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

Abstract

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' Δgra17 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 Δgra17 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 Δgra17 parasites. Furthermore, Δgra72 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.