Interferon-Inducible E3 Ligase RNF213 Facilitates Host-Protective Linear and K63-Linked Ubiquitylation of Toxoplasma gondii Parasitophorous Vacuoles

Abstract

The obligate intracellular protozoan pathogen Toxoplasma gondii infects a wide range of vertebrate hosts and frequently causes zoonotic infections in humans. Whereas infected immunocompetent individuals typically remain asymptomatic, toxoplasmosis in immunocompromised individuals can manifest as a severe, potentially lethal disease, and congenital Toxoplasma infections are associated with adverse pregnancy outcomes. The protective immune response of healthy individuals involves the production of lymphocyte-derived cytokines such as interferon gamma (IFN-γ), which elicits cell-autonomous immunity in host cells. IFN-γ-inducible antiparasitic defense programs comprise nutritional immunity, the production of noxious gases, and the ubiquitylation of the Toxoplasma-containing parasitophorous vacuole (PV). PV ubiquitylation prompts the recruitment of host defense proteins to the PV and the consequential execution of antimicrobial effector programs, which reduce parasitic burden. However, the ubiquitin E3 ligase orchestrating these events has remained unknown. Here, we demonstrate that the IFN-γ-inducible E3 ligase RNF213 translocates to Toxoplasma PVs and facilitates PV ubiquitylation in human cells. Toxoplasma PVs become decorated with linear and K63-linked ubiquitin and recruit ubiquitin adaptor proteins in a process that is RNF213 dependent but independent of the linear ubiquitin chain assembly complex (LUBAC). IFN-γ priming fails to restrict Toxoplasma growth in cells lacking RNF213 expression, thus identifying RNF213 as a potent executioner of ubiquitylation-driven antiparasitic host defense. IMPORTANCE Globally, approximately one out of three people become infected with the obligate intracellular parasite Toxoplasma. These infections are typically asymptomatic but can cause severe disease and mortality in immunocompromised individuals. Infections can also be passed on from mother to fetus during pregnancy, potentially causing miscarriage or stillbirth. Therefore, toxoplasmosis constitutes a substantial public health burden. A better understanding of mechanisms by which healthy individuals control Toxoplasma infections could provide roadmaps toward novel therapies for vulnerable groups. Our work reveals a fundamental mechanism controlling intracellular Toxoplasma infections. Cytokines produced during Toxoplasma infections instruct human cells to produce the enzyme RNF213. We find that RNF213 labels intracellular vacuoles containing Toxoplasma with the small protein ubiquitin, which functions as an "eat-me" signal, attracting antimicrobial defense programs to fight off infection. Our work therefore identified a novel antiparasitic protein orchestrating a central aspect of the human immune response to Toxoplasma.

Keywords: LUBAC; RNF213; TAX1BP1; Toxoplasma; cell-autonomous immunity; interferons; linear ubiquitin; mysterin; parasitology; parasitophorous vacuole; ubiquitination; xenophagy.

FIG 1

The LUBAC complex is dispensable for IFN-γ-mediated Toxoplasma PV ubiquitylation and clearance. (A) Western blotting showing absence of HOIP and HOIL-1 expression in CRISPR-generated deletion (KO) A549 cells. (B) WT, HOIP KO, and HOIL-1 KO A549 cells were cotransfected with NF-κB-Luc and hRluc/TK reporter constructs. At 24 h posttransfection, cells were left unprimed or primed with TNF-α (100 ng/mL) for 24 h, and cell extracts were analyzed for luciferase activity. (C) Unprimed or IFN-γ-primed (100 U/mL) WT, HOIP KO, and HOIL-1 KO A549 cells were infected with Toxoplasma type II (Pru) at an MOI of 3 for 6 h. Representative confocal images and quantification of ubiquitin (FK2) and M1 recruitment to PVs are shown. Statistical comparisons are shown for groups “both.” (D) Unprimed or IFN-γ-primed (100 U/mL) WT, HOIP KO, and HOIL-1 KO A549 cells were infected at an MOI of 2 with Toxoplasma type II (Pru), and parasites per vacuole were quantified at 24 hpi. (E and F) Unprimed and IFN-γ-primed (100 U/mL) WT A549, HOIP KO, and HOIL-1 KO cells were infected with luciferase-expressing strains RH and Me49 at an MOI of 1 or MOI of 2, respectively, and cell lysates were analyzed for luciferase activity at 24 hpi. Growth of each strain was normalized to growth in WT unprimed cells. All data depict the mean ± SEM from 3 to 4 independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 2

