Molecular Basis for Interferon-mediated Pathogen Restriction in Human Cells

Abstract

To define novel mechanisms for cellular immunity to the intracellular pathogenToxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to provide an unbiased assessment of genes important for IFN-γ-dependent growth restriction. We revealed a previously unknown role for the tumor suppressor NF-2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an additional focused ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as essential for IFN-γ mediated control ofT. gondii. RNF213 mediated ubiquitination of targets on the parasite-containing vacuole and growth restriction in response to IFN-γ in a variety of cell types, thus identifying a conserved factor that plays a prominent role in human cells. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a non-canonical autophagy pathway that has previously been implicated in control ofT. gondii. RNF213 was also important for control of unrelated intracellular pathogens in human cells treated with IFN, as shown here forMycobacterium tuberculosisand Vesicular Stomatitis Virus. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity to a broad spectrum of intracellular pathogens in human cells.

A heterotrimeric complex ofToxoplasmaproteins promotes parasite survival in interferon gamma stimulated human cells

“…Ubiquitination of the PVM is a marker for parasite clearance in IFNγ-activated human cells (Clough et al ., 2016; Yao et al ., 2021; Hernandez et al ., 2022; Matta et al ., 2022). We therefore assessed if deletion of GRA57, GRA70 or GRA71 leads to differences in ubiquitination of the PVM.…”

“…Recent work has found that RNF213, an IFNγ-induced host E3 ubiquitin ligase, mediates the majority of ubiquitin recruitment to both RH and PRU Toxoplasma vacuoles in HFFs (Hernandez et al ., 2022; Matta et al ., 2022); however, the target of this ligase is currently unknown. The observation that GRA57/GRA70/GRA71 deletion leads to reduced ubiquitination of the PV poses the intriguing question of whether the complex is required for RNF213 recruitment, is a direct target of ubiquitination, or is responsible for correct positioning of an RNF213 target at the PV.…”

“…Compared to type II & type III parasites, type I parasites are more resistant to ubiquitination and clearance in both HUVECs and HeLa cells. Recent evidence suggests a major function for the E3 ligase RNF213 in ubiquitination of the PVM and enhancing parasite clearance, in multiple human cell types and in a strain-independent manner (Hernandez et al ., 2022; Matta et al ., 2022). The importance of each E3 ligase and ubiquitin linkage therefore depends on the exact combination of host species, parasite strain and cell type studied, but nevertheless ubiquitination has emerged as a key process in the regulation of Toxoplasma clearance.…”

Host E3 ubiquitin ligase ITCH mediatesToxoplasma gondiieffector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity

“…These results indicate that ITCH does not exclusively mediate the ubiquitination of the vacuole in HFFs and may affect the dynamics of other host E3 ubiquitin ligases that mark the PVM with ubiquitin. RNF213, another host E3 ubiquitin ligase that is an IFNγ-stimulated gene and actively accumulates on the vacuole, mediates Toxoplasma growth inhibition (40, 41). We found that Δ gra35 parasites had significantly less RNF213 on the PVM, especially in IFNγ-activated Itch -knockout HFFs, compared to wild-type and Δ gra35 + GRA35HA parasites ( Fig 4H ).…”

A Combination of Four Nuclear Targeted Effectors ProtectsToxoplasmaAgainst Interferon Gamma Driven Human Host Cell Death During Acute Infection

“…These mechanisms include processes such as nutrient deprivation (29), endo-lysosomal destruction (30, 31), autophagy (32), and induction of host cell death (33). A comprehensive gain of function screen of hundreds of ISGs induced by IFNγ and a genome wide loss of function screen conducted in human cells uncovered only a few host effectors capable of restricting T. gondii growth including RARRES3 that acts only against type III strains (34) and the E-3 ubiquitin ligase RNF213 that acts against multiple strain types in a range of cell types (35). These findings emphasize the complexity of immunity to T. gondii , indicating that a multitude of factors are likely involved in influencing the outcome of infection.…”