The identification of a sequence related to apicomplexan enolase ...

Parasitol Res (2004) 94: 354–360 DOI 10.1007/s00436-004-1224-3

O R I GI N A L P A P E R

A. P. Wilson Æ J. J. Thelen Æ J. Lakritz Æ C. R. Brown A. E. Marsh

The identification of a sequence related to apicomplexan enolase from Sarcocystis neurona

Received: 5 May 2004 / Accepted: 19 August 2004 / Published online: 30 September 2004
Springer-Verlag 2004

Abstract Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona, an apicomplexan parasite. S. neurona is also associated with EPM-like diseases in marine and small mammals. The mechanisms of transmission and ability to infect a wide host range remain obscure; therefore, characterization of essential proteins may provide evolutionary information allowing the development of novel chemotherapeutics that target non-mammalian biochemical pathways. In the current study, two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry were combined to characterize and identify an enolase protein from S. neurona based on peptide homology to the Toxoplasma gondii protein. Enolase is thought to be a vestigial, nonphotosynthetic protein resulting from an evolutionary endosymbiosis event of an apicomplexan ancestor with green algae. Enolase has also been suggested to play a role in parasite stage conversion for T. gondii. Characterization of this protein in S. neurona and comparison to other protozoans indicate a biochemical similarity of S. neurona enolase to other tissue-cyst forming coccidians that cause encephalitis. A. P. Wilson Æ C. R. Brown Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA J. J. Thelen Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA J. Lakritz Department of Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA A. E. Marsh (&) Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, 1920 Coffey Road, Columbus, OH 43210, USA E-mail: [email protected] Tel.: +1-614-2921206 Fax: +1-614-2924142

Introduction Sarcocystis neurona, Neospora spp. and Toxoplasma gondii are related protozoan genera that can cause encephalomyelitis in a variety of animals (Dubey and Lindsay 1996; Dubey et al. 2001; Suzuki 2002). Equine protozoal myeloencephalitis (EPM) is caused by a protozoan infection of the CNS (Rooney et al. 1970; Cusick et al. 1974; Madigan and Higgins 1987; Marsh et al. 1996) and is one of the most commonly diagnosed neurologic disease of horses in North America (MacKay 1997). S. neurona has remained problematic to the equine industry due to difficulties in definitive diagnosis (Jones 2002) and lack of an effective vaccine. EPM produces non-specific neurologic symptoms that are compatible with a number of etiologies. The current method of parasite detection is through the evaluation of cerebrospinal fluid (CSF) for parasite-specific antibodies; however, compromise of the blood-brain barrier or blood contaminated CSF samples can interfere with CSF antibody analysis (Granstrom and Reed 1994). Common symptoms include, but are not limited to, asymmetrical ataxia, muscle wasting, and indistinct lameness (Jones 2002). Many aspects of the S. neurona life cycle are currently unclear, including the susceptibility of specific horses or other mammals to infection and the development of disease. Other members of the genus Sarcocystis have an obligatory, predator-prey, or scavenger-carrion life cycle which is based on the specific Sarcocystis species, meaning there are characterized defined definitive and characterized defined intermediate hosts for individual Sarcocystis species. For example, S. cruzi uses the canine as a definitive host and the bovine as intermediate host (Dubey et al. 1989). The opossum has been identified as the definitive host of S. neurona (Fenger et al. 1995), but the intermediate host range in which the tissue cysts form has continued to expand in recent years. To date, the intermediate and aberrant host range includes: the nine-banded armadillo (Cheadle et al. 2001a; Tanhauser

