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Journal of Science 113, 2409-2419 (2000) 2409 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1127

Toxoplasma gondii catalase: are there in Toxoplasma?

M. Ding, C. Clayton and D. Soldati Zentrum für Molekulare Biologie Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany *Author for correspondence (e-mail: [email protected])

Accepted 19 April; published on WWW 14 June 2000

SUMMARY

The intracellular protozoan parasite Toxoplasma gondii, catalase has -AKM at the C terminus, which falls within like all members of the phylum , is known to the consensus of the PTS1 peroxisomal targeting signal. possess many : in addition to mitochondria and Southern blot analysis confirmed the presence of a single the compartments of the secretory pathway, there is a copy gene. Northern and western blot analyses showed reduced (the ) and the phylum- that the catalase gene is transcribed and translated. specific components of the apical complex: dense granules, Immunofluorescence assays using an antibody raised and . Conspicuously missing so far against a catalase peptide identified a distinct structure are , organelles that can be found in nearly all towards the apical end, but other catalase-specific eukaryotic organisms. Microbodies show a large variation antibodies failed to confirm this localisation. Cell with regard to their size, number and contents, depending fractionations indicated that the majority of the on the organism and cell type. One marker enzyme of this was in the . The fusion of the C-terminal twelve single membrane-bound is catalase, which is amino acids, including AKM, or the canonical peroxisomal responsible for the degradation of hydrogen peroxide to targeting signal, -SKL, to GFP resulted in predominantly water and oxygen. The EST project in T. gondii revealed cytosolic localization in T. gondii. There was therefore no the existence of two overlapping clones which showed evidence for membrane-bound peroxisomes in Toxoplasma. similarity with catalase, and these were used to clone the corresponding gene. The predicted sequence of T. gondii Key words: Toxoplasma gondii, Catalase,

INTRODUCTION Sommer et al., 1992; Waterham et al., 1997) but all combinations have not been tested. Microbodies are enzyme-containing organelles bounded by a The phylum Apicomplexa includes several human and single unit membrane and lacking DNA. The family includes animal parasites including Toxoplasma gondii and the peroxisomes, and glycosomes. The organelles species that cause malaria. In the mammalian have a variety of metabolic functions, none of which is present host, both Toxoplasma and Plasmodium multiply in all members: beta-oxidation of fatty acids, the glyoxylate intracellularly, and spread from one cell to the next via invasive cycle, alcohol oxidation, and even have been found extracellular forms. Electron micrographs reveal an confined within microbodies. Animal cell peroxisomes were astonishing variety of organelles. At the apex of the parasites the first members to be identified. They were so named because are the rhoptries and micronemes, both involved in host cell of their activity in peroxide , and can be identified invasion; and the dense granules, which are on the secretory by the presence of catalase. pathway and involved in remodelling the into a The biogenesis of microbodies is not well understood. The metabolically active compartment. There are also the nucleus results of studies in a variety of systems, particularly in yeast, and a non-photosynthetic chloroplast derivative, the apicoplast have shown that the matrix are imported post- (Kohler et al., 1997b; McFadden et al., 1996). In addition, translationally, in a folded or even oligomerized state, while a mitochondrial DNA is present, and the single minority of the membrane proteins may be routed via the shows a tubular morphology (Seeber et al., 1998). Since endoplasmatic reticulum (Kunau and Erdmann, 1998; microbodies have been identified in nearly all eucaryotic Subramani, 1998; Titorenko and Rachubinski, 1998; van der species investigated, we were intrigued by the apparent Klei and Veenhuis, 1997). Two types of signal have been absence of evidence for them in Apicomplexa. A literature defined: the C-terminal PTS-1 and the N-terminal PTS-2. The search revealed no mention of peroxisomes in combination first PTS-1 signal to be identified was serine-lysine-leucine- with any apicomplexan species. We therefore searched for COOH. Subsequent work has demonstrated that this signal can evidence of peroxisomes in T. gondii. be very degenerate, and that the precise specificity varies A common marker enzyme for peroxisomes is catalase, between species. The spectrum of possibilities includes which, along with superoxide dismutase, is involved in defence (S/A/C/K/N)-(K/R/H/Q/N/S)-(L/F/I/Y/M) (Amery et al., 1998; against oxidative stress and oxidative metabolic by-products. 2410 M. Ding, C. Clayton and D. Soldati

