Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 12;7(1):5209.
doi: 10.1038/s41598-017-05487-7.

TRIM21 is critical for survival of Toxoplasma gondii infection and localises to GBP-positive parasite vacuoles

Clémence Foltz  1 , Anna Napolitano  1 , Rabia Khan  1 , Barbara Clough  1 , Elizabeth M Hirst  1 , Eva-Maria Frickel  2
Affiliations

TRIM21 is critical for survival of Toxoplasma gondii infection and localises to GBP-positive parasite vacuoles

Clémence Foltz et al. Sci Rep. .

Abstract

Interferon gamma (IFNγ) is the major proinflammatory cytokine conferring resistance to the intracellular vacuolar pathogen Toxoplasma gondii by inducing the destruction of the parasitophorous vacuole (PV). We previously identified TRIM21 as an IFNγ-driven E3 ubiquitin ligase mediating the deposition of ubiquitin around pathogen inclusions. Here, we show that TRIM21 knockout mice were highly susceptible to Toxoplasma infection, exhibiting decreased levels of serum inflammatory cytokines and higher parasite burden in the peritoneum and brain. We demonstrate that IFNγ drives recruitment of TRIM21 to GBP1-positive Toxoplasma vacuoles, leading to Lys63-linked ubiquitination of the vacuole and restriction of parasite early replication without interfering with vacuolar disruption. As seen in vivo, TRIM21 impacted the secretion of inflammatory cytokines. This study identifies TRIM21 as a previously unknown modulator of Toxoplasma gondii resistance in vivo thereby extending host innate immune recognition of eukaryotic pathogens to include E3 ubiquitin ligases.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
TRIM21-deficient mice are susceptible to Toxoplasma infection correlating with higher parasite burden and decreased levels of proinflammatory cytokines. (a) Survival curves of wild-type versus TRIM21-deficient mice infected intraperitoneally with 5 × 104 type II Toxoplasma (n = 18 wild-type, n = 22 TRIM21 knockout). Curves were compared by log-rank survival analysis of Kaplan-Meier curves, ****p < 0.0001. (b) The clinical score, assessed by piloerection, a hunched position and immobility, was recorded throughout the time of the experiment (n = 18 wild-type, n = 22 TRIM21 knockout). Mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001, 2-way ANOVA. (c) The evolution of the firefly luciferase-expressing parasite load in wild-type versus TRIM21-deficient mice was assessed by in vivo imaging after injection of luciferin at 3, 5 and 7 days post-infection (n = 18 wild-type, n = 22 TRIM21 knockout). Mean ± SEM, *p < 0.05, 2-way ANOVA. (d) Quantitative analysis of the number of GFP-expressing Toxoplasma tachyzoites in the brain of wild-type and TRIM21-deficient mice at 7d p.i. (n = 14 wild-type, n = 11 TRIM21 knockout). Data pooled from three independent experiments. Mean ± SEM, ***p < 0.001, unpaired t-test. (e) The serum of wild-type and TRIM21-deficient mice infected intraperitoneally with type II Toxoplasma was collected at 3 and 7 days post-infection. Cytokine levels were measured using multiplex. Mean ± SEM, *p < 0.05, unpaired t-test.
Figure 2
Figure 2
TRIM21 and ubiquitin are targeted to GBP1-positive type II Toxoplasma vacuoles in IFNγ-stimulated cells dependent on parasitic virulence factors. (a) Representative immunofluorescence confocal images of IFNγ-stimulated mouse embryonic fibroblasts infected 1 h with type II Toxoplasma and co-stained for GBP1 and TRIM21. Scale bar is 5 μm. (b) Quantification of GBP1 and TRIM21 co-recruitment to the parasite-containing vacuoles 1 h p.i. Data pooled from three independent experiments. (c) Representative immunofluorescence confocal images of IFNγ-stimulated mouse embryonic fibroblasts infected 1 h with type II Toxoplasma and co-stained for GBP1 and ubiquitin. Scale bar is 5 μm. (d) Quantification of ubiquitin- and/or GBP1-positive parasite-containing vacuoles in IFNγ-stimulated mouse embryonic fibroblasts infected 1 h with the canonical Toxoplasma type II (Pru) and type III (CEP) strains, as well as with Toxoplasma type II strain transgenic for the type I Rop16 (Pru Rop16I), and Toxoplasma type III strain transgenic for the type I version of Rop18 (CEP Rop18I). Data pooled from three independent experiments. Mean ± SEM, ****p < 0.0001, 2-way ANOVA. (e) Quantification of TRIM21- and/or GBP1-positive parasite-containing vacuoles in IFNγ-stimulated mouse embryonic fibroblasts infected 1 h with the canonical Toxoplasma type III (CEP) strain and Toxoplasma type III strain transgenic for the type I version of Rop18 (CEP Rop18I). Data pooled from three independent experiments. Mean ± SEM, *p < 0.05, **p < 0.005, 2-way ANOVA.