355

et al. 2001), striped skunk (Cheadle et al. 2001b), cat (Dubey et al. 2000), sea otter (Rosonke et al. 1999), harbor seal (Lapointe et al. 1998) and raccoon (Stanek et al. 2002). The horse is considered an aberrant, deadend host because S. neurona sarcocysts do not form in the horse (Dubey et al. 2001). However, Neospora hughesi, a minor parasite associated with EPM (Marsh et al. 1998), do form tissue cysts in the horse (Marsh et al. 1996). Tissue cyst development in the Sarcocystidae, which include Sarcocystis spp., Neospora spp., and T. gondii has been evaluated, but is still not fully understood (Dubey et al. 1989; Dubey and Lindsay 1996; Dubey 1998; Vonlaufen et al. 2002; Lyons et al. 2002). Immune status, parasite strain, and several host factors have been identified as playing a role in the transition from a rapidly dividing parasite stage to the more dominant bradyzoite encysted stage. Recently, enolase gene expression has been linked to T. gondii stage conversion. Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) is an enzyme responsible for catalyzing the only dehydration step in the glycolytic pathway (Van der Straeten et al. 1991). Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry were used to identify an enolase protein in S. neurona. Additional characterization and comparative sequence data was generated using the polymerase chain (PCR) reaction with S. neurona and Neospora genomic DNA.

sample buffer (Invitrogen, Carlsbad, Calif.), and 10% (v/v) 2-ME to each parasite pellet. Parasite and host cell preparations were heated for 10 min at 70C. Each sample was added to a 1.0 mm, 12-well 4–12% NuPAGE Bis-Tris gel (Invitrogen) along with a SeeBlue 2 Plus molecular weight standard and run on the onedimensional gel for 40 min at 200 V. Once separation was complete, protein was transferred to a nitrocellulose membrane by electrophoresis for 2 h at 324 mA. The nitrocellulose membrane was blocked in 5% (v/v) nonfat dry milk in Tris-buffered saline (TBS; 10 mM TrisHCl pH 8.0, 150 mM NaCl) with 0.5% (v/v) Tween (TBS-T) for 1 h and then incubated with 1:500 dilution of a polyclonal antibody specific for T. gondii enolase 1 (ENO1) or enolase 2 (ENO2) (rabbits immunized with either recombinant T. gondii ENO1 or ENO2 proteins) (Dzierszinski et al. 2001), overnight at 4C with gentle rocking. The nitrocellulose membrane was subjected to three 5 min washes with TBS-T, followed by a 2 h incubation with a 1:5,000 dilution of horse radish peroxidase-conjugated, goat anti-rabbit IgG secondary antibody (Caltag, Burlingame, Calif.). The nitrocellulose membrane was washed three times, 5 min each in TBST, after which it was incubated for 2 min in chemiluminescent reagents (Cell Signaling Technology, Beverly, Mass.). Reactive bands were visualized by exposure to light-sensitive film and their size determined by comparison to molecular weight standards. Protein isolation and sequence identification

Materials and methods Cell culture T. gondii (RH strain), N. hughesi (NE-1), N. caninum (VMDL) and S. neurona (SN-UCD1) were grown in equine dermal (ED) (American Type Culture Collection, CCL57) monolayers in RPMI-1640 media supplemented with 10% v/v heat inactivated fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 5·10 2 mM 2-mercaptoethanol (2-ME), 50 units penicillin/ml, and 50 lg streptomycin/ml, and incubated at 37C. At approximately 75% host cell infection or lysis, parasites were harvested, washed, counted, and immediately frozen at 80C. Host cells, including human foreskin fibroblast cells (HS-68), were grown under similar conditions except in heat-inactivated fetal bovine supplemented DMEM with penicillin and streptomycin, and the media was changed to supplemented RPMI after parasite inoculation. The HS-68 were used only as a source for DNA in the molecular studies. Enolase characterization using specific antibodies S. neurona, Neospora spp., T. gondii, and ED pellets were separated by one-dimensional electrophoresis by adding 25% (v/v) 4· lithium dodecyl sulfate (LDS)