Both activities had previously been detected in tachyzoites of Native gel T. gondii (Sibley et al., 1986). We used information from the A 75 cm2 flask of freshly lysed RHhxgprt− was washed with PBS, and T. gondii EST database to clone and sequence the T. gondii one quarter of the resulting pellet was resuspended in 150 µl PBS catalase gene and to determine the location of the . containing protease inhibitors. Cells were sonificated. A 25 cm2 flask While this work was under review, Kaasch and Joiner (2000) of confluent Vero cells was trypsinised and one half of the resulting also reported studies of catalase localization in Toxoplasma but pellet was treated likewise as a negative control. After sonification, ° came to contrasting conclusions. cells were centrifuged for 15 minutes at 4 C, 14000 rpm. The resulting supernatants were kept and split in two halves. One half of each was used for loading four lanes in the native gel. Samples for the native MATERIALS AND METHODS gels were diluted 1:1 with the running buffer and 10% glycerol containing bromophenol blue. The separating gel contained 0.375 M Tris-HCl, pH 8.8, and had a percentage of 7.5%, the stacking gel Toxoplasma strains and culture conditions 0.0625 M Tris-HCl, pH 6.8 and a percentage of 4.5%. The running In all experiments, the T. gondii tachyzoite WT strain RH, lacking the − buffer of the native gels contained 0.05 M Tris-HCl and 0.38 M HXGPRT gene (RHhxgprt ), was used. Human foreskin fibroblasts glycine, pH 8.3 and no SDS. The native gel ran at 15 V overnight. (HFF) were grown in Dulbecco’s modified Eagles medium (DMEM; Gibco) containing 10% fetal calf serum. Southern blotting For the preparation of T. gondii genomic DNA, a freshly lysed 75 cm2 Sequencing of EST clones and cosmids flask of tachyzoites RHhxgprt− (see above) was centrifuged at 1000 The sequencing was done by Toplab, 82152 Martinsried, Germany. rpm for 15 minutes. The pellet was resuspended in 500 µl of PBS and Sequences were analysed using the DNAStar software package centrifuged for 2 minutes at 13000 rpm. The resulting pellet was (DNAStar Madison, WI). resuspended in 40 µl PBS, 200 µl of lysis solution (120 mM NaCl, 10 mM EDTA, 25 mM Tris-HCl, pH 7.5, 1% Sarkosyl) and 0.1 mg/ml Probe for southern blotting, northern blotting and cosmid RNase A and incubated for 30 minutes at 37°C. After this, 1 mg/ml screening proteinase K was added and the mixture was incubated overnight at The catalase probe was synthesised by PCR using one of the EST 65°C. The solution was extracted twice with phenol:chloroform (1:1), clones as template and the primers 5′-ggtggcggccgctctagaact-3′ and twice with chloroform, precipitated with ethanol, washed with 70% 5′-tcgccgatgtgccaatgagc-3′ according to the DIG Systems User’s ethanol and resuspended in TE buffer. For Southern blotting, 5 µg of Guide for Filter Hybridisation (Boehringer Mannheim, Germany). T. gondii genomic DNA were used per digest, size-fractionated by After PstI digestion to remove bluescript polylinker sequences, the agarose gel electrophoresis on a 0.8% agarose gel and blotted onto resulting probe had a size of 355 bp. For the cosmid screen, the Boehringer Mannheim positively charged nylon membranes. unlabelled probe was sent to James W. Ajioka (Cambridge, UK). Hybridization and wash procedures were performed according to the DIG Systems User’s Guide for Filter Hybridization (Boehringer Generation of antisera specific for T. gondii catalase Mannheim, Germany). The antisera to T. gondii catalase were generated by immunizing rabbits with two different peptides, CVDGFPKEDRNAAVSGT or Northern blotting CHPGQEHPNSDFE (the N-terminal cysteine was added for To isolate T. gondii RNA, two 175 cm2 flasks of freshly lysed coupling). The peptides were coupled to keyhole limpet hemocyanin tachyzoites of RHhxgprt− were prepared according to the GibcoBRL (KLH) according to the instructions of the manufacturer (Pierce). Trizol protocol. Rabbits were first immunized with 150 µg of both peptides with Total RNA was loaded on a formaldehyde gel and northern blotting the complete Freunds’ adjuvant. Three successive subcutaneous was done according to the DIG Systems User’s Guide for Filter injections with 150 µg of peptides in incomplete adjuvant LQ Hybridization (Boehringer Mannheim, Germany). (GERBU) were performed at at intervals of 28 days (twice) then 21 days. Indirect immunofluorescence assay (IFA) For the indirect immunofluorescence assay, RHhxgprt− tachyzoites Antibody purification were used to infect human foreskin fibroblast (HFF) cells that were The sera were immunoaffinity purified against the peptides previously growing on cover slides in 24-well plates. Bradyzoites were obtained coupled to Affi-Gel 15 according to the manufacturer (Bio-Rad). 2 by cultivating the cyst forming strain Prugniaud in HFF. After 36 mg of each peptide were used to affinity-purify 2 ml of serum hours, cells were washed with PBS and fixed with 4% paraformaldehyde or 4% paraformaldehyde, 0.005% glutaraldehyde Electrophoresis and western blotting in PBS for 15 minutes. For the Dense granules/catalase double RHhxgprt− were collected from a freshly lysed 25 cm2 culture flask immunofluorescence assay, cells were fixed with 3% of Vero cells. T. gondii tachyzoites were recovered by centrifugation paraformaldehyde alone for 10 minutes. After fixation, cells were for 8 minutes at 1200 rpm and washed in 1 ml PBS, then lysed by washed with PBS and neutralized with PBS containing 0.1 M glycine addition of 50 µl of RIPA lysis solution (150 mM NaCl, 1% NP40, for 3 minutes. After PBS wash, the cells were permeabilized for 20 0.5% natriumdeoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1 minutes with PBS containing 0.2% Triton X-100. Fixed mM EDTA) and centrifuged for 15 minutes at 14000 rpm, 4°C. 3 µl permeabilized cells were incubated for 20 minutes with PBS of the resulting supernatant were diluted in 1× protein loading containing 0.2% Triton and 2% albumin fraction V, then with buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% antibodies diluted in PBS containing 0.2% Triton X-100 and 2% bromophenol blue, 10% glycerol) and loaded on 12% SDS-PAGE albumin fraction V for 1 hour. The primary antibodies were polyclonal gels. A 25 cm2 culture flask of Vero cells was trypsinated and treated rabbit anti-T. gondii catalase (dilution 1:500), mouse monoclonal anti- likewise. Western blotting onto nitrocellulose membranes (0.45 µm, MIC3 (dilution 1:1000), mouse monoclonal anti-GRA3 (dilution Schleicher and Schuell, Germany) was performed according to 1:1000) and mouse monoclonal anti-ROP2 (dilution 1:1000). After standard protocols and blots were processed for antigen detection three washing steps with PBS 0.2% Triton X-100, cells were with a chemiluminescence system (ECL, Amersham Buchler incubated with the second antibody for 1 hour. For the double Braunschweig). The antibodies used for detection were the rabbit immunofluorescence assay, Alexa™ 488 goat α-mouse IgG and polyclonal anti-T. gondii catalase peptide sera described above. Alexa™ 594 goat α-rabbit IgG antibodies (highly cross absorbed) Toxoplasma gondii catalase 2411 were