Figure 3
Figure 3
TRIM21 mediates Lys63-linked ubiquitination around type II Toxoplasma. (a) Representative immunofluorescence confocal images of IFNγ-stimulated wild-type (upper panel) or TRIM21 knockout (lower panel) mouse embryonic fibroblasts infected 1 h with type II Toxoplasma expressing GFP and stained for Lys48 ubiquitin (left panel) or Lys63 ubiquitin (right panel). Scale bar is 5 μm. (b) Quantification of ubiquitin- (Ub), Lys48 ubiquitin- and Lys63 ubiquitin-positive parasite-containing vacuoles in IFNγ-stimulated wild-type versus TRIM21 knockout MEFs infected 1 h with type II Toxoplasma. Data were pooled from three independent experiments. Mean ± SEM, *p < 0.05, **p < 0.005, 2-way ANOVA. (c) Quantification of Lys63 ubiquitin-positive parasite-containing vacuoles in IFNγ-stimulated mouse embryonic fibroblasts infected 1 h with the canonical Toxoplasma type III (CEP) strain and Toxoplasma type III strain transgenic for the type I version of Rop18 (CEP Rop18I). Mean ± SEM, **p < 0.005, 2-way ANOVA. (d) Quantification of GBP1 and GBP2 localisation to the parasite-containing vacuoles in IFNγ-stimulated wild-type versus TRIM21 knockout mouse embryonic fibroblasts 1 h p.i. Data pooled from three independent experiments, unpaired t-test.
Figure 4
Figure 4
TRIM21 restricts early parasite replication and modulates cytokine production during Toxoplasma infection. (a) Ultrastructural analysis of type II Toxoplasma PVM in IFNγ-stimulated wild-type versus TRIM21 knockout mouse embryonic fibroblasts. The lower images are enlarged views from the upper images. Red arrows indicate blebbing and disruption of the parasitophorous vacuole. Scale bar is 200 nm. (b) Percentage of infection with Toxoplasma in untreated or IFNγ-stimulated TRIM21 knockout and wild-type cells at 1 h p.i.. Data pooled from three independent experiments. Mean ± SEM, p < 0.05, 2-way ANOVA. (c) Quantification of the number of parasites per vacuole in IFNγ-stimulated MEFs infected for 15 h with type II Toxoplasma. Invaded Toxoplasma were assessed for GBP1 recruitment. Data pooled from three independent experiments. Mean ± SEM, ****p < 0.0001, 2-way ANOVA. (d) Representative confocal images of replicating type II Toxoplasma in IFNγ-stimulated MEFs at 15 h post-infection. GBP1 staining in red, type II Toxoplasma in green. White arrows indicate highly replicative Toxoplasma vacuoles. Scale bar is 10 μm. (e) Cytokine production in culture supernatants of IFNγ-stimulated wild-type versus TRIM21-deficient MEFs infected with type II Toxoplasma for 18 h. Data representative of three experiments. Mean ± SEM, **p < 0.01, ***p < 0.005, unpaired t-test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363:1965–1976. doi: 10.1016/S0140-6736(04)16412-X. - DOI - PubMed
    1. Munoz M, Liesenfeld O, Heimesaat MM. Immunology of Toxoplasma gondii. Immunological Review. 2011;240:269–285. doi: 10.1111/j.1600-065X.2010.00992.x. - DOI - PubMed
    1. Lingelbach K, Joiner KA. The parasitophorous vacuole membrane surrounding Plasmodium and Toxoplasma: an unusual compartment in infected cells. J. Cell. Sci. 1998;111(Pt 11):1467–1475. - PubMed
    1. Suss-Toby E, Zimmerberg J, Ward GE. Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc. Natl. Acad. Sci. USA. 1996;93:8413–8418. doi: 10.1073/pnas.93.16.8413. - DOI - PMC - PubMed
    1. Jones TC, Hirsch JG. The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 1972;136:1173–1194. - PMC - PubMed

Publication types

MeSH terms