Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine

Exp Parasitol. 1994 Nov;79(3):374-82. doi: 10.1006/expr.1994.1099.

Abstract

6-Thioxanthine caused 50% inhibition of the growth of Toxoplasma gondii in human fibroblasts at a concentration of 5 micrograms/ml. A mutant induced by treatment with ethylnitrosourea (ThxR-1) was 20-fold more resistant than the wildtype. Wild-type parasites grown in Lesch-Nyhan fibroblasts efficiently incorporated hypoxanthine, guanine, and xanthine, but ThxR-1 incorporated each of these precursors less than 2% as well as the wildtype did. Soluble extracts of wild-type parasites had potent phosphoribosyltransferase activities for hypoxanthine, guanine, and xanthine, while extracts of ThxR-1 had barely detectable activity with any of these substrates. The basis for the resistance of ThxR-1 to 6-thioxanthine is, therefore, the lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase. Thus, salvage pathways that employ this enzyme are not essential for the acquisition of purines, which the parasite must obtain from the host cell. Incubation in a medium containing mycophenolic acid and xanthine allowed the efficient recovery of wild-type T. gondii in the presence of many ThxR-1 parasites. Together with the use of 6-thioxanthine to detect resistant mutants in the presence of many wild-type parasites, this procedure provides a simple selection and back-selection for mutations that affect the hypoxanthine-guanine phosphoribosyltransferase gene of T. gondii.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cells, Cultured
  • Ethylnitrosourea
  • Fibroblasts / parasitology
  • Guanine / metabolism
  • Humans
  • Hypoxanthine
  • Hypoxanthine Phosphoribosyltransferase / metabolism
  • Hypoxanthines / metabolism
  • Mutagenesis
  • Toxoplasma / drug effects*
  • Toxoplasma / enzymology
  • Toxoplasma / genetics
  • Xanthine
  • Xanthines / metabolism
  • Xanthines / pharmacology*

Substances

  • Hypoxanthines
  • Xanthines
  • Xanthine
  • 6-thioxanthine
  • Hypoxanthine
  • Guanine
  • Hypoxanthine Phosphoribosyltransferase
  • Ethylnitrosourea