used (Molecular probes, Netherlands) at 1:1000 dilution. Cells (Sigma, 212-300 microns, unwashed). Cells were mechanically were washed three times with PBS 0.2% Triton X-100 and mounted broken using the Bead-beater. Breakage of the cells was judged by in a mounting solution containing 86.5% glycerol, 10% PBS, 1% microscopical analysis. After one minute of settling, broken parasites DAPI and 2.5% DAPCO. A Dapi wash was performed with a were transferred into a fresh tube and THB was added up to a final concentration of 0.1 µg Dapi/ml PBS before mounting the cells. volume of 1 ml. The lysate was centrifuged for 5 minutes at 4000 rpm, 4°C, to pellet large mitochondrial fragments, nuclei and Construction of plasmids unbroken cells. The resulting supernatant was centrifuged for 15 The plasmid pTGFP-AKM was generated by the insertion of double- minutes, 13000 rpm at 4°C. The pellet was resuspended in 200 µl stranded oligonucleotides between the PstI and PacI sites of the THB and loaded on a sucrose gradient, containing 70%, 60%, 50%, pTGFP vector. The mutated coding sequence of a temperature 40%, and 30% sucrose in 25 mM Tris-HCl, pH 7.8, 1 mM EDTA. resistant GFP mutant (Haseloff et al., 1997) was cloned between The tube was spun for 45 minutes at 45000 rpm, and 14 fractions of the NsiI and PacI sites of the pT230CAT vector (Soldati and 330 µl were collected. The fractions were TCA precipitated and Boothroyd, 1995). The sequences of the oligonucleotides were acetone washed. 20 µl 2× PP (containing 200 mM Tris-HCl, pH 7.8, 5′-ggtttgccgactgcggcgtgctacccagccaagatgtagttaat-3′ (sense primer) for neutralization) were added to the fourteen samples and 10 µl of and 5′-taactacatcttggctgggtagcacgccgcagtcggcaaacctgca-3′ (antisense each fraction loaded on the gel. primer), corresponding to the last C-terminal 12 amino acids of T. gondii catalase (GLPTAACYPAKM). The plasmid pTGFP-SKL was Immunofluorescence microscopy constructed in the same way, using the oligonucleotides 5′- The microscope used was a Leica DMRXA with Openlab 2.0.2 ggcccgagcaagctttaat-3′ (sense primer) and 5′-taaaagcttgctcgggcctgca- software. Immunofluorescence and Dapi pictures were processed 3′ (antisense primer), which results in the addition of -GPSKL to the using the supplied deconvolution algorithm. GFP C terminus (‘GP’ were introduced as helix breakers). Parasite transfection and selection of stable transformants RESULTS T. gondii tachyzoites (RHhxgprt−) were transfected by electroporation as previously described (Soldati and Boothroyd, 1993) using 107 Cloning and sequencing of T. gondii catalase µ freshly lysed-out tachyzoites, 80 g plasmid DNA and 100 units of A cDNA probe derived from a catalase EST sequence BamHI for restriction enzyme mediated integration (REMI) (Black et (accession number W63499) was used to screen a cosmid al., 1995). After electroporation, parasites were inoculated into HFF cells grown on glass coverslips (for immunofluorescence microscopy) library. The sequence of the gene was determined by primer or in 25 cm2 T-flasks for selection. Stable transformants containing walking across 6957bp (Toplab, München, Germany). The the transfected vectors were selected using medium containing 25 open reading frame of T. gondii catalase comprises 1506 µg/ml mycophenolic acid and 50 µg/ml xanthine and cloned by nucleotides (Fig. 1) and can encode a protein of 502 amino limited dilution in 96-well plates (Donald et al., 1996). acids with a predicted mass of 57 kDa. The putative start codon is preceded by the AAA that is very common at the site of Digitonin fractionation translation of T. gondii genes (Seeber and Boothroyd, 1996). Freshly released parasites were washed once in ice-cold phosphate The gene is interrupted by eight introns and the intron/exon buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, splice sites conform to the consensus. 1.8 mM KH2PO4, pH 7.4), once in STE (250 mM sucrose, 25 mM Two types of catalases are known: Mn-catalases (hexameric Tris-HCl pH 7.4, 1 mM EDTA) and resuspended in ice-cold STEN (STE containing 150 mM NaCl), 0.1 mM phenylmethylsulfonyl present only in prokaryotes) and the more common fluoride (PMSF). 5 samples (for the five different digitonin tetrameric haem catalases. The predicted T. gondii catalase concentrations), each containing 10 µg of Toxoplasma protein, were amino acid sequence contains the conserved His-64 that filled up to 98 µl with STEN (25°C). After adding 2 µl of digitonin participates in the catalysis and Asn-137 playing a role in (in dimethylformamide, DMF; five different starting concentrations binding of the substrate H2O2. The proximal ligand to the haem were used, namely 0.1, 0.2, 0.3, 0.4 and 0.5 mg digitonin per ml DMF) iron is Tyr-347. In catalases, the haem groups are deeply buried and 5 seconds of vortexing, the cells were incubated at 25°C for 4 in non-polar pockets and are connected with the surface by minutes, then centrifuged (2 minutes, 20000 g). Supernatant and rather narrow channels (Fita and Rossmann, 1985a). Most of pellets were separated, the supernatant being TCA precipitated and × the residues in the pocket are conserved in the apicomplexan the proteins being harvested by centrifugation; after adding 2 sample protein. Mammalian catalases are known to contain a tightly buffer, 1 M Tris-HCl, pH 8, was added to the supernatant fractions for neutralization. Both fractions of each sample (pellet and bound NADPH. The NADPH-binding sites demonstrated for supernatant fraction) were loaded on 12% SDS-PAGE, transferred to bovine catalase are also highly conserved in the deduced nitrocellulose and probed with anti-catalase, anti-GRA3 and anti-actin amino acid sequence of T. gondii catalase (Fig. 1; Fita and antibodies. The monoclonal anti-actin antibody was raised against Rossmann, 1985b; Hillar et al., 1994; Kitlar et al., 1994). The actin from Dictyostelium discoideum. This antibody crossreacts with C terminus of T. gondii catalase, AKM, matches the consensus T. gondii actin and shows no cross-reaction with human actin (D. PTS-1 signal which includes (S/A/C/K/N)-(K/R/H/Q/N/S)- Soldati, unpublished). (L/F/I/Y/M) (see e.g. Amery et al., 1998; Sommer et al., 1992; Waterham et al., 1997). Crude fractionation of T. gondii tachyzoite proteins To confirm the parasitic origin of the cDNA, Southern and Freshly lysed T. gondii tachyzoites were pelleted for 8 minutes at northern blot analyses were performed. The results of genomic 1200 rpm, 4°C. The pellet was resuspended in ice-cold Toxoplasma homogenization buffer (THB; 25 mM Tris-HCl, pH 7.8, 1 mM Southern blot analysis validated that the gene cloned and EDTA, 10% sucrose and 2 µg/ml leupeptin) and centrifuged 1 minute sequenced is not a contamination from host cells and is present at 14000 rpm at 4°C (microfuge). The following steps were as a single copy gene in the T. gondii parasite (Fig. 2). performed on ice. The pellet was resuspended in 1 ml of THB and The catalase gene is transcribed in tachyzoites and produces the suspension was transferred to a tube half-filled with glass beads transcripts of about 2800 nucleotides (Fig. 3). 2412 M. Ding, C. Clayton and D. Soldati