Frozen parasite pellets were resuspended in four volumes of 100% ice-cold acetone, mixed well and incubated at 20C for 1 h. The mixture was then centrifuged for 15 min at 5,000 g at 20C. The supernatant was removed and the pellets were resuspended in rehydration buffer containing 8 M urea, 2% (v/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.5% (v/v) Zoom Carrier Ampholytes pH 4–7 (Invitrogen), 20 mM dithiothreitol (DTT) and 0.002% (v/v) bromophenol blue. The sample was incubated overnight at room temperature with a 7-cm immobilized pH gradient (IPG) (Invitrogen). The rehydrated IPG strip was focused by step voltage protocol at 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and 2,000 V for 1 h. After focusing, the strips were equilibrated in two 15-min incubations using disposable rehydration/equilibration trays (Biorad, Hercules, Calif.), the first with 10% (v/v) of 2-ME in 0.5 ml of 1· LDS sample buffer (Invitrogen) per strip with gentle rocking at room temperature, and the second with 0.125 M of iodoacetamide (Sigma, St. Louis, Mo.) in 0.5 ml of 1· LDS sample buffer per strip with gentle rocking at room temperature. Proteins were further separated using two-dimensional gel electrophoresis following the manufacturer’s recommended protocol (Invitrogen). Briefly, the strip was placed into a one-well Invitrogen Bis-Tris Zoom gel

356

(4–12%), and proteins were separated by molecular weight using 200 V for 40 min. SeeBlue2 Plus molecular weight standard (Invitrogen) was used to compare molecular weights. After protein size separation, gels were either transferred to nitrocellulose for immunoblot analysis as earlier described, or stained with Simply Blue Coomassie blue stain (Invitrogen) for total protein visualization and selection of protein spots for MALDIToF protein preparation. For MALDI-ToF, Coomassie blue-stained gels were imaged and visible spots were excised with a sterile disposable 500-ll pipette tip and transferred to a 1.5-ml Eppendorf tube. Separate pipette tips were used for each spot excised. Spots were destained by adding 0.5 ml destain solution [50% (v/v) acetonitrile, 50 mM ammonium bicarbonate in water] and incubated at room temperature for 15 min with gentle agitation. The destain solution was removed, and the gel spots were destained two more times, for 15 min each time, with 500 ll of wash solution, until the Coomassie dye was completely removed. Finally, 100% acetonitrile was added for 5 min and the sample was completely dried at room temperature in a vacuum centrifuge. Dried gel plugs were rehydrated in 50 ll of sequencing grade porcine trypsin (Promega, Madison, Wis.), diluted to working stock according to the manufacturer’s instructions, and incubated for 16 h at 37C. Tryptic peptides were extracted by adding 100 ll of 60% (v/v) acetonitrile, 1% (v/v) formic acid, followed by gentle agitation for 15 min at room temperature. Extracted peptides were concentrated to approximately 5 ll and co-spotted with a-cyano hydroxy cinnamic acid [10 mg ml-1 in 60% (v/v) acetonitrile, 1% (v/v) formic acid]. MALDIToF analysis was performed at the University of Missouri Proteomics Center using a Voyager DE-Pro mass spectrometer in the positive ion reflector mode. Mass spectra were searched against the database using Protein Prospector (http://prospector.ucsf.edu/). Peptide mass fingerprint analysis revealed enolase as the top-scoring hit. Confirmation of peptide sequence was made by tandem mass spectral analysis of the four most abundant ions using a Q-star quadrupole-time of flight mass spectrometer (Applied Biosystems, Foster City, Calif.)

