ARTICLE |
Correspondence to: Annie Bonhomme, INSERM U.314, IFR53, CHU Maison Blanche, 45, rue Cognacq Jay, 51092 Reims Cédex, France..
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Summary |
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Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:14111421, 1998)
Key Words: Toxoplasma gondii, GRA7, dense granules, antigen, parasitophorous vacuolar membrane
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Introduction |
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Toxoplasma gondii, a coccidian protozoan, is an obligate intracellular parasite of vertebrates and an important opportunistic pathogen in humans. T. gondii has a powerful capacity to infect a wide range of cell types, both phagocytic and nonphagocytic cells. Host cell invasion is an active process that leads to parasite internalization in a new membrane-bounded intracellular compartment termed the parasitophorous vacuole (PV). This specialized vacuole prevents acidification (
T. gondii has two main secretory organelles that are involved in the invasion process, the rhoptries and the dense granules (
Dense granule proteins of T. gondii have been described as highly immunogenic molecules that may be involved in humoral immunity (
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Materials and Methods |
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Parasite Culture
Tachyzoites of T. gondii RH strain were maintained by twice-weekly passages of peritoneal fluid into female Swiss mice carrying TG sarcoma cells. Tachyzoites were recovered from the peritoneal cavity 3 days after infection, washed with PBS, pH 7.2, collected by centrifugation, and resuspended (1200 x g, 4 min) at the desired cell density. The tachyzoite population was on the order of 109/ml.
Isolation of T. gondii DNA, RNA, and Protein, and Synthesis of cDNA
A 10-liter secondary suspension culture of HeLa cells infected with the RH strain of T. gondii was grown to a tachyzoite density of approximately 1 x 107/ml and filtered through a 10-µm Millipore (Bedford, MA) Polygard cartridge filter to remove HeLa cells from the tachyzoites. The tachyzoite filtrate obtained contained less than 1% HeLa cells. The tachyzoites were then concentrated by centrifugation, washed, and resuspended in Hank's buffer. The tachyzoite concentrate was then pipetted dropwise into liquid nitrogen and the frozen tachyzoite pellets were recovered and stored at -80C until further use. The tachyzoite pellets were converted to tachyzoite powder by grinding the pellets to a fine powder using a mortar and pestle chilled with dry ice and liquid nitrogen. The tachyzoite powder was subsequently used for the isolation of tachyzoite nucleic acid as described below.
T. gondii DNA was isolated from the tachyzoite powder using the Stratagene DNA extraction kit (La Jolla, CA). The tachyzoite powder was dissolved in Solution 2 and total DNA was isolated according to the kit's protocol. After ethanol precipitation and resuspension of the DNA in TE buffer, undissolved DNA and contaminating polysaccharides were removed by centrifugation at 200,000 x g for 1 hr.
T. gondii RNA was isolated from the tachyzoite powder using the Stratagene RNA isolation kit. The tachyzoite powder was dissolved in Solution D and total RNA was isolated according to the kit's protocol. After ethanol precipitation and resuspension of the RNA in diethyl pyrocarbonate (DEPC)-treated water, polyA+ RNA was selected with an oligo-dT column using a Pharmacia mRNA isolation kit (Piscataway, NJ). The purified mRNA was concentrated by ethanol precipitation and stored in DEPCwater at -80C until further use.
T. gondii protein was isolated from filtered and concentrated tachyzoites. Approximately 2.4 x 108 tachyzoites were dissolved in 1 ml sample buffer (1% glycerol, 1.5% SDS, 125 mM Tris-HCl, pH 6.8, 0.05% bromophenol blue) and boiled for 5 min. The final protein concentration was approximately 1 mg/ml. The protein preparation was stored at -20C until further use.
Purified T. gondii mRNA was used as a template for the synthesis of cDNA using the ZAP-cDNA Synthesis kit (Stratagene). The cDNA was then ethanol-precipitated, resuspended in water, and stored at -20C until further use as a template for construction of a Toxoplasma cDNA library.