Fig. 1. Comparison of the predicted T. gondii catalase protein with catalases from other species. Jotun-Hein-based alignment of amino acid sequences of catalase from Saccharomyces cerevisiae (AC X13028), Nicotinia tabacum (accession number U93244), Methanosarcina barkeri (AC AJ005939), Homo sapiens (accession number AC 4557013), Drosophila melanogaster (AC U00145) and Bacillus subtilis (AC X85182) with the catalase from T. gondii (GenBank accession number NCBI: AF161267). Boxes indicate that all amino acids are identical at this site. The symbols ‘<’ and ‘>’ mark the amino acids which are supposed to be involved in the binding of NADPH in the T. gondii sequence; amino acids marked with ‘<’ primarily interact with the nicotinamide moiety, amino acids marked with ‘>’ primarily interact with the adenine. The catalytically important residues are marked with asterisks: His-64, which participates in catalysis, Asn-137, which plays a role in binding of H2O2 and Tyr-347, which is the proximal ligand to the haem iron. Toxoplasma gondii catalase 2413

Antibodies to T. gondii catalase recognize a protein also tested against sonificated parasite extracts after native gel of 57 kDa in tachyzoites electrophoresis, to check for reactivity with native catalase. To confirm the presence of the protein, polyclonal antisera The pattern obtained was identical with all four antisera were raised against two independent peptides predicted from (Fig. 4) and no cross-reactivity with any other proteins was the T. gondii catalase gene sequence. Each peptide was used detected. to immunize two rabbits, yielding four antisera in all. Rabbits #84 and #85 were immunized with the peptide CVDGFPKEDRNAAVSGT and rabbits #96 and #97 were immunized with the peptide CHPGQEHPNSDFE. The original antisera recognized a band migrating approx. 57 kDa kb on immunoblots of tachyzoite lysates (data not shown). The western blot analysis was repeated using the same antisera after immunoaffinity purification and delivered the same result (Fig. 4). The sera did not react with host cell lysates (HFF and Vero cells) and the preimmune sera showed no reactivity on T. gondii lysate (data not shown). Fainter lower molecular mass products were visible with sera #84, #85 and #97; these could be degradation products, as upon longer exposure of the autoradiogram, the same pattern of additional bands was detected with all four sera (data not shown). The antisera were

