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[Display omitted] • Toxoplasma gondii dense granule proteins, GRA47 and GRA72, exhibit interactivity. • GRA47 and GRA72 are required for normal parasitophorous vacuole (PV) morphology. • GRA47 plays a critical role in maintaining the permeability of the PV membrane. • GRA47 and GRA72 affect the growth and virulence of T. gondii. • Simultaneous loss of any two of GRA47, GRA72 or GRA17 may be lethal to T. gondii. Establishing an intact intracellular parasitophorous vacuole (PV) that enables efficient nutrient uptake and protein trafficking is essential for the survival and proliferation of Toxoplasma gondii. Although the PV membrane (PVM)-localized dense granule protein 17 (GRA17) and GRA23 mediate the permeability of the PVM to small molecules, including nutrient uptake and excretion of metabolic by-products, the molecular mechanism by which T. gondii acquires nutrients remains unclear. In this study, we showed that the secreted protein GRA47 contributed to normal PV morphology, PVM permeability to small molecules, growth, and virulence in T. gondii. Co-immunoprecipitation analysis demonstrated potential interaction of GRA47 with GRA72, and the loss of GRA72 affected PV morphology, parasite growth and infectivity. To investigate the biological relationship among GRA47, GRA72, GRA17 and GRA23, attempts were made to construct strains with double gene deletion and overexpressing strains. Only Δ gra 23Δ gra72 was successfully constructed. This strain exhibited a significant increase in the proportion of aberrant PVs compared with the Δ gra23 strain. Overexpressing one of the three related GRAs partially rescued PVs with aberrant morphology in Δ gra47 , Δ gra72 and Δ gra17 , while the expression of the Plasmodium falciparum PVM protein Pf Exp2, an ortholog of GRA17 and GRA23, fully rescued the PV morphological defect in all three Δ gra strains. These results suggest that these GRA proteins may not be functionally redundant but rather work in different ways to regulate nutrient acquisition. These findings highlight the versatility of the nutrient uptake mechanisms in T. gondii , which may contribute to the parasite's remarkable ability to grow in different cellular niches in a very broad range of hosts. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dense granular protein GRA76 was more highly expressed in tachyzoites than in bradyzoites of Toxoplasma gondii. • GRA76 is important for the growth and virulence of T. gondii. • Deletion of gra76 in the Pru strain (PruΔ gra76) increased expression of bradyzoite-associated genes. • PruΔ gra76 exhibited increasd propensity for forming bradyzoites in vitro. Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49_299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔ gra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔ gra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔ gra76 as a candidate for a live-attenuated vaccine. [ABSTRACT FROM AUTHOR]