TgDDP is a novel DIX domain protein that plays an essential role in Toxoplasma endodyogeny.

UNLABELLED: Apicomplexan parasites are protozoan pathogens responsible for major human diseases, including toxoplasmosis, malaria, and cryptosporidiosis. Toxoplasma gondii has emerged as a model for studying cell division in tissue coccidians. Unlike higher eukaryotes that divide by binary fission, Toxoplasma replicates via internal budding (endodyogeny), yet many factors governing daughter cell biogenesis remain poorly understood. Here, we characterize TgDDP (T. gondii DIX-DnaJ domain protein), a previously unannotated protein containing both DIX and DnaJ domains-a unique combination restricted to stramenopiles and apicomplexans. Endogenous tagging revealed that DDP is constitutively expressed and localizes predominantly to the trans-Golgi network in extracellular parasites, with a more dispersed distribution in intravacuolar stages. To assess function, we generated a conditional knockdown line using the auxin-inducible degron system. DDP depletion resulted in a complete block in parasite replication, demonstrating its essential role in the lytic cycle. Microscopy analyses showed that loss of DDP disrupts daughter cell scaffold formation, while nuclear division remains largely intact, indicating a specific role after karyokinesis. Complementation with a wild-type TgDDP allele fully restored parasite replication, and orthologs from related apicomplexans functionally substituted for DDP. Biochemical analysis (SEC-MALS) demonstrated that DDP forms dimers, consistent with DIX domain-containing proteins. Interactome profiling further identified associations with trafficking-related proteins, including clathrins. Together, these findings establish DDP as a critical regulator of daughter cell formation and a potential therapeutic target to inhibit parasite propagation. IMPORTANCE: Toxoplasma gondii is a protozoan parasite that can cause life-threatening disease in humans with immunodeficiency conditions; hence, identifying key factors required for parasite propagation is important to develop novel therapeutics. In this study, we characterize a novel DIX domain-containing protein, DDP, expressed in Toxoplasma. We show that DDP mostly localizes to the trans-Golgi network in extracellular stages and exhibits dispersed localization in the intracellular form. To determine the function of DDP, we generated a conditional knockdown strain and showed that loss of this protein results in abrogation of parasite division. Specifically, parasites lacking DDP are unable to form daughter parasites. Further, the DDP interactome includes trafficking proteins clathrins, along with IMC proteins, thus suggesting that DDP plays an essential role in building daughter cells during endodyogeny.