Fig. 3. Northern blot analysis of T. gondii catalase. Northern blot analysis of catalase expressed in T. gondii tachyzoites harvested from freshly and completely lysed HFF cells. Using the same probe as for Southern blot analysis, a unique transcript is detectable with a size of about 2800 nucleotides.

kDa

Fig. 2. Structure of the catalase genomic locus and Southern blot analysis. (A) A schematic drawing of the exon and intron regions of the T. gondii catalase gene, to scale. (B) Schematic illustration of the restriction map of T. gondii catalase genomic locus. The position of Fig. 4. Western analysis of catalase expression in T. gondii the probe used for the Southern blot analysis is indicated above. The tachyzoites. Toxoplasma (T) and host Vero cell (V) Ripa lysates were probe includes exon 7 and parts of exons 6 and 8. The restriction loaded on an SDS gel (upper panel), and blotted after sites and the predicted sizes of the fragments are depicted below. electrophoresis. Similar membranes bearing control (V) and parasite (C) Genomic DNA from T. gondii tachyzoites (5 µg per lane) was (T) samples were incubated with each of the four anti-peptide digested with HindIII, BamHI, PstI and EcoRI. Southern blot antibodies #84, #85, #96 and #97. The lower panel shows the results analysis was done using a DNA probe derived from the T. gondii with the native gel with Toxoplasma (T) and Vero cell (V) EST coding for catalase (W 63499) via PCR. Size standards are homogenate, incubated with the four antibodies in the same way as indicated in base pairs on the right. The faint band in the EcoRI described for the SDS gel. Regions of the gel that are not shown gave digest is probably the predicted 770 bp fragment, although the part of no signal even upon prolonged exposure. Catalase staining only the probe which is able to detect this fragment is very small. occured in lanes bearing parasite samples. 2414 M. Ding, C. Clayton and D. Soldati

a c e g

b d f h

a b c

Fig. 5. Localization of T. gondii catalase by indirect immunofluorescence. HFF infected with RH tachyzoites were fixed by the standard paraformaldehyde 4% and 0.005% glutaraldehyde procedure and analyzed by indirect immunofluorescence microscopy with two sera raised against peptides of T. gondii catalase. (A) Panels a and b show differential interference contrast images for antibodies #84 and #96, respectively, with tachyzoites forming rosettes. Panels c (#84) and d (#96) show the catalase staining, whereas panels e (#84) and f (#96) show the corresponding DAPI staining of the nucleus and the apicoplast. Panels g (#84) and h (#96) correspond to the merge images of immunofluorescence staining and DAPI. (B) Panel b shows bradyzoites stained with bradyzoite-specific anti-Sag4 antibodies. Panel a shows the same cyst, stained with anti-catalase antibody (#96). Panel c shows the merge image of a and b.

Immunolocalization of catalase in T. gondii The concentrated staining obtained with antiserum #96 was tachyzoites and bradyzoites similar to that obtained by Kaasch and Joiner (2000) using a The sera were used to determine the subcellular distribution of polyclonal antibody to recombinant T. gondii catalase, and did catalase by indirect immunofluorescence microscopy of not overlap with previously characterized organelles. Staining tachyzoites. Strangely, the four affinity-purified antibodies for the dense granules was obtained by using anti-GRA3 gave different patterns despite their identical reactivities on antibodies (Leriche and Dubremetz, 1991), an anti-MIC3 was western blots. Two sera (from rabbits #85 and #97) gave a used to detect the micronemes (Achbarou et al., 1991) and diffuse cytosolic staining when used at rather low dilutions an anti-ROP2 for the rhoptries (Fig. 6). Attempts to localise (1:100; not shown). With serum #84, the major staining catalase to a membrane-bounded organelle at the electron appeared to be over the apicoplast (Fig. 5A, panel g), a microscopic level failed. chloroplast-like DNA-containing organelle (Kohler et al., 1997a) made visible by DAPI staining. Antibodies to other Distribution of GFP fused to the last 12 amino acids peptides coupled with the same carrier protein give no of T. gondii catalase in recombinant tachyzoites apicoplast staining in T. gondii. In contrast, antiserum #96 To determine if the last three amino acids of the catalase can revealed an elongated structure that appeared next to but not function as targeting signal to putative T. gondii peroxisomes, coincident with the apicoplast (Fig. 5A, panel h). The we constructed a vector in which the green fluorescent protein persistent Prugniaud strain of T. gondii shows a high propensity GFP was fused to the last 12 amino acids of the T. gondii to build cysts in vitro and was used to examine the localization catalase. Stable recombinant parasites expressing GFP or GFP- of catalase with #96 in bradyzoites, which could be identifed AKM were generated and cloned. The distribution of GFP was by using antibodies recognizing the bradyzoite-specific antigen analyzed by direct fluorescence microscopy (Fig. 7, panel d). SAG4 (Ödberg-Ferragut et al., 1996). Patterns were similar to The GFP-AKM appeared to be predominantly cytosolic. Since those obtained in tachyzoites (Fig. 5B, panel a). –AKM is not a canonical targeting signal we repeated the Toxoplasma gondii catalase 2415

