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.

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

“…Despite much effort from the research community, many apicomplexan proteins still need to be annotated owing to their uniqueness and absence in other model organisms. The CRISPR screens enabled the researchers to investigate hundreds to thousands of genes in Toxoplasma at once in vitro culture or in vivo infection to identify essential genes for parasite growth, metabolism, drug resistance, differentiation, host-pathogen interaction, and virulence (10)(11)(12)(13)(14)(15)(25)(26)(27)(28)(29)(30)(31)(32)(33). Most in vivo CRISPR screens in Toxoplasma have been focused on identifying secreted virulence effectors such as rhoptry bulb proteins (ROPs) and dense granule proteins (GRAs) (11)(12)(13).…”

GRA47 and GRA72 areToxoplasma gondiipore-forming proteins that influence small molecule permeability of the parasitophorous vacuole

“…S2). Endotagging, knockout generation and complementation of GRA72 has been described previously (7).…”

“…To uncover additional genes encoding for proteins involved in nutrient acquisition from the host at the PVM, or the correct trafficking to and insertion into the PVM, in the absence of GRA17, we recently conducted a genome-wide synthetic lethality screen within a parasite background lacking GRA17 (7). This screen discovered multiple GRA proteins (GRA57, GRA70, GRA71, GRA72) that are required for the correct localization of GRA17/GRA23 to the PVM (7,8) while other studies have identified that GRA42, GRA43, and the chaperone-like GRA45 protein are also involved in this process (8,9). GRA72 was confirmed to be synthetically lethal with GRA17 and deletion of GRA72 in a wild-type background resulted in the formation of 'bubble' vacuoles with reduced permeability similar to GRA17 knockout parasites (7).…”

“…This screen discovered multiple GRA proteins (GRA57, GRA70, GRA71, GRA72) that are required for the correct localization of GRA17/GRA23 to the PVM (7,8) while other studies have identified that GRA42, GRA43, and the chaperone-like GRA45 protein are also involved in this process (8,9). GRA72 was confirmed to be synthetically lethal with GRA17 and deletion of GRA72 in a wild-type background resulted in the formation of 'bubble' vacuoles with reduced permeability similar to GRA17 knockout parasites (7).…”

“…AlphaFold2-multimer (10,11) (12) predicted that GRA72 can form a hexameric pore (7). Because deletion of GRA72 also led to mislocalization of GRA17/GRA23, it was unclear if the phenotype of GRA72 knockout parasites was due to mislocalization of GRA17/GRA23 or from GRA72 itself possessing a crucial role as a pore-forming protein.…”