Genome-wide CRISPR screen identifies ELFN2 as a key regulator of host autophagy and lipid metabolism important for Toxoplasma gondii proliferation

Toxoplasma gondii is a globally prevalent foodborne zoonotic pathogen that threatens animal production and human health. Consumption of undercooked meat like pork and lamb is a major route of human Infection. In this study, we performed a genome-wide CRISPR knockout screen in the porcine cell line PK15 to identify host factors that are critical for T. gondii replication. The results showed that disrupting the ELFN2 (extracellular leucine rich repeat and fibronectin type III domain containing 2) gene in PK15 did not affect host cell growth, but significantly reduced the proliferation of Toxoplasma parasites. Loss of ELFN2 decreased macroautophagy/Autophagy in PK15 cells and impaired lipid metabolism, resulting in reduced lipid availability for the parasites and consequent suppression of T. gondii proliferation. Exogenous lipid supplementation or pharmacological activation of Autophagy could fully restore the replication of parasites in ΔELFN2 cells. The requirement of host ELFN2 for optimal Parasite proliferation in vivo was validated by constructing elfn2^-/- mice, which showed increased resistance to T. gondii Infection and reduced Parasite burden, highlighting the value of ELFN2 in breeding Toxoplasma-resistant Animals. Notably, naturally occurring loss-of-function mutations in ELFN2 could be found in certain pig breeds, further indicating the feasibility of breeding T. gondii-resistant Animals like pigs, to reduce the transmission of Toxoplasma.Abbreviations: 3-MA: 3-methyladenine; ATG5: Autophagy related 5; ATG7: Autophagy related 7; BODIPY-C12: BODIPY FL C₁₂; CCK-8: Cell Counting Kit-8; ComN2: complemented with an ectopic copy ofELFN2; ELFN2: extracellular leucine rich repeat and fibronectin type III domain containing 2;elfn2^-/-:elfn2homozygous knockout; FASII: type II synthesis pathway; GFP: green fluorescent protein; KO: knockout; LD: lipid droplets; MG: monoacylglycerol; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; PV: parasitophorous vacuole; PVM: parasitophorous vacuole membrane; T. gondii: Toxoplasma gondii; WT: wild type.