Functional analyses of Toxoplasma gondii dihydroorotase reveal a promising anti‐parasitic target

Abstract Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine‐5′‐monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of Tg DHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L‐dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHO H35A and DHO D284E mutant strains proliferated much slower than DHO‐expressing parasites, suggesting an essential role of both Tg DHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO‐deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that Tg DHO contributes to parasite growth both in vitro and in vivo. The significantly differences between Tg DHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of Tg DHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of Tg DHO in parasite growth and reveal that it is a promising anti‐parasitic target for future control of toxoplasmosis.