In vivoCRISPR screens identify GRA12 as a transcendent secreted virulence factor acrossToxoplasma gondiistrains and mouse subspecies

Abstract

SummaryToxoplasma gondiiparasites exhibit extraordinary host promiscuity owing to over 250 putative secreted proteins that disrupt host cell functions, enabling parasite persistence. However, most of the known effector proteins are specific toToxoplasmagenotypes or hosts. To identify virulence factors that function across different parasite isolates and mouse strains that differ in susceptibility to infection, we performed systematic pooledin vivoCRISPR-Cas9 screens targeting theToxoplasmasecretome. We identified several proteins required for infection across parasite strains and mouse species, of which the dense granule protein 12 (GRA12) emerged as the most important effector protein during acute infection. GRA12 deletion in IFNγ-activated macrophages results in collapsed parasitophorous vacuoles and increased host cell necrosis, which is partially rescued by inhibiting early parasite egress. GRA12 orthologues from related coccidian parasites, includingNeospora caninumandHammondia hammondi,complement TgΔGRA12in vitro, suggesting a common mechanism of protection from immune clearance by their hosts.