The IFN-γ-inducible E3 ligase RNF213 colocalizes with Toxoplasma PVs in A549 and HFF-1 cells. (A) Western blotting showing expression of RNF213 during infection in unprimed or IFN-γ-primed (100 U/mL) WT A549 and HFF-1 cells. Cells were infected with Toxoplasma strains RH or Pru at an MOI of 3, and protein lysates were collected at 6 hpi. (B and C) Representative confocal images and quantification of RNF213 recruitment to Toxoplasma RH and Pru PVs in WT A549 and HFF-1 cells. Unprimed or IFN-γ-primed (100 U/mL) WT A549 and HFF-1 cells were infected with RH or Pru at an MOI of 3. RNF213 recruitment was quantified at 2 hpi, 4 hpi, and 6 hpi. All data depict the mean ± SEM from 3 independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 3

RNF213 colocalizes with ubiquitin at the Toxoplasma PV and facilitates M1- and K63-linked ubiquitylation. (A and B) Representative confocal images of RNF213 and ubiquitin (FK2) localization to Toxoplasma type II Pru PVs in IFN-γ-primed WT A549 (A) and HFF-1 (B) cells at 6 hpi. Colocalization was quantified at 2 hpi, 4 hpi, and 6 hpi, and combined data from three independent experiments are shown. Statistical comparisons are shown for groups “both.” (C) Western blotting for RNF213 expression in unprimed and IFN-γ-primed (100 U/mL) WT deletion (KO) A549 pools. (D) Representative confocal images of ubiquitin (FK2) and ubiquitin linkages M1, K63, and K48 found on the PV in WT A549 cells at 6 hpi. Quantification of ubiquitin linkages detectable on Toxoplasma Pru PVs in IFN-γ-primed (100 U/mL) WT and RN213 KO A549 cells infected at an MOI of 3 for 6 h. All data depict the mean ± SEM from 3 independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 4

RNF213 facilitates recruitment of ubiquitin adaptors to the Toxoplasma PV. (A) Representative confocal images and quantification of ubiquitin adaptors (p62, TAX1BP1, NDP52, and OPTN) and ubiquitin (FK2) colocalization to Toxoplasma Pru PVs in WT A549 cells. Unprimed and IFN-γ-primed (100 U/mL) cells were infected with Toxoplasma Pru at an MOI of 3 for 6 h. Statistical comparisons is shown for groups “both.” (B) Unprimed and IFN-γ-primed (100 U/mL) WT and RN213 KO A549 cells were infected with Toxoplasma Pru at an MOI of 3, and the localization of p62, TAX1BP1, NDP52, and OPTN to Toxoplasma PVs was quantified at 6 hpi. All data depict the mean ± SEM from 3 to 4 independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 5

RNF213 mediates restriction of Toxoplasma in IFN-γ-primed A549. (A and B) Unprimed and IFN-γ-primed (100 U/mL) WT and RNF213 KO A549 cells were infected with Toxoplasma type I RH and type II Pru at an MOI of 1 or MOI of 2, respectively, and parasites per vacuole were quantified at 24 hpi. Statistical comparisons are shown for groups “1 parasite per vacuole.” (C and D) Toxoplasma plaque number in unprimed or IFN-γ-primed (100 U/mL) WT and RNF213 KO A549 cell pools. Cell monolayers in 96-well plates were infected with 50 Toxoplasma RH or Pru tachyzoites per well, and plaque formation was quantified at 72 hpi. All data depict the mean ± SEM from 3 to 4 independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.