sequence with few mismatches was chosen for primer design to amplify an appropriate size product of 304 base pairs. The primer pair selected, forward (5¢-G CGT CTA CGG GTA TTT ATG AGG C-3¢) and reverse (5¢GAA AAA CGG TAC AGG CAT AAC CAT C-3¢) primers, was used to amplify the partial ENO2 gene region. These primers correspond to 146–167 nt (forward) and 471–495 nt (reverse) using AF123457 as a reference sequence. Genomic DNA was added to 50 ll PCR reactions containing thermophilic DNA polymerase 10· buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton ·-100), 1.5 mM MgCl2, 250 lM each of dATP, dCTP, dGTP, and dTTP, 20 pmol of oligonucleotide primers, and 0.5 units of Taq DNA polymerase (Promega). The templates were subjected to an initial denaturation cycle of 95C for 5 min followed by 30 cycles of 95C for 30 s, 55C for 30 s, and 72C for 2 min, and followed by a single 7-min extension cycle at 72C. A second reaction was also utilized in which the MgCl2 concentration was increased to 2 mM. All other conditions remained the same. The PCR products were analyzed on an ethidium-bromide-stained 2% v/v NuSieve 3:1 agarose gel to visualize the expected 304 base pair product. After confirmation of product size, PCR products were purified through the use of QIAquick PCR purification kit (Qiagen). The purified PCR product was added to 6 pmol of either forward or reverse primers and the complete double-stranded nucleotide sequences were obtained by PRISM dye terminator cycle sequencing using the manufacturer’s recommended protocol for the ABI 377 automated seqencer (Applied Biosystems). Final DNA sequence construction, alignments, and comparisons were facilitated using Vector NTI (Invitrogen), and S. neurona (AY563177), N. caninum (AY563178), and N. hughesi (AY563179) nucleotide sequences were submitted to GenBank.

PCR and DNA sequencing of the enolase gene

The proteins of T. gondii, N. caninum, N. hughesi, S. neurona and ED cells all reacted with the polyclonal antibodies against ENO2 (Fig. 1A). The parasite preparations showed a reactive band at approximately 47 kDa, and the host cell preparation indicated a relatively weak band at approximately 48 kDa. ENO1-specific antibody showed low reactivity in the tachyzoite stage of N. caninum, N. hughesi, T. gondii and in the merozoite stage of S. neurona; therefore, further analyses of ENO1 were not pursued (data not shown). S. neurona two-dimensional gels that were initially focused in the 3–10 pH range showed a number of proteins in the center of the gel, more so than on the peripheral edges. Therefore, the conditions were

The genomic DNA (gDNA) of parasite and host cell pellets was isolated by the use of DNeasy tissue kit (Qiagen, Valencia, Calif.) and analyzed on a 0.8% agarose gel. Primers designed for enolase, 5¢-ATGGTGGCCATCAAGGACATCACT-3¢ and 5¢-GTAGTCGAAACTCCGTTTCCACTTAG-3¢ and conditions were used as described (Dzierszinksi 2001). In addition, primers were constructed by aligning and comparing S. neurona MALDI-ToF protein sequence data and a published T. gondii ENO2 DNA sequence (GenBank, Accession no. AF123457). An area in which the MALDI-ToF sequence aligned to the ENO2 translated

Results Immunodetection of enolase on one- and two-dimensional gels

357

Fig. 2 Two dimensional SDS-PAGE gel separation of S. neurona. The proteins were isoelectric focused between pH 4 and 7, size separated on a 4–12% Bis-Tris ZOOM gel, and stained with Coomassie blue overnight. The arrow denotes spot no. 3, which was excised from the gel, dehydrated, and submitted for MALDI-ToF/ MS spectral analysis which identified it as an enolase-like protein

was excised between approximately 38 and 49 kDa and had an approximate pI between 4 and 6. Fig. 1 a One-dimensional separated protein immunoblot probed with anti-enolase 2 (ENO2) antibody. Proteins loaded were as follows: Lane 1 Toxoplasma gondii, lane 2 Neospora caninum, lane 3 N. hughesi, lane 4 Sarcocystis neurona, and lane 5 equine dermal cells. Parasite enolase is distinguishable from the host cells as a smaller molecular size protein. b Two-dimensional separated S. neurona merozoite proteins immunoblot probed with anti-ENO2 antibody

