Toxoplasma gondii secreted effectors co-opt host repressor complexes to inhibit necroptosis [I]

During infection, Toxoplasma gondii translocate effector proteins directly into the infected host cells to subvert various immune signaling pathways. We identified a novel secreted effector-TgNSM that localizes to the host cell nucleus. Mechanistically, TgNSM drives increased NCoR/SMRT repressor complex levels and enhances transcriptional repression of interferon regulated genes (ISGs). Type I and type II interferons can induce necroptosis by upregulating protein kinase PKR that induces the formation of a necrosome complex consisting of the RIPK1 and RIPK3. The necrosome then activates the pro-necroptotic protein MLKL to execute necrotic cell death. TgNSM acts together with another secreted effector TgIST, previously shown to down-modulate IFN-γ signaling. TgNSM and TgIST block IFN driven expression of PKR and MLKL, thus preventing host cell necroptotic death and assuring survival of intracellular cysts. The mechanism of action of TgNSM highlights a previously unappreciated role of NCoR/SMRT in regulation of necroptosis. We created single and double mutant strains in the type II (ME49) line (i.e. WT, ∆nsm, ∆ist and ∆nsm/∆ist) carrying mCardinal (mCard) driven by a constitutive tubulin promoter and the bradyzoite specific promoter BAG1 driving mNeonGreen (mNG). The strains were grown in HFF cells under alkaline stress to induce T. gondii differentiation into bradyzoites for 5 days. Infected HFF monolayers were then stimulated with IFN-γ for 6 hr, harvested, and infected cells were purified by fluorescence-activated cell sorting (FACS) based on the expression of both mCard and mNG. RNA was then extracted and sequenced using Illumina chemistry on a NovaSeq-6000 using paired end reads extending 150 bases. Three biological replicates were done for each sample.