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Article |
Address correspondence to Con Beckers, Dept. of Cell and Developmental Biology, University of North Carolina at Chapel Hill, 108 Taylor Hall, CB# 7090, Chapel Hill, NC 27599. Tel.: (919) 966-1464. Fax: (919) 966-1856. email: cbeckers{at}med.unc.edu
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Abstract |
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Key Words: Toxoplasma; Plasmodium; class XIV myosin; motility; apicomplexa
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Introduction |
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The cell wall or pellicle of apicomplexan parasites consists of the plasma membrane and the closely associated, flattened cisternae of the inner membrane complex. Both actin and the myosin-A homologues have been localized to the space between the plasma membrane and the inner membrane complex of Toxoplasma and Plasmodium (Dobrowolski et al., 1997; Pinder et al., 1998). In Toxoplasma, actin filaments appear to be associated with the plasma membrane of the parasite through an interaction with the cytoplasmic tail of the transmembrane adhesin MIC2, which in turn may be mediated by the glycolytic enzyme aldolase (Jewett and Sibley, 2003). TgMyoA was originally also believed to be associated with the plasma membrane (Dobrowolski et al., 1997). However, more recent evidence in Plasmodium yoelii suggests that its myosin-A is associated with the inner membrane complex, as judged by the localization of MTIP, a myosin light chain-like protein that interacts with the myosin-A tail (Bergman et al., 2003).
The manner in which the apicomplexan myosins associate with membranes is not known. A dibasic motif in the carboxy terminus of TgMyoA has been found to be essential for localization to the pellicle and has been proposed to mediate association with membranes (Hettmann et al., 2000). As TgMyoA is easily extracted using high pH, it appears to be a peripheral membrane protein (Hettmann et al., 2000). However, it is not known whether its membrane association is mediated by an integral membrane protein or through a direct interaction with phospholipid head groups, in a manner analogous to that proposed for myosin IC and Iß (Doberstein and Pollard, 1992; Reizes et al., 1994). In the case of two class V myosins, Myo2p in Saccharomyces cerevisiae and myosin-Va in melanocytes, specific receptors have been identified on the membranes of cargo organelles, the yeast vacuole and melanosome, respectively. In the melanocyte, myosin-Va is linked by melanophilin to Rab27a that is, in turn, associated with the melanosome membrane through two geranylgeranyl moieties at its carboxy terminus (Wu et al., 2002a,b). In S. cerevisiae, Myo2p is linked by Vac17p to Vac8p that is associated with the vacuole membrane through an N-myristoyl and multiple palmitoyl groups at its amino terminus (Ishikawa et al., 2003).
Here, we show that the two known components of the Toxoplasma glideosome, TgMyoA and TgMLC1, are associated with two novel proteins, TgGAP45 and TgGAP50. Although the function of TgGAP45 is not clear at this time, TgGAP50 is an integral membrane glycoprotein that anchors the glideosome in the inner membrane complex of T. gondii, and thus performs a critical function in parasite motility.
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Results |
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The antiserum to one of the clones detected with antisera to the DOC-soluble fraction labels the periphery of parasites in permeabilized (Fig. 1 A, GAP45) but not intact parasites (unpublished data), suggesting it is associated with a cytoplasmic aspect of the Toxoplasma pellicle. However, the staining pattern of this protein, TgGAP45, does not overlap precisely with that of the plasma membrane marker SAG1. Specifically, discontinuities are observed at the anterior end of the parasite, suggesting the protein is associated with the inner membrane complex rather than the plasma membrane. To confirm this, immunofluorescence was performed on parasites treated with Clostridium septicum -toxin, which causes their plasma membrane to swell away from the inner membrane complex (Wichroski et al., 2002). After toxin treatment, the staining patterns for TgGAP45 and SAG1 are clearly distinct (Fig. 1 A), also suggesting that the protein is present in the inner membrane complex of the parasite rather than the plasma membrane.