changed to achieve better separation of proteins by using a narrower 4–7 pH range. Like the one-dimensional immunoblot membrane, the two-dimensional S. neurona immunoblot membrane showed reactivity with the ENO2 antibody (Fig. 1B). There were four distinct reactive spots, just larger than approximately 38 kDa, and two distinct spots at appropriately 62 kDa. The spots span a pH range across the membrane from appropriately pH 4 to 7. Identification of enolase protein using MALDI-ToF mass spectrometry and peptide mass fingerprinting Three spots were excised from Coomassie-stained gels digested with trypsin and subjected to MALDI-ToF mass spectrometry for peptide mass fingerprinting analysis. Two of the spots were not matched definitively to other potential parasite protein homologs and further analysis of these spots was not pursued (data not shown). Alternatively, a third spot (Fig. 2) had the best match, in which 10 of 30 peptide ions (125 of 444 amino acids) matched a published T. gondii enolase protein (Table 1, Fig. 3). The T. gondii enolase protein has a molecular weight of 48 kDa and an isoelectric point (pI) of 5.67, whereas the putative S. neurona enolase protein

PCR and DNA sequence of the enolase gene PCR was performed on parasite gDNA in an attempt to amplify the ENO2 gene from S. neurona. The DNA obtained from S. neurona, N. caninum, N. hughesi, T. gondii and mammalian cells (ED and HS-68) each produced PCR bands. The parasite DNA samples produced a single bright band appearing at between 300 and 400 bp as well as several faint bands greater than 1,000 bp as seen in Fig. 4A. The bands greater than 1,000 bp are most likely due to mammalian host cell contamination as ED cell and HS-68 DNA produced a single faint band visible at a size greater than 1,000 bp (Fig. 4B) at the higher MgCl2 concentration. Figure 5 shows the predicted amino acid sequence alignment of T. gondii, N. caninum, N. hughesi, and S. neurona. The alignment shows two different motifs, EWGYS and EWGWC, suggesting that two different enolases were amplified from the parasite genomic DNA. S. neurona showed 80% similarity to T. gondii enolase whereas the N. hughesi sequence showed 93% similarity. There was only a single amino acid difference between N. hughesi and N. caninum.

Discussion Enolase is a protein thought to be evolutionarily derived from a photosynthetic lineage by a secondary endosymbiosis event between green algae and apicomplexans (Dzierszinski 1999), and has been described in a number of other parasite species (Hannaert et al. 2000; Keeling

358 Table 1 MALDI-ToF data summary for excised spot 3 on the two-dimensional gel (Fig. 2) which indicates the 10 of 30 ions (125 of 444 amino acids) matched to a published Toxaplasma gondii protein, enolase protein (GenBank AF123457) M/z submitted

MH+ matched

Delta ppm

Start

End

Peptide sequence

824.4175 1606.8484 1622.8529 1639.9625 1696.0130 1805.0041 1849.9398 1849.9398 2095.0732 2111.0718 2116.1394

824.4089 1606.7868 1622.7817 1639.9019 1695.9468 1804.9445 1849.8754 1849.9451 2094.9629 2110.9578 2116.0472

10.3976 38.3890 43.8921 36.9449 39.0564 33.0405 34.7722 2.8608 52.6573 54.0027 43.5659

419 111 111 325 73 34 17 109 92 92 150

424 125 125 339 87 51 33 125 108 108 169

(K)YNQLMR(I) (K)LGANAILAVSMACCR(A) (K)LGANAILAVSMACCR(A) (K)VQIVGDDLLVTNPTR(I) (K)IIKPALIGKDPCDQK(G) (R)AAVPSGASTGIYEALELR(D) (R)GNPTVEVDLLTDGGCFR(A) (K)SKLGANAILAVSMACCR(A) (K)LMVEELDGTKNEWGWCK(S) (K)LMVEELDGTKEWGWCK(S) (K)MVMPVPFFNVINGGSHAGNK(V)

