GRA47 and GRA72 are pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole.

, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified as synthetically lethal with . Deleting produced similar phenotypes to parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of resulted in an aberrant 'bubble vacuole' morphology with reduced small molecule permeability, mirroring the phenotype observed in and knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δ parasites and parasites expressing GRA47 with a histidine mutation had reduced proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against infections.