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Sequence analysis of full-length clones of TgGAP45 revealed a single ORF of 735 bp, predicted to encode a protein of 245 amino acids with a molecular mass of 27.3 kD (Fig. 1 C). Comparison of the predicted amino acid sequence to those of proteins with known function revealed no strong homologies. However, we did find orthologues in databases of genomic and EST sequences of the related parasites Neospora caninum, Plasmodium falciparum, Plasmodium yoelii, and Cryptosporidium parvum. The predicted amino acid sequences of these proteins demonstrate a high degree of sequence homology at their carboxy termini (Fig. 1 C). Further analysis of the predicted amino acid sequences of all orthologues revealed regions with a high probability to form a coiled-coil structure (unpublished data) and a potential N-myristoylation site (Fig. 1 C).
When analyzed by immunoblot analysis, the TgGAP45 antiserum reacted with a single protein in Toxoplasma lysates with an apparent molecular mass of 45 kD (Fig. 2 A), larger than the predicted molecular mass of 27.3 kD. As recombinant fusion proteins containing the putative coiled-coil domain (but not the remainder of the protein) display a similar anomalous migration behavior during SDS-PAGE, we believe this to be due to an elongated structure or the high content of charged residues (unpublished data).
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Considering the fact that TgGAP45 is found in the Toxoplasma pellicle and that the associated 93- and 32-kD proteins are similar in size to the pellicle-associated myosin-A (TgMyoA) and its associated light chain (TgMLC1), we analyzed the immunoprecipitates with antisera to both proteins. As is shown in Fig. 2 C, the 93- and 32-kD proteins do indeed react with antisera to TgMyoA and TgMLC1, respectively. All TgMyoA and TgMLC1 in the parasite is apparently associated with TgGAP45, as judged by immunoblot analysis of parasite extracts after immunodepletion of TgGAP45 (unpublished data). However, the 50-kD protein does not react with any of the antisera used, suggesting it represents a novel protein, and is henceforth referred to as TgGAP50.
As the complex of TgMyoA and TgMLC1 has been demonstrated to participate in the gliding motility of Toxoplasma and other apicomplexan parasites, it has been named the glideosome (Opitz and Soldati, 2002). Based on their association with this complex and their apparent molecular mass, the two novel proteins will therefore be referred to as gliding-associated protein (GAP) 45 and 50, or TgGAP45 and TgGAP50.
To determine the relative composition of this complex, we labeled parasite proteins to steady state with [35S]-labeled methionine and cysteine and quantitated the amount of each subunit by phosphorimaging analysis, correcting for the number of methionine and cysteine residues in the predicted sequence of each protein. TgMyoA, TgGAP50, TgGAP45, and TgMLC1 were found to be present in a ratio of 1:1.42 (±0.21):1.31 (±0.21):1.38 (±0.21) (n = 2, ± SD), respectively. Although these data are consistent with the glideosome being a heterotetrameric complex of one copy of each protein, further analysis is needed to confirm this.
TgGAP50 is an integral membrane glycoprotein of the inner membrane complex
TgGAP50 was purified by preparative two-dimensional gel electrophoresis, and tryptic digests were analyzed by mass spectroscopy. The amino acid sequences of four tryptic fragments were obtained and used to identify a candidate gene in the database of Toxoplasma genomic DNA. The complete ORF was subsequently identified by RT-PCR analysis and isolation of a full-length cDNA clone. The TgGAP50 ORF predicts a 431-residue protein with a predicted molecular mass of 46.6 kD. Analysis of the predicted amino acid sequence reveals the presence of putative transmembrane domains at the amino terminus (residues 2545) and the extreme carboxy terminus (residues 402426), suggesting that TgGAP50 is an integral membrane protein (Fig. 3).
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The amino-terminal transmembrane domain of TgGAP50 appears to act as a cleavable signal peptide, as direct amino acid sequencing of purified protein reveals that the amino terminus of the mature protein corresponds to residue 51 in the predicted sequence. As the predicted molecular mass of the mature protein was smaller than that observed in SDS-PAGE and as the TgGAP50 sequence is predicted to have three N-linked glycosylation sites (Fig. 3), we subjected immunoprecipitates to digestion with the endoglycosidase PNGase-F. As can be seen in Fig. 4, this results in a substantial decrease in molecular mass of TgGAP50 to a value close to the one predicted for the mature protein. This result demonstrates that TgGAP50 is indeed N-glycosylated in Toxoplasma. The decrease in molecular mass is consistent with the removal of about three N-linked glycans, suggesting that all three predicted glycosylation sites are indeed used.