Fig. 6. Immunolocalization of catalase and markers of T. gondii secretory organelles. HFF infected with RH tachyzoites were fixed and examined for colocalization of catalase with several organellar markers. A, B and C show staining of catalase with #96 in red, whereas D, E and F correspond to the organellar markers stained in green; merged images are panels G (A+D), H (B+E) and I (C+F). (D) Micronemes stained with anti-MIC3 (Achbarou et al., 1991); (E) dense staining with anti-GRA3 (Dubremetz et al., 1993); (F) rhoptries stainied with anti- ROP2 (Beckers et al., 1994). experiment with the GFP-SKL fusion. Exactly the same result under the permeabilization conditions used. Actin, the - cytosolic distribution - was obtained (Fig. 7, panel c). As cytoplasmic marker, was mostly in the soluble fraction as control, we expressed the non-fusion GFP which has the expected, but a significant fraction remained insoluble even tendency to accumulate in the nucleus as previously described with higher levels of detergent, probably indicating association (Hettmann et al., 2000). with larger cytoskeletal structures. These results indicate that either the catalase is largely cytosolic, or it is within a structure Catalase distributes mainly in the in T. that is extremely sensitive to low levels of digitonin. gondii tachyzoites To see if we could detect organellar catalase by other means, Given the rather contradictory and unexpected results obtained we subjected the cells to mechanical breakage. Again, the vast by microscopy, we attempted to define the localization of majority of catalase was in the supernatant. Since this could catalase by other means. First, we undertook a differential have been a consequence of organelle breakage, we subjected permeabilization with digitonin, as peroxisomal membranes the crude organellar pellet to fractionation on a sucrose are usually considerably more resistant to this detergent than gradient (Fig. 9). Some pellet-associated catalase was indeed plasma membranes. The catalase clearly fell exclusively into found associated with particulate fractions, but more remained the soluble cytosolic fraction by this criterion using either at the top of the gradient. antibodies #84 or #96 (Fig. 8). Under the permeabilization These results indicate that most catalase is in the cytosol, but conditions used, GRA3, which is soluble within the dense it is nevertheless possible that a small proportion of the enzyme granules (Ossorio et al., 1994) was essentially in the organellar might be localized to digitonin-sensitive organelles in an apical fraction, indicating that the dense granules were still intact location. 2416 M. Ding, C. Clayton and D. Soldati

Fig. 7. Fluorescence microscopy analysis of recombinant tachyzoites expressing GFP fusion proteins. HFF cells were infected with recombinant parasites stably expressing GFP fused to the final 12 amino acids of the C-terminal end of the T. gondii catalase (GFP-AKM) or GFP-SKL. The cells were fixed and analyzed by direct fluorescence for the detection of GFP. (C) Tachyzoites expressing GFP-SKL, (D) tachyzoites expressing GFP-AKM. A (GFP-SKL) and B (GFP-AKM) show the same cells stained with anti-catalase antibodies (#96). (E and F) The overlay of GFP-SKL (C) and GFP-AKM (D) with the corresponding catalase stainings (A and B). (G) A control – direct fluorescence of parasites stably expressing non-fusion GFP, which has the tendency to accumulate in the nucleus.

DISCUSSION homologues were short DNAs that appeared to be contaminations of bacterial origin. The presence of catalase We have shown that T. gondii contains a gene coding for activity in these parasites has been reported (Clarebout et al., catalase and that the protein is expressed in tachyzoites and 1998) but is controversial because of the possibility of bradyzoites. Antioxidant enzymes are responsible for erythrocyte contamination or by the parasites. neutralizing reactive oxygen species such as superoxide anion However, Plasmodium does possess superoxide dismutase, radicals, hydrogen peroxide and hydroxyl radicals; they which could provide anti-oxidant defense and is related to include superoxide dismutases, catalases and peroxidases. typical Fe-superoxide dismutases (Becuwe et al., 1996). Catalases are a central component of the enzymatic Although a Cu, Zn-containing superoxide dismutase is located detoxification pathways that prevent the formation of the in the matrix of watermelon peroxisomes (Bueno et al., 1995), hydroxyl radical by decomposing H2O2. Parasites may be Fe-superoxide dismutases are generally cytosolic. exposed not only to the reactive species that are produced as Antisera raised against two completely different peptides of normal by-product of aerobic metabolism, but also those T. gondii catalase identified the same major band on western produced by activated host cells or macrophages. T. gondii blots, but analysis by indirect immunofluorescence revealed invades cells by a mechanism that does not provoke an striking contradictory results. Two of the antibodies (one to oxidative burst, and reside in a vacuole that cannot fuse with each peptide) gave faint cytosolic staining. One of the . Nevertheless, the parasites probably have aerobic antibodies gave staining that colocalized perfectly with the metabolism and (together with the host cells) are exposed to recently described -like organelle, made visible by immune mechanisms. A cytosolic catalase could, for example, staining of the multiple copies of circular 35 kb DNA genome be of advantage for intracellular parasites as a first line of with DAPI. This location is improbable. We have found no defense against the oxidative burst of attacking macrophages. previous report of catalase in the of plants or Interestingly, we could not find a catalase gene in the very . Targeting into the apicoplast requires the presence of a extensive P. falciparum genome database – the only bipartite signal at the N terminus, a secretory pathway signal Toxoplasma gondii catalase 2417

