TOXOPLASMA-GONDII - CHARACTERIZATION OF A MUTANT RESISTANT TO 6-THIOXANTHINE

6-Thioxanthine caused 50% inhibition of the growth of Toxoplasma gondii in human fibroblasts at a concentration of 5 mu g/ml. A mutant induced by treatment with ethylnitrosourea (Thx(R)-1) was 20-fold more resistant than the wildtype. Wild-type parasites grown in Lesch-Nyhan fibroblasts efficiently incorporated hypoxanthine, guanine, and xanthine, but Thx(R)-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 Thx(R)-1 had barely detactable activity with any of these substrates. The basis for the resistance of Thx(R)-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 Thx(R)-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. (C) 1993 Academic Press, Inc.