Molecular Basis for Interferon-mediated Pathogen Restriction in Human Cells

  1. Sumit K. Matta
  2. Hinissan P. Kohio
  3. Pallavi Chandra
  4. Adam Brown
  5. John G. Doench
  6. Jennifer A. Philips
  7. Siyuan Ding
  8. L. David Sibley

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

To define novel mechanisms for cellular immunity to the intracellular pathogen Toxoplasma 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 NF2/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 of T. 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 of T. gondii . RNF213 was also important for control of unrelated intracellular pathogens in human cells treated with IFN, as shown here for Mycobacterium tuberculosis and 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.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The Authors do not wish to provide a response at this time.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary: In this study, the authors attempt to discover new effector mechanisms of IFN-gamma mediated cell autonomous immunity using a human malignant lung cell line infected with T. gondii. A genome-wide screen discovered NF2 (neurofibromatoris 2) as a transcriptional modulator of IRF-1 dependent cell autonomous response induced by IFN-gamma. To increase the chance of discovering true effectors, a focused screed was performed, which yielded the E3 ligase RNF213. This E3 ligase is constitutively expressed but its levels are further upregulated upon exposure to IFN-gamma. Functional studies indicate that RNF213 plays a role in both the basal restriction and the induced/enhanced restriction of T. gondii growth, which occurs inside a well-defined vacuole. Data further showed that RNF213 associates with the parasite vacuole, both at basal and activated states, and is associated with molecular players involved in non-canonical autophagy. However, further analysis indicated that non-canonical autophagy was itself not required for growth restriction mediated by RNF213. Additional studies also indicated a role for RNF213 in cell autonomous immunity to an intracellular bacterium and a virus. In summary, the screens identified a regulators of the antimicrobial transcriptional and effector programs induced by interferons.

    Major comments:

    The title of the article seems misleading as the experiments and data described in the study does not truly provide a mechanistic basis for how pathogen growth restriction occurs. A new title that better reflects the limited extent of the advance reported here should be selected.

    Because RNF213 is constitutively expressed, it is possible that it could independently downregulate parasite growth without the need for other interferon-inducible effectors. Have the authors determined whether overexpression is sufficient in cells that are not exposed to interferon treatment?

    Minor comments:

    Figure 4F. Labelling to highlight key structures in this EM photograph would be

    Referees cross commenting

    The comment by Reviewer 1 regarding lack of ubiquitin staining of parasitophorous vacuole should be reconsidered, because it is shown in Figure 4 by use of FK2 antibody.

    Significance

    Knowledge of how human cells execute cell-autonomous growth restriction of intracellular parasites remains rudimentary. Thus, by identifying regulators of the antimicrobial and effector programs induced by interferons, this study represents an notable advance. However, it did not elucidate a novel effector mechanism of pathogen growth restriction.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript "Molecular Basis for Interferon-mediated Pathogen Restriction in Human Cells" by Sumit Matta et al. describes the identification of RNF213 (ring finger protein 213), an E3 ubiquitin ligase, as essential for IFNg mediated control of T. gondii in human cells (A549, THP-1, HFF). RNF213 was found by a CRISPR/Cas9 based screen of IFNg stimulated genes in A549 cells. Additional data obtained from a genome wide CRISPR/Cas9 screen (using the Brunello library from Addgene) found previously known essential genes for Toxoplasma control such as IRF1, STAT1, JAK2, IFNGR1/2 as well as one novel gene, NF-2, as being important for IFNg mediated Toxoplasma control. Functional data reveal that RNF213 is recruited to the T. gondii PV and that ubiquitination is found at the RNF213 positive PVs. For RNF213 function, ATG5 appears not to be of critical importance. Finally, functional assays determined that RNF213 is also required for the IFNg mediated control of the intracellular pathogen M. tuberculosis and the IFNb mediated control of VSV.

    The study is very well performed and executed, the findings are of broad interest and advance our understanding of host-pathogen relationship on a molecular level.