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The glideosome is assembled in two stages
Immunofluorescence analysis of Toxoplasma expressing the TgGAP50-YFP fusion protein reveals a distinct difference in the localization of the different components of the glideosome. TgGAP50 is found in the inner membrane complex of both mature parasites and immature daughters, as judged by its colocalization with the marker TgIMC1 (Fig. 5). In contrast, TgMyoA, TgMLC1, and TgGAP45 are only found associated with the inner membrane complex of mature parasites and are entirely absent from immature daughters (Fig. 5). This observation suggests that the glideosome may be assembled in multiple stages during cell division in T. gondii.
To test this hypothesis, we used pulse-chase analysis and immunoprecipitation with TgGAP45 antiserum to determine if there were any changes in glideosome composition over time. As can be seen in Fig. 7 A, a complex containing TgMyoA, TgMLC1, and TgGAP45 can be isolated after a 15-min pulse labeling, but TgGAP50 is absent. In contrast, after a 4-h chase, all glideosome subunits, including TgGAP50, are present in the complex. Together with the data in Fig. 5, these observations demonstrate that the glideosome complex is assembled in two stages. During or shortly after their synthesis, the three glideosome subunits synthesized on cytoplasmic ribosomes, TgMyoA, TgMLC1, and TgGAP45, associate with each other into a complex, the proto-glideosome. TgGAP50, on the other hand, is cotranslationally inserted into the parasite ER and is transported to the inner membrane complex where, in mature parasites, it associates with the TgMyoA/TgGAP45/TgMLC1 proto-glideosome to form the functional, membrane-associated glideosome.
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Because the association of the proto-glideosome with TgGAP50 most likely occurs through the latter's short carboxy-terminal cytoplasmic domain, we generated a mutant TgGAP50-YFP fusion protein, TgGAP50(427-431)YFP, in which this entire domain was deleted. Although expression of the full-length TgGAP50-YFP is not deleterious to the parasite and stable transfectants can be obtained with ease, we were unable to obtain stably transfected parasites expressing TgGAP50
(427-431)YFP. In fact, at 48 h after transfection 2040% of parasites expressed normal TgGAP50-YFP, but no parasites expressing the mutant protein were observed at that time. This observation indicates that expression of TgGAP50
(427-431)YFP exerts a dominant lethal effect on Toxoplasma.
To determine whether this was due to an effect on assembly of the glideosome, Toxoplasma transiently expressing this construct were metabolically labeled 24 h after transfection and subjected to immunoprecipitation with antisera to TgGAP45 or GFP. Unlike full-length TgGAP50-YFP, TgGAP50(427-431)YFP does not associate with the other glideosome subunits (Fig. 6 B), suggesting that the carboxy-terminal cytoplasmic domain is indeed essential for that process.
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Discussion |
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The role of TgGAP45 is not clear at this time, although the failure to obtain viable parasites with a disrupted TgGAP45 gene using various strategies (Kim et al., 1993; Donald and Roos, 1994, 1998; Meissner et al., 2002) suggests it is essential (unpublished data). Expression of various TgGAP45 mutants under the control of inducible (Meissner et al., 2001) and constitutive promoters have thus failed to affect normal glideosome assembly or parasite infectivity, possibly due to the low level of expression (<23% of the wild-type protein) typically observed (unpublished data). TgGAP50 is an essential protein by the same criteria (unpublished data). Biochemical and genetic analysis of TgGAP50 suggests that this protein serves to anchor the complex of TgMyoA, TgMLC1, and TgGAP45 in the membrane of the inner membrane complex that faces the parasite plasma membrane.
Conflicting observations have been published on the localization of apicomplexan myosin-A homologues. TgMyoA was originally believed to be associated with the plasma membrane (Dobrowolski et al., 1997), although the same group has since suggested these observations may have been erroneous (Jewett and Sibley, 2003). Recent evidence in P. yoelii suggests that its myosin-A is associated with the inner membrane complex (Bergman et al., 2003). Our observations support the latter model in that the complex of TgMyoA, TgMLC1, TgGAP45, and TgGAP50 appears to be associated with the inner membrane complex of Toxoplasma rather than the plasma membrane.
