Lewis rat NLRP1 inflammasome activation is mediated by three Toxoplasma gondii dense granule proteins

The Lewis rat is the only known warm-blooded animal that has sterile immunity to Toxoplasma. Upon invasion of Lewis rat macrophages Toxoplasma rapidly activates the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 1 (NLRP1) inflammasome resulting in interleukin (IL)-1β secretion and a form of cell death known as pyroptosis, which prevents Toxoplasma replication. Using a chemical mutagenesis screen we identified Toxoplasma mutants that no longer induced pyroptosis. Whole genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42 and GRA43 that are individually required for inflammasome activation in Lewis rat macrophages. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites leads to greatly reduced cell death and reduced IL-1β secretion. Lewis rat macrophage infected with parasites containing single, double or triple deletion of these GRAs showed similar levels of cell viability suggesting the three GRAs function in the same pathway that activates the inflammasome. Deletion of GRA42 and GRA43 resulted in GRA35, and other GRAs, being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Toxoplasma deficient in GRA35, GRA42 or GRA43 do not establish chronic infection in Lewis rats, but have reduced cyst number in parasite-susceptible F344 rats, in which Toxoplasma does not activate the NLRP1 inflammasome, revealing these GRAs determine parasite in vivo fitness independent of their role in inflammasome activation. Overall, our data suggest that Toxoplasma dense granule proteins that localize to the parasitophorous vacuole membrane are novel mediators of host NLRP1 inflammasome activation.