Cloning and Expression of the Gene Encoding the Novel Antigen
Standard methods were used for isolation and analysis of plasmid DNA, DNA sequence analysis, and Southern analysis (
The 1.3-KB gene fragment containing the novel antigen was cloned into an E. coli CMP-2-keto-3-deoxyoctulosonic acid synthetase (CKS) expression vector as described previously (
Cloning and Expression of Other Toxoplasma Antigens
Using the published DNA sequences, PCR primers were generated to clone the genes encoding the following Toxoplasma antigens previously described in the literature into the CKS expression vector (
Generation of the 5-241-178 Mouse Monoclonal Antibody Directed Against the Novel Antigen
Swiss mice (Charles River Laboratories; Wilmington, MA) were infected intraperitonally with 2.5 x 107 tachyzoites of T. gondii strain TS4 (American Type Tissue Collection; Rockville, MD). Five days later the mice were treated orally with 10 mg pyrimethamine and 200 mg sulfamethoxazole per kg daily for 10 days. After 12 additional weeks, the mice were injected i.v. with 1.2 x 107 sonicated tachyzoites 3 days before fusion. Resulting hybrids from the PEG-mediated fusion of splenocytes and the SP2/0 myeloma cells were screened with sonicated tachyzoites, sonicated E. coli lysates containing the CKSnovel antigen, CKS-P22, P24, P25, P28, P30, P35, P41, P54, P66, and P68 CKS fusion proteins and unfused CKS using standard procedures (
Gel Electrophoresis and Western Blot Analysis
Approximately 10 mg of Toxoplasma lysate and 10 mg E. coli extract containing the CKSnovel antigen fusion protein were loaded on a 420% gradient SDS-polyacrylamide gel in the presence of ß-mercaptoethanol using a Daiichi precast gel (Integrated Separation Systems; Natick, MA). Prestained protein molecular weight markers (10618.5 kD) were included on the gel for molecular weight determination (BioRad; Richmond, CA). After electrophoresis, the proteins were transferred to nitrocellulose (Schleicher & Schuell; Keene, NH) using the ISS SemiDry Electroblotter and reagents (Integrated Separation Systems). After transfer, the membrane was saturated overnight with membrane blocking solution [1% bovine serum albumin (BSA), 1% casein acid hydrolysate, 0.05% Tween-20 in Tris-buffered saline (TBS; 20 mM Tris, 0.5 M NaCl, pH 7.5)] at room temperature (RT). The next day the membrane was briefly rinsed with water and immersed in monoclonal antibody diluent (100 mM Tris, 135 mM NaCl, 10 mM EDTA, 0.2% Tween-20, 0.01% thimerosal, 4% bovine calf serum, pH 7.5). The 5-241-178 monoclonal antibody was diluted into monoclonal antibody diluent, added to the membrane, and then incubated for 1 hr at RT. The membrane was washed once with water and twice with TBS and then immersed in conjugate diluent (100 mM Tris, 135 mM NaCl, 0.01% thimerosal, 10% bovine calf serum, pH 7.5). The goat anti-mouse IgGhorseradish peroxidase conjugate (Kirkegaard & Perry Laboratories) was diluted into conjugate diluent (1:1,000), added to the membrane, and then incubated for 1 hr at RT. The membrane was washed with water and twice with TBSTween-20 and then developed using BioRad HRP Color Development Reagent until the desired band intensity was achieved. Color development was stopped by rinsing the membrane with water.
Immunocytochemistry
After two washes with PBS, pH 7.2, freshly isolated tachyzoites were fixed for 2 hr with 2% paraformaldehyde, 0.5% glutaraldehyde in PBS (pH 7.2) at 4C. Tachyzoites were washed with PBS, then pre-embedded in BSA (diluted 1:4) and glutaraldehyde (25%) in the ratio 4:1, dehydrated with increasing concentrations of ethanol, and embedded in LR White resin. Ultrathin sections (thickness 80 nm) placed on gold grids were immunolabeled as follows. They were incubated first with PBS1% BSA, pH 7.2, for 30 min at RT and then with the 5-241-178 monoclonal antibody, diluted 1:10,000 in PBS1% BSA for 1 hr at RT. After three washes with PBS1% BSA, thin sections were incubated for 1 hr with goat anti-mouse IgGgold (10 nm) (Biocell; Tebu, France) diluted 1:50 for 1 hr at RT. After two washes in PBS and distilled water, the sections were contrasted with uranyl acetate. Controls without monoclonal antibody were prepared and observed.