Fig. 9. Fractionation of mechanically disrupted T. gondii. T. gondii Fig. 8. Digitonin fractionation of T. gondii tachyzoites. Freshly tachyzoites were broken mechanically. An organellar pellet was released tachyzoites were treated with 5 different starting subjected to sucrose gradient fractionation and proteins identified by concentrations of digitonin (0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 western blotting using anti-T. gondii catalase antibodies or anti- mg/ml, 0.5 mg/ml in dimethylformamide). Supernatant and pellets GRA3 antibodies. The lighter fractions are towards the left. The were separated on SDS-PAGE, transferred on nitrocellulose and catalase can be found both in the uppermost fractions representing probed with anti-T. gondii catalase, anti-GRA3 and anti- either cytosolic protein or leakage from broken organelles, and in the Dictyostelium discoideum actin antibodies. The supernatant fractions more dense organellar fractions. In contrast, the dense granules are are marked with an ‘S’, whereas the pellet fractions are marked with concentrated mainly in more dense fractions (5-12). a ‘P’. Catalase is mainly found in the supernatant, regardless of the digitonin concentration while GRA3, the dense granule marker trypanosomes to man, but our digitonin fractionation of protein, is mainly detected in the pellet fraction. Actin was found in parasites provided no evidence whatsoever for a peroxisomal both fractions. location for catalase. If the enzyme is in a peroxisome, the membrane would have to be considerably more sensitive followed by a chloroplast signal (Waller et al., 1998). This to digitonin than any other membrane so far signal is not present on the T. gondii catalase. If the signal were tested. Interestingly, preliminary results from detergent encoded by an upstream exon, and the cytosolic and apicoplast permeabilisation of P. falciparum also indicate that firefly forms were generated by alternative splicing, we would expect luciferase, an -SKL-targeted peroxisomal protein, to see two mRNAs. Instead, only one population of transcript cofractionates with a cytosolic marker (K. Lingelbach and seems to be present. While this work was in progress, a catalase P. Burghaus, personal communication). After mechanical cDNA sequence was placed in the database by another group breakage of T. gondii, most of the catalase was in the soluble (Kaasch and Joiner, 2000), and this too lacks an apicoplast fraction but a small proportion of the protein did sediment with targeting sequence. We can not exclude the existence of a other organelles in sucrose gradients. This could have been second, divergent gene coding for catalase, but it seems more caused by a small amount of protein sticking to membranes or likely that this staining pattern is an artifact. In other organisms aggregates, but would also be consistent with an organellar where catalase is found in the cytosol and peroxisomes, such location for a very minor fraction of the protein. as in C. elegans and mammals, two distinct copies of the The predicted amino acid sequence of catalase includes a catalase gene are present, one of which lacks the PTS1 signal peroxisomal targeting signal, -AKM. Kaasch and Joiner (2000) (Bulitta et al., 1996; Taub et al., 1999). have demonstrated that this signal is functional in mammalian The structure detected by the other antiserum was at the cells. As an alternative method to find peroxisomes in T. gondii, anterior of the nucleus but shows an elongated form composed we fused the last 12 amino acids of catalase to GFP. We used of closely associated beads. This structure failed to colocalize this long sequence because the presence of the amino acids with any of the characterized organelles, and resembled that preceding the C-terminal tripeptide can facilitate localization recently published by Kaasch and Joiner (2000) using (Blattner et al., 1992). The GFP fusion protein was antibodies to recombinant catalase. This is the most likely predominantly in the cytosol. This could be a consequence of candidate for peroxisomes in T. gondii. Peroxisomes can take over-expression, but another possible explanation, which on a wide variety of forms in cross-section, including not only would fit with the observed partial cytosolic localization of circular structures but also elongated vesicles or a peroxisomal catalase, is that the peroxisome targeting signal is inefficient. reticulum (Lazarow and Fujiki, 1985). Unfortunately, we have Inefficient targeting with weak signals has been observed been unable to see any structure that might correspond to the previously in other systems (Wiemer et al., 1995). However, staining pattern of catalase in published electron micrographs, when we replaced the -AKM signal with the very efficient and attempts at immuno-gold labeling yielded no evidence canonical targeting signal, -SKL, there was again no indication for organellar compartmentation. Kaasch and Joiner (2000) whatsoever of any localization to the structures visualized by showed some catalase localization at the ultrastructural level immunofluorescence. by cytochemical labeling, there were no clear indications of Given the equivocal nature of the evidence presented above, surrounding membranes and immuno-electron microscopical we tested for peroxisomes in other ways. A search in the T. evidence was again lacking. gondii EST database for acyl CoA oxidase was fruitless. We Despite the indications from immunofluorescence that attempted to express chloramphenicol acetlytransferase catalase might be localized within the cell, extensive efforts to bearing the canonical PTS1 signal -SKL, but the protein was find evidence for a microbody membrane yielded consistently so poorly expressed that localization experiments were negative results. Subcellular fractionation using digitonin is an impossible, both after transient transfection and after the extremely reliable method for isolation of microbodies, from generation of permanent cell lines (data not shown). 2418 M. Ding, C. Clayton and D. Soldati

