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

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 HeLa stable line expressing TgNSM-TY upon DOX treatment. For RNA seq experiments in HeLa cells, HeLa-tet and HeLa-tet-TgNSM-TY cell lines were seed at a density of 0.06x10e6 /ml in T25 flasks. The next day DOX (Sigma) was added to a final concentration of 1µg/ml. After 48 hr culture media was replaced for fresh DOX media ±IFNγ 100U/ml. Cells were collected 6 hr post ±IFNγ media swap and their RNA was extracted and sequenced using Illumina chemistry on a illumina NovaSeq 6000 using paired-end reads extending 150 bases. Three biological replicates were done for each sample.