Fig. 3 Protein sequence from T. gondii ENO2 protein. The underlined amino acids indicates tryptic peptides that matched 100% between the S. neurona two-dimensional SDS-PAGE gel excised protein and the published T. gondii sequence

Fig. 4 a Analysis of PCR products observed under UV light after electrophoresis in an ethidium bromide stained 2% NuSieve 3:1 agarose gel. Lane 1 T. gondii, lane 2 N. caninum, lane 3 N. hughesi, and lane 4 S. neurona. b Secondary reaction with higher, 2 mM MgCl2 using the following DNA templates: lane 1 T. gondii, lane 2 equine dermal cell, and lane 3 water blank

and Palmer 2001). Previous studies have established two distinct isoforms of enolase, ENO1 and ENO2, in T. gondii. ENO1 primarily exists in the bradyzoite stage Fig. 5 Predicted amino acid sequence of the enolase PCR amplified fragment for N. caninum, N. hughesi, and S. neurona as compared to the published T. gondii sequence. Bolded C denotes conserved cysteine residues and shading indicates domain I, the plant motif or plant-like motif

Modifications

1Met-ox

2Cys-am 1Met-ox

of T. gondii whereas ENO2 is more highly expressed in the tachyzoite stage (Dzierszinski 2001). When an unknown event triggers a stage conversion in apicomplexan parasites from tachyzoite to bradyzoite, enolase genes are either overexpressed or exclusively expressed at both the transcriptional and protein levels within the bradyzoite stage (Dzierszinski 1999) as compared to levels detected in the tachyzoite. Enolase has previously been classified as an important part of the glycolytic pathway in that it is responsible for the conversion of 2-phosphoglycerate to phosphoenolpyruvate, the only dehydration step in glycolysis (Tomavo 2001). This is significant considering that related parasites like T. gondii rely on anaerobic glycolysis during their bradyzoite encystment period, due to non-functioning mitochondria (Tomavo and Boothroyd 1995). These two enzyme isoforms share significant amino acid homology but are still antigenically distinct (Dzierszinski 2001; Ferguson 2002). In this study we report the identification of an ENO2 protein in S. neurona merozoites by protein and molecular methods and compare the results to molecular data from Neospora spp. and T. gondii. The combination of protein analysis using two-dimensional gel electrophoresis with MALTI-ToF and molecular detection using PCR and DNA sequencing provide complementary tools for protein discovery and identification. The S. neurona MALDI-ToF protein sequence data acquired was used to design primers to amplify an enolase gene from S. neurona. These primers also proved satisfactory for acquiring PCR products from Neospora spp. and T. gondii that were easily differentiated from the minor, larger products produce by host cell DNA in the PCR reaction. The PCR assay and sequencing reaction did not target the entire genetic sequence for S. neurona nor