All apicomplexan parasites for which sequence data are available possess highly similar orthologues of TgGAP45 and TgGAP50, suggesting that their function is conserved throughout the entire phylum. The apicomplexan TgGAP45 proteins all possess amino-terminal myristoylation motifs and a highly conserved carboxy-terminal domain, separated by poorly conserved domains that share a high propensity to form coiled coils. The apicomplexan GAP50 proteins also demonstrate a high degree of sequence similarity, except in their amino-terminal signal sequences. The predicted lumenal, cytoplasmic, and even transmembrane domains are highly conserved amongst these proteins. The lumenal domains of the apicomplexan GAP50 proteins are also surprisingly similar to the purple acid or tartrate-resistant phosphatases, a family of secreted enzymes found in animals and plants (Oddie et al., 2000; Schenk et al., 2000). However, the amino acid residues critical to phosphatase activity (Oddie et al., 2000) are mostly not conserved in the apicomplexan GAP50 proteins, suggesting they are not phosphatases. This notion is supported by our inability to detect phosphatase activity in preparations of recombinant TgGAP50 (unpublished data). However, the high degree of similarity does suggest that there may be substantial similarities in the secondary and tertiary structure of the lumenal domains of apicomplexan GAP50 proteins and the purple acid phosphatases. The high degree of sequence identity observed in the transmembrane domains of the GAP50 proteins is surprising and may indicate that this domain interacts in a specific manner with other membrane-embedded components of the inner membrane complex. The near identity of the short carboxy-terminal cytoplasmic domains of the GAP50 proteins probably reflects its role in the interaction with other members of the glideosome complex (see below).
The glideosome complex is assembled in two stages. Pulse-chase analysis and subcellular fraction revealed that TgMyoA, TgMLC1, and TgGAP45 are first assembled into a soluble complex, the proto-glideosome. Subsequently, this complex associates with the integral membrane protein TgGAP50 and becomes anchored in the membrane, forming the glideosome proper. The two-stage assembly of the glideosome is also reflected in the subcellular distribution of the different subunits. TgGAP50 is found in the inner membrane complex of immature daughter parasites as well as mature parasites. However, The proto-glideosome subunits TgGAP45, TgMyoA, and TgMLC1 are only found associated with the inner membrane complex of the mature parasites. This suggests that the soluble proto-glideosome and membrane-associated TgGAP50 are transported separately from their site of synthesis in the cytoplasm and ER, respectively, and are assembled into the glideosome in the inner membrane complex of mature parasites.
The proto-glideosome most likely associates with the conserved carboxy-terminal cytoplasmic domain of TgGAP50, as the interaction is prevented by the deletion of this domain. It is not known at this time which glideosome subunit interacts with TgGAP50 and the manner in which this occurs. Although TgMyoA and TgMLC1 could be shown to interact after in vitro translation (unpublished data), we were unsuccessful in our attempts to detect interaction of TgGAP45 and TgGAP50 with the other glideosome subunits using in vitrotranslated proteins and recombinant proteins (unpublished data). This suggests that one or more of the subunits may need to undergo cell cycle or parasite-specific post-translational modifications for complex assembly to occur. These modifications may, in fact, be critical for controlling the two-stage assembly of the glideosome we observed in living parasites.
The assembly of the glideosome in two stages may serve several purposes for Toxoplasma. Most likely, it limits assembly of the fully active glideosome to the location where it is needed, the outer face of the inner membrane complex of the mature parasite. However, it is also possible that the proto-glideosome association with the cytoplasmic tail of TgGAP50 is reversible, and thus offers a mechanism for control of parasite motility.