Quantitative Immunolabeling Analysis
Conditions for the preparation of samples were designed at the same time to preserve the ultrastructure, to identify labeled organelles, and to retain immunoreactivity. The monoclonal antibody was tested at increasing concentrations up to optimal immunolabeling response, i.e., when the ratio of total signal to nonspecific signal was the highest possible. Under these conditions, we could carry out immunolabeling assays quantitatively, the antibody being bound to almost all accessible epitopes of the antigen. Thus, we could compare the distribution of the antigens quantitatively by evaluating the immunolabeling density (number of gold particles/µm2).
Quantitation was performed with an image analyzer (Bio 500; Biocom, Les Ulis, France) using transmission electron micrographs of ultrathin sections and a semiautomatic quantitative method (
The average number of gold particles/µm2 was calculated by subtracting the background determined in the resin (LR White) from the labeling density of the experimental specimens. Quantitation was determined on 13 negatives for extracellular tachyzoites and 20 negatives for intracellular tachyzoites on 50 observed negatives. Only those having a good contrast were taken into account.
State 1 and State 2 of the intracellular state of the tachyzoites are discriminated from the parasitophorous vacuole membrane (PVM) labeling, PVM being the second target after the reticular network of the dense granule exocytosis.
The quantitative evaluation of the immunolabeling is expressed by averages with the standard deviation (SD). To define the significant difference in the immunolabeling among the different states (extracellular, State 1, and State 2), among five different compartments (DG, dense granules; R, rhoptries; MC, membrane complex; RN, reticular network; PVM, parasitophorous vacuole membrane), and among the different compartments during the different states, we used the U-test of
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Results |
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Identification of the Novel Antigen as the P29 Antigen of T. gondii
The 5-241-178 monoclonal antibody was used to probe a Western blot containing total Toxoplasma protein and the recombinant CKSnovel antigen fusion protein, which is shown in Figure 1. The 5-241-178 monoclonal antibody reacted specifically with a 29-kD protein present in Toxoplasma lysate (Figure 1, Lane 2), thus identifying the novel antigen as the P29 antigen of T. gondii. The monoclonal antibody also reacted with the recombinant fusion protein (Figure 1, Lane 3) which was used to isolate the monoclonal antibody during hybridoma screening. The 5-241-178 monoclonal antibody did not react with CKS recombinant proteins containing the SAG1, SAG2, GRA1, GRA2, GRA4, ROP1, or ROP2 proteins (data not shown). The possibility that this monoclonal antibody reacts with the one of the other three dense granule proteins not tested (GRA 3, GRA5, and GRA6) is extremely remote, for the following two reasons. First, the 5-241-178 monoclonal antibody reacts with a single protein of 29 kD. The dense granule proteins GRA3 (30 kD), GRA5 (21 kD), and GRA6 (32 kD) (
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The distribution of the labeling density of the P29 antigen using the monoclonal antibody 5-241-178 examined in the five compartmentsdense granules, membrane complex, rhoptries, reticular network of the parasitophorous vacuole, and the parasitophorous vacuole membraneis shown in Figure 2 Figure 3 Figure 4. The results of the quantitative estimation of the immunolabeling are given in Table 1.
