Targeted in vivo screens identify GRA12 as a strain and mouse-transcendent secreted virulence factor of Toxoplasma gondii

Toxoplasma survives in a remarkably vast repertoire of host cell types and species. This ability is granted by over 200 rhoptry and dense granule proteins secreted by the parasite during or after invasion. Most effectors have been shown to be either host- and/or parasite strain-specific, such as the ROP18 kinase which is a key virulence factor only in the context of virulent parasites infecting laboratory mouse strains. However, effector proteins required to colonise more resistant mice such as the M. m. castaneus and the M. m. musculus subspecies, or virulence factors that are shared across Toxoplasma lineages are still unknown.

Here, we performed CRISPR-Cas9 in vivo screens of the Toxoplasma secretome in type I, II, III and the South American VAND isolate in mouse strains that differ in their susceptibility to infection. We identified the dense granule protein 12 (GRA12) as a prominent strain-transcendent virulence factor during acute infection in resistant and susceptible mice. A CRISPR screen in IFNγ-stimulated macrophages in vitro confirmed the importance of GRA12 for Toxoplasma survival and suggests the protein protects the parasite from cell-autonomous immune responses.

We established GRA12 KO and complemented strains in the RH ΔKU80 background and in a newly created VAND ΔKU80 strain and confirmed GRA12 role as a virulence factor in resistant murine subspecies in vitro and in vivo. Lack of GRA12 results in loss of vacuolar space and ruffling of the parasitophorous vacuole membrane as observed by transmission electron microscopy. Macrophages infected with GRA12-KO parasites, but not with complemented or parental strains, undergo a IFNγ-dependent cell death with features of necrosis. GRA12 homologs of the closely related species Neospora caninum and Hammondia hammondi rescue the restriction of GRA12-KO parasites, suggesting a conserved function beyond Toxoplasma.

In summary, we identified the first Toxoplasma virulence factor to date that is shared across parasite and host murine strains, and which is critical for cell-autonomous survival. Further investigation on GRA12 will elucidate a Toxoplasma core mechanism to evade the host-mediated clearance.