In our working model of Toxoplasma motility, glideosomes on the inner membrane complex interact with actin filaments that are associated with the adhesin MIC2 in the parasite plasma membrane. This arrangement would allow the glideosome to move with respect to the MIC2 anchored on a host cell or other substrate. However, in order for this to result in parasite motility it is critical that the glideosome is not only attached to the inner membrane complex, but also that it is immobilized within the plane of the membrane. This could be accomplished most easily by direct or indirect interaction of the glideosome with stable elements of the Toxoplasma cytoskeleton. Such structures have not been described on the side of the inner membrane complex that faces the plasma membrane. However, on the cytoplasmic side a dense fibrillar network forms a membrane skeleton along the length of the parasite (Mann and Beckers, 2001). In addition, 22 microtubules are present on that side, although these extend only partly along the length of the parasite (Nichols and Chiappino, 1987). As the inner membrane complex consists of two membranes, association of the glideosome with either cytoskeletal element would require the presence of structures that span both membranes. Freeze fracture analysis of the inner membrane complex has in fact revealed candidates that could fulfill this function in the form of large numbers of intramembranous particles present in both membranes and distributed in a manner suggesting they are associated with both the membrane skeleton and the microtubules (Morrissette et al., 1997). Experiments are in progress addressing, in general, the immobilization of the glideosome and specifically its association with the intramembranous particles. The possibility that TgGAP50 may, in fact, interact directly or indirectly with the cytoskeleton is suggested by the observation that expression of a mutant TgGAP50 lacking its cytoplasmic domain is lethal for Toxoplasma. As expression of this protein does not disrupt assembly of the glideosome on endogenous TgGAP50, its lethality may be explained by a dominant disruption of the glideosomecytoskeleton interaction.
The identification in T. gondii of TgGAP50 as the receptor for a complex of TgMyoA, TgMLC1, and TgGAP45 in the inner membrane complex provides the first evidence that the class XIV myosins in apicomplexan parasites associate with membranes through interaction with a transmembrane protein. Further analyses are needed to determine how the different subunits interact with each other and how complex assembly is controlled in the parasite. Moreover, identification of the manner in which TgGAP50, and therefore the glideosome as a whole, is immobilized in the plane of the inner membrane complex is of critical importance in understanding the mechanism of gliding motility in Toxoplasma and the other apicomplexan parasites.
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Materials and methods |
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Library screening with anti-pellicle antisera
A T. gondii cDNA library in ZAPII (AIDS Research and Reference Reagent Program, McKesson Biosciences, Rockville, MD) was screened using antisera to pellicle proteins with the ProtoBlot® Immunoscreening System (Promega). Positive clones were initially grouped based on restriction enzyme digestion patterns, and a single representative of each group was sequenced (Keck Biotechnology Resource Laboratory, Yale University, New Haven, CT).
Preparation of monospecific antisera to recombinant TgGAP45
For production of monospecific antisera to TgGAP45, a BamHI and XhoI fragment containing the carboxy-terminal 72 amino acids of the predicted ORF was inserted in-frame behind GST in pGEX2 (Amersham Biosciences). GST-GAP45 fusion protein was expressed in Escherichia coli JM109 and purified on glutathione-agarose (Sigma-Aldrich). The fusion protein was used to immunize mice and rabbits (Cocalico Biologicals, Inc.).
Metabolic labeling of T. gondii
Parasites were allowed to invade a monolayer of human foreskin fibroblast (HFF) cells. After 1416 h, cells were incubated in methionine/cysteine-free medium (Mediatech) for 1 h before addition of 0.1 mCi [35S]-labeled methionine/cysteine (Amersham Biosciences) per ml medium. Parasites were harvested on ice after a 2024-h incubation at 37°C.
To perform pulse-chase experiments, HFF cells grown in multiple flasks were infected and starved in methionine/cysteine-free medium as described above. To each flask, 0.25 mCi [35S]-labeled methionine/cysteine was added per ml of label medium. After 15 min at 37°C, one flask (pulse) was placed on ice. Unlabeled methionine and cysteine were added to the other flasks to final concentrations of 1 and 0.2 mM, respectively, and these were incubated at 37°C for the time indicated.
Immunoprecipitation of the glideosome
Parasites (510 x 107/ml) were lysed with 1% TX100 or 1% SDS in the presence of protease inhibitors (P8340; Sigma-Aldrich). For TX100 lysis, cells were resuspended in IP buffer (1% TX100, 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 5 mM EDTA). For SDS lysis, cells were resuspended in 100 µl 1% SDS in water plus protease inhibitors and heated for 5 min at 95°C, followed by ninefold dilution in IP buffer. SDS- or TX100-lysed cells were incubated on ice for 10 min, followed by centrifugation at 14,000 rpm for 10 min at 4°C. The supernatant was incubated at 4°C for 1 h with anti-GAP45 or anti-GFP antisera. Protein ASepharose (Zymed Laboratories) was added and the incubation was continued for 30 min at 4°C. Immune complexes were washed three times in IP buffer and separated by SDS-PAGE. Gels containing radiolabeled immune complexes were subjected to fluorography using Enhance (NEN Life Science Products), dried, and exposed to film.
