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Item The Dually Localized EF-Hand Domain-Containing Protein TgEFP1 Regulates the Lytic Cycle of Toxoplasma gondii(MDPI, 2022-05-21) Dave, Noopur; LaFavers, Kaice; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of MedicineThe propagation of the obligate intracellular parasite Toxoplasma gondii is tightly regulated by calcium signaling. However, the mechanisms by which calcium homeostasis and fluxes are regulated in this human pathogen are not fully understood. To identify Toxoplasma’s calcium homeostasis network, we have characterized a novel EF-hand domain-containing protein, which we have named TgEFP1. We have determined that TgEFP1 localizes to a previously described compartment known as the plant-like vacuole or the endosomal-like compartment (PLV/ELC), which harbors several proteins related to ionic regulation. Interestingly, partial permeabilization techniques showed that TgEFP1 is also secreted into the parasitophorous vacuole (PV), within which the parasite divides. Ultrastructure expansion microscopy confirmed the unusual dual localization of TgEFP1 at the PLV/ELC and the PV. Furthermore, we determined that the localization of TgEFP1 to the PV, but not to the PLV/ELC, is affected by disruption of Golgi-dependent transport with Brefeldin A. Knockout of TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress. Thus, our work has revealed an interplay between the PV and the PLV/ELC and a role for TgEFP1 in the regulation of calcium-dependent events.Item A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii(Wiley, 2017-09) LaFavers, Kaice A.; Nogueras, Karla M. Márquez; Coppens, Isabelle; Moreno, Silvia N.J.; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of MedicineToxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.