Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma

Author summaryPathogens that reside in a membrane-bound vacuole inside host cells need to overcome the vacuole membrane barrier to acquire host nutrients. The vacuolar parasite Toxoplasma gondii makes pores in the vacuole membrane using two proteins, GRA17 and GRA23, that are required for the diffusion of small host molecules, such as nutrients, across the membrane. GRA17 and GRA23 are exocytosed from the parasite's dense granule secretory organelles into the vacuolar lumen. However, the exact mechanism that subsequently determines their proper localization into the vacuole membrane is not known. In parasites that are missing the GRA17 gene (& UDelta;gra17), GRA23 becomes a critical factor for parasite survival. We exploited this vulnerability by conducting a screen that determined what parasite genes are essential in & UDelta;gra17 but not in wild-type parasites. This screen identified multiple Toxoplasma genes that encode for proteins that are required for the correct localization of multiple dense granule proteins, including GRA17 and GRA23. Because dense granule proteins can mediate host-parasite interactions, the parasite proteins that are needed for the correct localization of dense granule proteins are critical for parasite fitness. Our screen also identified a dense granule protein with the predicted ability to form pores in the vacuole membrane. These pores are likely to play a role in facilitating the acquisition of host nutrients. Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' & UDelta;gra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and & UDelta;gra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for & UDelta;gra17 parasites. Furthermore, & UDelta;gra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.