyl-1-thio-ß-D-galactopyranoside. Positive plaques were identified using a 1:4000 dilution mouse anti-Lac+ preparation antiserum, 1:2000 alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Gibco BRL), and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Gibco BRL). Two positive plaques denoted E1 and E7 were submitted to three rounds of purification. The isolated
phage clones were then converted into pBluescript SK () phagemids with the ExAssist helper phage (Stratagene) according to the manufacturers instructions. Double-stranded sequencing of clones was determined using an automatic DNA sequencer (ALFexpress DNA Analysis System, Amersham Pharmacia Biotech) and a Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit (Amersham Pharmacia Biotech). Sequence homologies were obtained by using Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990
).
Protein quantification by amino acid analysis
Amino acid analyses were performed using the phenylthiocarmyl derivative method (Bidlingmeyer et al., 1984) after acid hydrolysis with 6 N HCl containing 1% phenol in the vapor phase, at 110°C for 22 h. A mixture containing 2.5 nmol of each amino acid (Standard H, Pierce Chemical Co., Rockford, IL) was derivatized, and 4% (100 pmol) was injected.
Edman degradation
The N-terminal protein sequences of PVDF-blotted proteins were determined with a Procise sequenator (Procise Microsequencer, model 491, Applied Biosystem, Foster City, CA) using gas-phase chemistry with online identification of phenylthiohydantoin derivatives. The phenylthiohydantoin amino acid standard contained 10 pmol of each derivatized amino acid, and 10 pmol of lactalbumin was used to determine the efficiency of the system.
RP-HPLC
Protein separations were carried out using a Thermo Separation Products system (Riviera Beach, FL) employing two pumps (model constaMetric 3500 and 3200) and a dual wavelength spectromonitor 4200 controlled by the LC-Talk (version 2) software. The proteins retained on the column were eluted with a gradient (10% B for 10 min, 1050% in 10 min, 5075% in 35 min, and 75100% in 5 min) using trifluoroacetic acid (TFA)/acetonitrile (ACN) (solvent A was 0.1% TFA and solvent B was 80% ACN, 20% water, and 0.085% TFA) at 0.2 ml/min flow at room temperature. The column was reequilibrated with 10% B for 20 min. HPLC peaks of the Lac+ fraction were identified by N-terminal sequencing.
Fluorescence confocal microscopy
T. gondii tachyzoites were adsorbed to glass slides to obtain 1020 parasites/field at a magnification of 400x and fixed with 1% (v/v) formaldehyde in PBS for 30 min at room temperature. The slides were washed three times with PBS to remove excess formaldehyde and the parasites were permeabilized with 0.01% (w/v) saponin (Amersham Pharmacia Biotech) in PBS. After incubation with 1% (w/v) BSA in PBS for 30 min at room temperature, slides were then incubated with 1:1000 mouse polyclonal anti-STAg serum, 1:1000 mouse polyclonal anti-60/70-kDa serum, or 1:1000 nonimmunized mouse serum for 1 h at room temperature. They were washed three times with PBS and developed with 1:1500 fluorescein-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h at room temperature. After washing twice in PBS, slides were incubated with propidium iodide (0.5 µg/ml; Sigma) and after three washes with PBS, the slides were mounted in glycerol buffered with 50 mM carbonate, pH 9.5. Samples were imaged on a Leica TCN-NT confocal system, using a 100x N.a 1.4 Plan-Apochromatic (DIC) oil-immersion objective.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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