The PEX11 protein of Trypanosoma brucei is localized to Bulitta, C., Ganea, C., Fahimi, H. D. and Volkl, A. (1996). Cytoplasmic and the peroxisomal membrane when it is expressed in both peroxisomal catalases of the guinea pig liver: evidence for two distinct Saccharomyces cerevisiae and mammalian cells (Lorenz et al., proteins. Biochim. Biophys. Acta 1293, 55-62. Clarebout, G., Slomianny, C., Delcourt, P., Leu, B., Masset, A., Camus, D. 1998). When we constructed transgenic T. gondii designed to and Dive, D. (1998). Status of Plasmodium falciparum towards catalase. Br. express this protein, in contrast, TbPEX11 could not be J. Haematol. 103, 52-59. detected. Mutant versions of TbPEX11 are unstable in Donald, R., Carter, D., Ullman, B. and Roos, D. S. (1996). Insertional trypanosomes (Maier et al., submitted). Pex11p is unstable in tagging, cloning and expression of the Toxoplasma gondii hypoxanthine- xanthine-guanine phosphoribosyltransferase gene. Use as a selectable mutants of yeast that lack peroxisomal membranes (Hettema marker for stable transformation. J. Biol. Chem. 271, 14010-14019. et al., 2000) so the lack of detectable expression of TbPEX11 Dubremetz, J. F., Achbarou, A., Bermudes, D. and Joiner, K. A. (1993). in T. gondii is most likely caused by the failure to find an Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host- appropriate membrane. cell interaction. Parasitol. Res. 79, 402-408. PEX proteins are essential for peroxisome biogenesis in Fita, I. and Rossmann, M. G. (1985a). The active center of catalase. J. Mol. Biol. 185, 21-37. every species so far examined, and homologies are detectable Fita, I. and Rossmann, M. G. (1985b). The NADPH binding site on beef liver from trypanosomes to man. In a final effort to find evidence catalase. Proc. Nat. Acad. Sci. USA 82, 1604-1608. for microbodies in the Apicomplexa, we searched the very Haseloff, J., Siemering, K. R., Prasher, D. C. and Hodge, S. (1997). extensive Plasmodium databases for homologues of eleven Removal of a cryptic intron and subcellular localization of green fluorescent PEX proteins (2,5,7,11,13,14, 15,17,18,19,20). There were no protein are required to mark transgenic Arabidopsis plants brightly. Proc. −3 Nat. Acad. Sci. USA 94, 2122-2127. hits of less than 10 probability apart from two likely human Hettema, E. H., Girzalsky, W., van den Berg, M., Erdmann, R. and Distel, contaminations. Only 10,000 T. gondii EST sequences are B. (2000). Saccharomyces cerevisiae Pex3p and Pex19p are required for available, but again no PEX homologues were found. The proper localization and stability of peroxisomal membrane proteins. EMBO absence of PEX genes suggests that peroxisomes cannot be J. 19, 223-233. Hettmann, C., Herm, A., Geiter, A., Frank, B., Schwarz, E., Soldati, T. formed. Thus although there are indications that catalase may and Soldati, D. A. (2000). Dibasic motif in the tail of an apicomplexan be organized within a particular region of the T. gondii is an essential determinant of plasma membrane localization. Mol. cytoplasm, the majority of the evidence suggests that there are Biol. Cell (in press). no membrane-bounded microbodies in this parasite. Hillar, A., Nicholls, P., Switala, J. and Loewen, P. C. (1994). NADPH binding and control of catalase compound II formation: comparison of This work was funded by the BMBF (Forschungsschwerpunkt bovine, yeast and Escherichia coli enzymes. Biochem J. 300, 531-539. Tropenmedizin in Heidelberg) and Deutsche Forschungsgemeinschaft Kaasch, A. J. and Joiner, K. A. (2000). Targeting and subcellular localization of Toxoplasma gondii catalase. J. Biol. Chem. 275, 1112-1118. (SFB 544). We are indebted to Dr J. Ajioka for his assistance in the Kitlar, T., Doring, F., Diedrich, D. F., Frank, R., Wallmeier, H., Kinne, R. screening of the T. gondii genomic libraries. 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