359

Neospora spp. However, enough of the DNA sequence was acquired in order to differentiate the two from T. gondii. Attainment of a larger sequence was attempted using S. neurona DNA in order to view another critical plant motif DK/EK using the reported PCR parameters (Dzierszinski 2001); however, the conditions failed to yield product for S. neurona, although they did produce a PCR product of the appropriate size for N. caninum, N. hughesi, and T. gondii. This failure was most likely due to the sequence differences between S. neurona and T. gondii. The sequencing and predicted amino acid sequence of the enolase PCR products from N. hughesi and N. caninum indicate the presence of a plant-like motif, EWGYS, previously identified in T. gondii ENO1, whereas the predicted amino acid sequence of the enolase from S. neurona shows the true plant motif EWGWC, previously identified in T. gondii ENO2 (Dzierszinski et al. 2001). These two motifs have none to complete conservation across phylum (Hannaert et al. 2000) with S. neurona and Neospora spp. being similar to other parasitic protozoa characterized. Overall, protein characterization using one- and two-dimensional gels with immunoblot analysis suggests conserved enolase protein structure amongst T. gondii, Neospora and S. neurona that is not shared with mammalian host cells. One significant protein region is pentapeptide EWGWC or EWGYS, a plant or plant-like motif important because it greatly contributes to enolase enzymatic activities (Dzierszinski 2001). The limitations of this study include the exclusion of bradyzoites from the corresponding parasites used in the antigenic studies, and the lack of using more traditional methods for gene cloning such as screening a tachyzoite, merozoite, or bradyzoite protein expression library for ENO1 or ENO2 cDNA to determine additional differences between the enolases. There is limited information from comparative studies for tissue cyst formation between Neospora spp., T. gondii, and Sarcocystis spp. Sarcocystis spp. readily form easily detected tissue cysts in the appropriate host (Dubey et al. 1989), whereas T. gondii and Neospora tissue cysts are smaller and more limited in distribution, making detection difficult in natural infections (Dubey and Beattie 1988; Dubey and Lindsay 1996). It would be of interest to determine whether the levels of anaerobic glycolysis or associated enzyme or enzyme mRNA levels were significantly different between enolase isotypes and could be related to tissue cyst size formation, bradyzoite replication, or duration of the tissue cyst. Two-dimensional gel electrophoresis coupled with MALDI-ToF are useful methods to characterize protein components of parasites. Using these methods, we were able to identify an interesting and important protein, enolase, that had previously been missed using established the PCR method for a related organism. Additional comparative studies should provide more information on enolase structure, which may in turn provide insight into stage conversion and apicomplexan

evolutionary history and perhaps identify a unique target for drug therapy against these important parasitic diseases. Acknowledgements This project was supported by the University of Missouri College of Veterinary Medicine, NIH through a Minority Biomedical Research Training Initiative (MBRTI) Fellowship (R25 GM56901-06) and AREA funding (R15 AI44781-01). The authors thank Stanislas Tomavo for providing T. gondii enolase-specific antibodies and Mary K. Cockrell for technical assistance.

References Cheadle MA, Tanhauser SM, Dame JB, Sellon DC, Hines M, Ginn PE, MacKay RJ, Greiner EC (2001a) The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona. Int J Parasitol 31:330–335 Cheadle MA, Yowell CA, Hines M, Ginn PE, Marsh AE, Dame JB, Greiner EC (2001b) The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona. Int J Parasit 31:843–849 Cusick PK, Sells DM, Hamilton DP, Hardenbrook HJ (1974) Toxoplasmosis in two horses. J Am Vet Med Assoc 164:77–80 Dubey JP (1998) Advances in the life cycle of Toxoplasma gondii. Int J Parasitol 7:1019–1024. Dubey JP, Beattie CP (1988) Toxoplasmosis of animals and man. CRC Press, Boca Raton, Fla Dubey JP, Lindsay DS (1996) A review of Neospora caninum and neosporosis. Vet Parasitol 67:1-59 Dubey JP, Speer CA, Fayer R (1989) Sarcocystosis of animals and man. CRC Press, Boca Raton Dubey J, Saville W, Lindsay D, Stich R, Stanek J, Speer C, Rosenthal B, Njoku C, Kwok O, Shen S, Reed S (2000) Completion of the life cycle of Sarcocystis neurona. J Parasitol 86:1276– 1280 Dubey JP, Lindsay DS, Saville WJA, Reed SM, Grandstrom DE, Speer CA (2001) A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet Parasitol 95:89–131 Dzierszinski F, Popescu O, Toursel C, Slomianny C, Yahiaoui B, Tomavo S (1999) The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes: implications for evolutionary origin of apicomplexans. J Biol Chem 35:24888–24895 Dzierszinski F, Mortuaire M, Dendouga N, Popescu O, Tomavo S (2001) Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii. J Mol Biol 309:1017–1027 Fenger CK, Granstrom DE, Langemeier JL, Stamper S, Donahue JM, Patterson JS, Gajadhar AA, Marteniuk JV, Xiaomin Z, Dubey JP (1995) Identification of opossums (Didelphis virginiana) as the putative definitive host of Sarcocystis neurona. J Parasitol 81:916–919 Ferguson DJP, Parmley SF, Tomavo S (2002) Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication. Int J Parasitol 32:1399–1410 Granstrom DE, Reed SM (1994) Equine protozoal myeloencephalitis. Equine Pract 16:23–26 Hannaert V, Brinkman H, Nowitzki U, Lee JA, Albert M, Sensen CW, Gaasterland T, Mu¨ller M, Michels P, Martin W (2000) Enolase from Trypanosoma brucei, from the amitochondriate protist Mastigamoeba balamuthi, and from the chloroplast and cytosol of Euglena gracilis: pieces in the evolutionary puzzle of the eukaryotic glycolytic pathway. Mol Biol Evol 17:989–1000 Jones WE (2002) EPM update. J Equine Vet Med 22:528–34 Keeling, PJ, Palmer JD (2001) Lateral transfer at the gene and subgenic levels in the evolution of eukarytic enolase. Proc Natl Acad Sci U S A 98:10745–10750

