Abstract
The malaria parasite Plasmodium falciparum depends entirely on de novo pyrimidine synthesis, as it is unable to salvage these essential nucleotides. This reliance makes the pyrimidine biosynthesis pathway a compelling target for antimalarial drugs, with several inhibitors targeting its rate-limiting enzyme, dihydroorotate dehydrogenase (PfDHODH), already in clinical development. In this study, we investigated the roles of three other pathway enzymes - aspartate transcarbamoylase (PfATC), carbamoyl phosphate synthetase II (PfCPSII), and dihydroorotase (PfDHO). PfATC features a unique N-terminal extension predicted to serve as an apicoplast trafficking peptide. However, using antibodies against the native protein and an epitope-tagged version, we found no evidence of apicoplast localization. Knockdown of PfATC expression proved lethal and could not be rescued by an apicoplast metabolic bypass. Complementation assays further revealed that truncation of the N-terminal domain impaired parasite growth, suggesting that this region is important for PfATC function or stability in vivo. PfCPSII, which harbors large Plasmodium-specific insertions between its catalytic domains, was likewise found to be essential for parasite proliferation. To assess the role of PfDHO, we engineered parasites to salvage uracil via heterologous expression of a yeast enzyme. Deletion of PfDHO in this parasite line resulted in uracil auxotrophy, confirming the enzyme's essential function in pyrimidine synthesis. Together, these findings reveal multiple vulnerable nodes within the pyrimidine biosynthesis pathway.