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Immunolocalization of the P29 antigen in extracellular tachyzoites demonstrated that the P29 antigen was present inside the dense granules. The P29 antigen was found to be about 25 times more abundant in the dense granules than in either the rhoptries or membrane complex. With the nonparametric U-test of
Immunolocalization of the P29 antigen in intracellular tachyzoites as shown in Figure 3 and Figure 4 and in Table 1 demonstrated that there was a differential distribution of the P29 antigen and, from all the negatives observed, we divided the intracellular state of the parasite into two states. In State 1, the P29 antigen present in the tachyzoite dense granules (DG), as in the extracellular state, was also redistributed principally in the membrane complex (MC) of the tachyzoite and in the reticular network (RN) inside the parasitophorous vacuole. In State 2, the P29 antigen distribution was extended and was found in association with the parasitophorous vacuole membrane (PVM). The quantitative P29 immunolabeling repartition is shown on Table 1. The dense granule labeling differences were not significant between the extracellular state and State 1 but were very significant between the extracellular state and State 2 (p<0.01) (the immunolabeling decreases from 1092 ± 280 to 494 ± 290) and were also significant between State 1 and State 2. The immunolabeling of the membrane complex increases from the extracellular state to State 1 and the observed labeling difference was significant (p<0.01). The immunolabeling of the PVM from State 1 to State 2 significantly increases from 23.8 ± 19.2 to 81.5 ± 47.5 (p<0.01), suggesting an inverse immunolabeling relationship between the DG and PVM from State 1 to State 2. There was probably a flux of P29 antigen secretion via the membrane complex from dense granules to the PVM once tachyzoites were inside the host cell. We observed a decrease in the immunolabeling of the membrane complex from State 1 to State 2 but the labeling difference was not significant. There were no significant immunolabeling differences in the RN between States 1 and 2. Outward extension of immunolabeling was observed from the parasitophorous vacuole into the host cell cytoplasm and plasma membrane (Figure 4). In necrosed cells (nucleus corpus observed was typical of dying cells), tachyzoites were seen inside the nucleus. Immunolabeling was always present in the dense granules and also in the nucleus (Figure 2B).
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Discussion |
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Dense granule organelles of protozoan parasites Sarcocystis (
Dense granule secretion has all the hallmark features of regulated exocytosis, including storage of se-cretory proteins in specialized organelles, fusion with the plasma membrane, and release of their contents. This burst of exocytosis occurs soon after internalization. Dense granule antigens, termed GRA proteins, are differentially targeted to either the vacuolar space, the network, or the surrounding membrane of the parasitophorous vacuole.
Sequencing of six genes encoding dense granule proteins has shown typical hydrophobic signal sequences that target them in the secretory pathway. Except for the canonical signal peptide at the N-terminus, their structural features are different and may be related to their differential distribution in the parasitophorous vacuole (
Immunolabeling of tachyzoites with the new monoclonal antibody 5-241-178, which binds specifically to the P29 antigen of T. gondii, intensely stained the electron-dense granules and characterized a new dense granule protein of 29 kD termed GRA7. Ultrastructural immunolocalization indicates that after invasion of the host cell by the tachyzoite, the P29 antigen (GRA7) was found associated with the parasite membrane complex and with the tubular elements of the intravacuolar network (State 1) (Figure 3; Table 1). Furthermore, this antigen was also incorporated into the membrane of the parasitophorous vacuole (State 2) (Figure 4). Electron micrographs suggest that the intravacuolar network of membranous tubules is connected in localized regions to the vacuolar membrane. Our immunolabeling data shows trafficking of the P29 antigen from the dense granules via the intravacuolar network to the PVM. We also observed an outward expansion of the P29 antigen from the vacuole into the host cell cytoplasm and into the cell plasma membrane.
Analysis of the primary amino acid sequence determined from the DNA sequence of the gene encoding the P29 (GRA7) antigen reveals several interesting structural features consistent with this antigen being a member of the GRA family. First, the P29 antigen contains a canonical signal peptide at the N-terminus to permit the protein to enter the secretory pathway. Second, this antigen contains two hydrophobic domains of 15 amino acids and 29 amino acids. The larger hydrophobic domain has a high degree of -helical character and is probably the transmembrane region that anchors the protein to the parasitophorous membrane. Third, this antigen contains a potential N-linked glycosylation site just downstream of this transmembrane region, which may also be important in targeting this protein to the membrane. Fourth, no amino acid homology exists between GRA7 and the other GRA proteins. The structural features of the P29 (GRA7) antigen are similar to those of the GRA4, GRA5, and GRA6 proteins in that all of these proteins contain amino acid sequences that fulfill the requirement of transmembrane regions (
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Note Added in Proof |
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During review of this work, the same gene and protein, and localization of this protein to the dense granules, have been described by