A novel ENTH domain-containing protein TgTEPSIN is essential for structural maintenance of the plant-like vacuolar compartment and bradyzoite differentiation in toxoplasma gondii.

Toxoplasma gondii is an intracellular and parasitic protozoon that harbors specialized cellular structures and molecular mechanisms, including the Plant-like Vacuolar Compartment (PLVAC). The PLVAC performs multifaceted roles in the parasite, contributing to ion homeostasis, proteolysis, pH regulation, and autophagy. Despite significant efforts over the past decade to characterize the PLVAC, the proteins localized to this organelle remain largely unidentified. In this study, we utilized TurboID and genetic engineering techniques to uncover additional biological characteristics and the conferring components in the PLVAC. By exploiting the bait PLVAC proteins cathepsin L (CPL) and chloroquine resistance transporter (CRT), we identified 9 novel PLVAC-associated proteins in the compartment. Further essentiality screening reveals that TgTEPSIN is required for the parasite lytic cycle. Further phenotypic analysis demonstrated the depletion of TgTEPSIN resulted in defects in the maintenance of PLVAC, virulence in mice as well as bradyzoite differentiation. Collectively, our findings broaden the repertoire of PLVAC proteins and provide new insights into the essential component and roles of the PLVAC in T. gondii.