PNGase-F treatment of immunoprecipitates was performed as suggested by the manufacturer (Calbiochem). The protein A beads were resuspended after the last wash in 25 µl 50 mM sodium phosphate, pH 7.5, 50 mM 2-mercaptoethanol, and 0.1% SDS, and were heated for 5 min at 95°C. After cooling and addition of TX100 to a final concentration of 1%, 5 U PNGase-F was added and the mixture was incubated for 4 h at 37°C followed by SDS-PAGE and fluorography as described above.
Subcellular fractionation
Toxoplasma-infected HFF cells were pulse labeled with [35S]-labeled methionine/cysteine as described above and either placed on ice or chased for an additional 4 h at 37°C. Cells and parasites were collected as described above and frozen in liquid nitrogen. After thawing on ice, cell pellets were resuspended in 500 µl TBS with 5 mM EDTA and protease inhibitors, and were subjected to five 10-s bursts in a cell disrupter (Misonix, Inc.). Half of each homogenate was placed on ice and the remainder was centrifuged for 30 min at 150,000 g in a rotor (SW55; Beckman Coulter). TX100 was added to the total homogenate sample and the soluble fraction to a final concentration of 1%. The particulate fraction was resuspended in 250 µl IP buffer. After addition of protease inhibitors and a 10-min incubation on ice, all fractions were clarified by centrifugation for 10 min at 14,000 g and analyzed by immunoprecipitation with TgGAP45 antiserum as described above.
Mass spectroscopy, protein sequencing, and cloning of TgGAP50
The glideosome complex was isolated from 109 parasites using anti-GAP45 antibodies covalently attached to CnBr-activated Sepharose-4B (Amersham Biosciences). Bound proteins were eluted using 7 M urea, 2 M thiourea, 2% ASB-14, 2% ampholytes 3-10, and 50 mM Tris, and were separated by two-dimensional electrophoresis using a pH 47 IPG strip (Bio-Rad Laboratories) in the first dimension and SDS-PAGE in the second dimension. Proteins of interest were detected by staining with Coomassie brilliant blue, excised, and subjected to in-gel digestion using trypsin. Tryptic peptides were analyzed by lipid chromatography coupled with tandem mass spectroscopy at the Mass Spectrometry Shared Facility in the UAB Comprehensive Cancer Center (University of Alabama at Birmingham, Birmingham, Alabama).
For direct sequencing of the TgGAP50 amino terminus, the glideosome proteins were transferred, after two-dimensional electrophoresis, to PVDF membrane (Bio-Rad Laboratories). The TgGAP50 spot was excised after staining with Coomassie brilliant blue and subjected to direct amino acid sequencing at the Protein Chemistry Facility of the UAB Comprehensive Cancer Center.
Toxoplasma genomic DNA and mRNA sequences in ToxoDB encoding TgGAP50 were identified using the peptide sequences obtained by mass spectroscopy. The complete predicted ORF and stop codon of TgGAP50 was amplified from total Toxoplasma RNA by RT-PCR using Pfu Turbo DNA polymerase (Stratagene) and the primers 5'-gcagatctaaaATGGCAGGCGCCCCCGTCGCGGCCGCC-3' and 5'-gccctaggTTATTTCATGTAGCGAGAGAGACCGTTC-3'. This PCR product was cloned into the TOPO®-TA vector (Invitrogen) and sequenced in its entirety. Several cDNA clones encoding the TgGAP50 ORF and flanking regions were isolated from a library of Toxoplasma cDNA in ZAPII (AIDS Research and Reference Reagent Program) and sequenced in their entirety.
Plasmid construction and expression in Toxoplasma
To generate the TgGAP50-YFP construct, the ORF was amplified using the primers 5'-gcagatctaaaATGGCAGGCGCCCCCGTC-3' and 5'-gccctaggTTTCATGTAGCGAGAGAG-3'. The PCR product was digested with BglII and AvrII and inserted between the BglII and AvrII sites in ptubß-IMC1-YFP/sagCAT (Hu et al., 2002). To generate the TgGAP50(427-431)YFP construct, the ORF minus the six carboxy-terminal residues was amplified using the same forward primer as above and the reverse primer 5'-gccctaggACCGTTCGCGACAGAGAG-3', digested with BglII and AvrII, and inserted between the BglII and AvrII sites in ptubß-IMC1-YFP/sagCAT.