360 Lapointe JM, Duignan PJ, Marsh AE, Gulland FM, Barr BC, Naydan DK, King DP, Farman CA, Huntingdon KA, Lowenstine LJ (1998) Meningoencephalitis due to a Sarcocystis neurona-like protozoan in Pacific harbor seals (Phoca vitulina richardsi). J Parasitol 6:1184–1189 Lyons RE, McLeod R, Roberts CW (2002) Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends Parasitol 18:198– 201 MacKay RJ (1997) Equine protozoal myeloencephalitis. Vet Clin North Am Equine Pract 13:79–96 Madigan JE, Higgins RJ (1987) Equine protozoal myeloencephalitis. Vet Clin North Am Equine Pract 3:397–403 Marsh AE, Barr BC, Madigan J, Lakritz J, Nordhausen R, Conrad PA (1996) Neosporosis as a cause of equine protozoal myeloencephalitis. J Am Vet Med Assoc 209:1907–1913 Marsh AE, Barr BC, Packham AE, Conrad PA (1998) Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). J Parasitol 84:983–991 Rooney JR, Prickett ME, Delaney FM, Crowe MW (1970) Focal myelitis-encephalitis in horses. Cornell Vet 60:494–501 Rosonke BJ, Brown SR, Tornquist SJ, Snyder SP, Garner MM, Blythe LL (1999) Encephalomyelitis associated with a Sarcocystis neurona-like organism in a sea otter. J Am Vet Med Assoc 12:1839–1842 Stanek J, Dubey J, Oglesbee M, Reed S, Lindsay D, Capitini L, Njoku C, Wittitow K, Saville W (2002) Life cycle of Sarcocystis

neurona in its natural intermediate host, the raccoon, Procyon lotor. J Parasitol 88:1151–1158 Suzuki Y (2002) Immunopathogenesis of cerebral toxoplasmosis. J Infect Dis 186:234–240 Tanhauser SM, Cheadle MA, Massey ET, Mayer BA, Schroedter DE, Dame JB, Greiner EC, MacKay RJ (2001) The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona. Int J Parasitol 31:325–329 Tomavo S (2001) The differential expression of multiple isoenzyme forms during stage conversion of Toxoplasma gondii: an adaptive developmental strategy. Int J Parasitol 31:1023–1031 Tomavo S, Boothroyd JC (1995) Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii. Int J Parasitol 25:1293– 1299 Van der Straeten D, Rodrigues-Pousada RA, Goodman HM, Montagu MV (1991) Plant enolase: gene structure, expression, and evolution. Plant Cell 3:719–735 Vonlaufen N, Muller N, Keller N, Naguleswaran A, Bohne W, McAllister MM, Bjorkman C, Muller E, Caldelari R, Hemphill A (2002) Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures. Int J Parasitol 32:1253–1265