    There are some critical points that should be addressed by the authors:

    The authors use a vacuolar size growth assay. The authors should verify / compare their assay to determine Toxoplasma control, to e.g. qPCR analysis or 3H-Uracil incorporation in the RNF213ko A549 and THP-1 cells.

    All experiments were conducted with the CTG strain of T. gondii which is a type III strain, the authors should investigate whether RNF213 can also restrict more virulent type II and type I toxoplasma strains.

    The induction (RNA / protein) of RNF213 by titrated amounts of IFNg and IFNb should be investigated and compared in A549 cells.

    Minor points:

    What is the induction of RNF213 in NF-2 deficient cells after IFN stimulation?

    Fig. 1E There are three genes indicated in the top left quadrant (PTEN/TSC1/TSC2) but only 2 green data points shown? Why?

    Significance

    The presented data corroborate and extend a study by Hernandez et al. (mBio. 2022 Oct26;13(5):e0188822. doi: 10.1128/mbio.01888-22. Epub 2022 Sep 26.) with regard to cell autonomous T. gondii defense and add information with regard to immunity against M. tuberculosis and VSV.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Matta et al. investigated, via an initial crispr screen, the host cellular factors involved in T. gondii growth restriction. In the past, different pathways have been implicated in parasite growth suppression including GBPs and IDO1 via tryptophan restriction. As the authors carefully note, the prior studies have notable caveats, and in human cells, other pathways must be involved. To find genes required for T. gondii growth suppression, the authors set a screen to read out parasite vacuole size after IFNg treatment, in a model cell system (A549). In this way, IFNg is the ultimate upstream cytokine that triggers parasite growth restriction and loss of components downstream of the IFNg-IFNgR-STAT1 pathway should be implicated as host anti-parasite effectors. Thus, the screen has the potential to uncover new pathways involved in host resistance.

    Following the screen, the authors initially found NF2 (see below) and then refining their approach to use an ISG-targeted screen, following which they focused on RNF213, recently described as an LPS E3 ligase. The authors chose to divide their manuscript into these two parts. The main critique of the manuscript concerns the fact that neither of the two parts is fully developed.

    Significance

    Critique:

    1. NF2 was clearly a top hit in the genome-wide screen and loss of NF2 by targeted knockout clearly recapitulated the screen result. However, (i) what the mechanism of growth restriction by NF2? After Figure 2, NF2 is dropped and the authors focus on RNF213. (ii) NF2 is not regulated (obviously) by IFNg (Fig. 2A, WT +/- IFNg). But what is the link between the IFN signaling pathway and NF2? It seems that the NF2 KO has less ISG expression (heat map, 2D) although this data is not convincingly shown: Proteomics seems essential here in addition to the transcript measurements. If NF2 regulates an "upstream" event in the IFNg pathway (implied in 2F, secondary screen), the authors should be able to track down the point at which it exerts its effect.
    2. RNF213 clearly plays an unexpected and important role in parasite restriction. However, the mechanisms involved are not clear. (i) The authors state in Figure 4, that RNF213 co-localized with ubiquitinated parasite-contained vacuoles, but this is not shown (there is no Ub staining in Figure 4). (ii) The effects of RNF213 are independent of ATG5. However, what is missing is the overall quantification of Ub +/- IFNg in control, RNF213 and ATG5 KO cells (di-Gly MS seems essential here). (iii) What is the effect of parasitophorous vacuole UB in the RNF213 WT vs. KO cells? (iv) The authors explain that RNF213 is not an obvious ISG in that its transcript does not fit with canonical ISG expression. Therefore, how do the authors link RNF213 activity with the IFNg pathway? (v) Finally, since we now know that RNF213 ubiquitinates LPS, further controls using this pathway may be useful (especially as LPS activates the type 1 IFN response).

    Further comments:

    1. The microscopy images are too small in my view (throughout).
    2. 2E should be should as bar graphs.
  5. Version published to 10.1101/2022.10.21.513197v2 on bioRxiv
  6. Version published to 10.1101/2022.10.21.513197v1 on bioRxiv