Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

Abstract

ABSTRACT Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii , disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii ( Tg DHODH) failed. To further address the essentiality of Tg DHODH, mutant gene alleles deficient in Tg DHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient Tg DHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that Tg DHODH is essential for the synthesis of pyrimidines and suggest that Tg DHODH is required for a second essential function independent of its role in pyrimidine biosynthesis.