TgGAP50-YFP and TgGAP50(427-431)YFP constructs were transfected into Toxoplasma by electroporation, and stable transfectants expressing TgGAP50-YFP were obtained by selection with chloramphenicol (Mann et al., 2002) and cloning by limiting dilution.
The HA-TgMyoA construct was amplified using primers 5'-cgggatccATGGCGAGCAAGACCACGTC-3' and 5'-GTCTAGAACGCCGGCTGAACAAGTCG-3'. The resulting PCR product was digested with BamHI and cloned between the BamHI and filled-in NotI sites of pEXP-NtermHA (Mann et al., 2002). The HA-TgMyoA construct was transfected into Toxoplasma expressing TgGAP50-YFP as described above, and parasites were analyzed by immunofluorescence 24 h after transfection.
Immunofluorescence and immuno-EM
Untreated extracellular parasites, parasites treated with C. septicum -toxin, or infected cells were fixed and permeabilized for 5 min in cold (20°C) methanol or for 15 min in 3% PFA and 0.25% glutaraldehyde in PBS, and for 5 min in 1% TX100 in PBS. Epifluorescence microscopy was performed as described previously (Mann et al., 2002) using a microscope (model BX60; Olympus), and images were collected with a SPOT2 camera (Diagnostic Instruments) and processed in Adobe Photoshop®.
For the -toxin experiments, parasites were treated with 20 nM toxin for 4 h and processed as described previously (Wichroski et al., 2002), except that fixed and permeabilized parasites were incubated first with primary antibody diluted at 1:2,000, followed by incubation with a 1:400 dilution of Alexa Fluor®conjugated secondary antibody (Molecular Probes, Inc.).
For preembedding immuno-EM, isolated pellicles were incubated with primary antibody (anti-TgGAP45 at 1:500) diluted in 3% BSA in PBS for 3 h on ice. The sample was washed in PBS by sedimentation (10,000 rpm for 10 min) and resuspension. Incubation with 10-nm goldconjugated goat antimouse IgG secondary antibody (Sigma-Aldrich) diluted 1:25 in 3% BSA in PBS was performed overnight at RT. The sample was washed in PBS as described above and fixed in 1% glutaraldehyde in PBS for 20 min. After another PBS wash, the sample was fixed in 1% osmium tetroxide in PBS for 20 min, washed in PBS, dehydrated in increasing concentrations of ethanol, embedded in Spurr's resin (Electron Microscopy Sciences), and polymerized overnight at 70°C. Thin sections were prepared, stained with uranyl acetate and lead citrate, and observed with an electron microscope (model H700; Hitachi).
SDS-PAGE and immunoblotting
Protein preparations were separated by SDS-PAGE on 12% polyacrylamide mini gels (Bio-Rad Laboratories). To obtain optimal resolution between TgGAP45 and TgGAP50, electrophoresis was continued for 15 min at 150 V after the bromophenol blue front had run off the gel. Transfer to nitrocellulose and immunoblot analysis was performed as described previously (Mann and Beckers, 2001).
Analysis of TgGAP45 and TgGAP50 sequences
DNA and predicted protein sequences were analyzed using BLAST and FASTA. Multiple sequence alignments were performed using ClustalW. Putative transmembrane domains were identified using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and HMMTOP (http://www.enzim.hu/hmmtop/). Potential N-linked glycosylation sites were identified using NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). Preliminary genomic and cDNA sequence data were accessed via http://ToxoDB.org. Sequence data for the P. falciparum genome were accessed via http://plasmodb.org/ (Bahl et al., 2003).
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Acknowledgments |
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Genomic data were provided by the Institute for Genomic Research (supported by National institutes of Health [NIH] grant #AI05093) and by the Sanger Center (Wellcome Trust). EST sequences were generated by Washington University (NIH grant #1RO1AI045806-01A1). This work is supported by NIH grants AI01719 (G. Ward) and AI41765 (C. Beckers), the Vermont EPSCoR programs under National Science Foundation grant EPS-9874685 (G. Ward), and the Burroughs Wellcome Fund (C. Beckers).
Submitted: 25 November 2003
Accepted: 6 April 2004
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