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Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites

Published online by Cambridge University Press:  13 June 2014

SRIVENY DANGOUDOUBIYAM ,
ZIJING ZHANG  and
DANIEL K. HOWE
SRIVENY DANGOUDOUBIYAM*
Affiliation:
M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA
ZIJING ZHANG
Affiliation:
M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA
DANIEL K. HOWE
Affiliation:
M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA
*
*Corresponding author: Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA. E-mail: [email protected]
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Summary

Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

Keywords

Sarcocystis neuronaApicomplexaHXGPRTpurine salvagetransfection
Type
Special Issue Article
Information
Parasitology , Volume 141 , Special Issue 11: ApiCOWplexa 2013 – 2nd International Meeting on Apicomplexan Parasites in Farm Animals , September 2014 , pp. 1399 - 1405
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Copyright
Copyright © Cambridge University Press 2014 

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References

REFERENCES

Beech, J. (1974). Equine protozoan encephalomyelitis. Veterinary Medicine, Small Animal Clinical 69, 1562, 1564–1566.Google ScholarPubMed
Berens, R. L., Krug, E. C. and Marr, J. J. (1995). Purine and pyrimidine metabolism. In Biochemistry and Molecular Biology of Parasites (ed. Marr, J. J. and Müller, M.), pp. 323–336. Academic Press, London, UK.Google Scholar
Bhatti, M. M. and Sullivan, W. J. Jr. (2005). Histone acetylase GCN5 enters the nucleus via importin-alpha in protozoan parasite Toxoplasma gondii . Journal of Biological Chemistry 280, 5902–5908.Google Scholar
Chaudhary, K., Darling, J. A., Fohl, L. M., Sullivan, W. J. Jr., Donald, R. G., Pfefferkorn, E. R., Ullman, B. and Roos, D. S. (2004). Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii . Journal of Biological Chemistry 279, 31221–31227.Google Scholar
Cheadle, M. A., Yowell, C. A., Sellon, D. C., Hines, M., Ginn, P. E., Marsh, A. E., Dame, J. B. and Greiner, E. C. (2001). The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona . International Journal for Parasitology 31, 843–849.CrossRefGoogle ScholarPubMed
Cheadle, M. A., Ginn, P. E., Lindsay, D. S. and Greiner, E. C. (2002). Neurologic disease in gamma-interferon gene knockout mice caused by Sarcocystis neurona sporocysts collected from opossums fed armadillo muscle. Veterinary Parasitology 103, 65–69.Google Scholar
Donald, R. G. and Roos, D. S. (1998). Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis. Molecular and Biochemical Parasitology 91, 295–305.Google Scholar
Donald, R. G. K., Carter, D., Ullman, B. and Roos, D. S. (1996). Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Journal of Biological Chemistry 271, 14010–14019.Google Scholar
Dubey, J. P., Saville, W. J., Lindsay, D. S., Stich, R. W., Stanek, J. F., Speer, C. A., Rosenthal, B. M., Njoku, C. J., Kwok, O. C., Shen, S. K. and Reed, S. M. (2000). Completion of the life cycle of Sarcocystis neurona . Journal of Parasitology 86, 1276–1280.Google Scholar
Dubey, J. P., Lindsay, D. S., Saville, W. J., Reed, S. M., Granstrom, D. E. and Speer, C. A. (2001 a). A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Veterinary Parasitology 95, 89–131.CrossRefGoogle ScholarPubMed
Dubey, J. P., Saville, W. J., Stanek, J. F., Lindsay, D. S., Rosenthal, B. M., Oglesbee, M. J., Rosypal, A. C., Njoku, C. J., Stich, R. W., Kwok, O. C., Shen, S. K., Hamir, A. N. and Reed, S. M. (2001 b). Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and experimental induction of neurologic disease in raccoons. Veterinary Parasitology 100, 117–129.Google Scholar
Fenger, C. K. (1998). Treatment of equine protozoal myeloencephalitis. Compendium on Continuing Education for the Practising Veterinarian 20, 1154–1157.Google Scholar
Gaji, R. Y. and Howe, D. K. (2009). The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona . Molecular and Biochemical Parasitology 166, 85–88.CrossRefGoogle ScholarPubMed
Gaji, R. Y., Zhang, D., Breathnach, C. C., Vaishnava, S., Striepen, B. and Howe, D. K. (2006). Molecular genetic transfection of the coccidian parasite Sarcocystis neurona . Molecular and Biochemical Parasitology 150, 1–9.CrossRefGoogle ScholarPubMed
Gherardi, A. and Sarciron, M. E. (2007). Molecules targeting the purine salvage pathway in Apicomplexan parasites. Trends in Parasitology 23, 384–389.CrossRefGoogle ScholarPubMed
Hoane, J. S., Carruthers, V. B., Striepen, B., Morrison, D. P., Entzeroth, R. and Howe, D. K. (2003). Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development. International Journal for Parasitology 33, 671–679.Google Scholar
Howe, D. K. and Sibley, L. D. (1997). Development of molecular genetics for Neospora caninum: a complementary system to Toxoplasma gondii . Methods: A Companion to Methods in Enzymology 13, 123–133.Google Scholar
Krug, E. C., Marr, J. J. and Berens, R. L. (1989). Purine metabolism in Toxoplasma gondii . Journal of Biological Chemistry 264, 10601–10607.Google Scholar
MacKay, R. J. (2006). Equine protozoal myeloencephalitis: treatment, prognosis, and prevention. Clinical Techniques in Equine Practice 5, 9–16.Google Scholar
Pfefferkorn, E. R. and Borotz, S. E. (1994). Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine. Experimental Parasitology 79, 374–382.CrossRefGoogle ScholarPubMed
Pfefferkorn, E. R. and Pfefferkorn, L. C. (1978). The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii . Journal of Parasitology 64, 486–492.Google Scholar
Pfefferkorn, E. R., Bzik, D. J. and Honsinger, C. P. (2001). Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine. Experimental Parasitology 99, 235–243.CrossRefGoogle ScholarPubMed
Vaishnava, S., Morrison, D. P., Gaji, R. Y., Murray, J. M., Entzeroth, R., Howe, D. K. and Striepen, B. (2005). Plastid segregation and cell division in the apicomplexan parasite Sarcocystis neurona . Journal of Cell Science 118, 3397–3407.CrossRefGoogle ScholarPubMed
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