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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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