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. 2013 Jan 2;110(1):294-9.
doi: 10.1073/pnas.1205635110. Epub 2012 Dec 17.

Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication

Daniel Degrandi  1 , Elisabeth Kravets, Carolin Konermann, Cornelia Beuter-Gunia, Verena Klümpers, Sarah Lahme, Eva Rasch, Anne K Mausberg, Sandra Beer-Hammer, Klaus Pfeffer
Affiliations

Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication

Daniel Degrandi et al. Proc Natl Acad Sci U S A. .

Abstract

IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFN-γ-induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the C-terminal domain is sufficient and essential for recruitment to the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of mGBP2. (A) mGBP2 protein expression in BMDM after 16-h stimulation with the indicated stimuli. (B) Protein expression of mGBP2 in various cell subtypes. Splenocytes of C57BL/6 mice were stained using B220+- or CD90+-specific antibodies (BD) and purified by cell sorting (BD FACS Aria), then stimulated for 16 h with the indicated stimuli. (C) Induction of mGBP2 is IRF1 dependent. Embryonic fibroblasts of IRF1-deficient mice were stimulated with IFN-γ for 16 h. (D) Protein expression of mGBP2 in the brain of T. gondii infected mice. C57BL/6 mice were infected i.p. with 20 cysts of freshly prepared T. gondii (ME49). After 5, 7, or 12 d, mice were killed and the brains were prepared. As positive control for Western blotting, cell lysate of IFN-γ–stimulated WT MEFs was used (+). All experiments were done in triplicate; one representative experiment is shown.
Fig. 2.
Fig. 2.
Infection of mGBP2-deficient mice and cells with T. gondii. (A) C57BL/6, IRF1−/−, and mGBP2−/− mice were infected i.p. with T. gondii (40 cysts of strain ME49) and monitored for 60 d. The survival rate of WT (n = 14), IRF1−/− (n = 5), and mGBP2−/− (n = 19) mice is shown. A combination of two independent experiments is shown. (B) Cysts per brain of WT and mGBP2−/− mice at day 30 after i.p. infection with 40 cysts T. gondii (ME49) 30 d after infection (n = 3). ***P < 0.0005. (C) WT and mGBP2−/− BMDMs pretreated overnight with IFN-γ and infected with T. gondii ME49 for 32 h. T. gondii were stained with anti-SAG1 mAb and anti–mouse-Cy3 (red). The amount of intracellular T. gondii rosettes and the amount of PVs containing a single parasite were enumerated microscopically, and the ratio was built (n = 3). ***P < 0.0005. (D) Astrocytes isolated from the brain of newborn WT and mGBP2−/− mice were treated as described in C (n = 3). ***P < 0.0005. (E) C57BL/6 and mGBP2−/− MEFs transduced with GFP were infected with T. gondii ME49 for 5 h and stained with anti-SAG1 mAb and anti–mouse-Cy3 (red). One representative image is shown. Arrows indicate PV disruption. (Scale bars: 5 μM.) (F) WT and mGBP2−/− MEFs transduced with GFP were infected with T. gondii ME49 for the indicated time periods and stained with anti-SAG1 mAb and anti–mouse-Cy3. The percentage of disrupted intracellular PVs out of total intracellular PVs from approximately 100 infected cells was enumerated microscopically (n = 6). **P < 0.005; ***P < 0.0005.
Fig. 3.
Fig. 3.
Subcellular localization of mGBP2 truncation mutants. (A) mGBP2−/− MEFs were transduced with the indicated GFP–mGBP2 constructs. Transduced cells were pretreated o/n with IFN-γ and infected with T. gondii ME49 for 2 h. T. gondii were stained with anti-SAG1 and anti–mouse-Cy3 (red). One representative experiment out of two is shown. (B) mGBP2−/− MEFs were transduced with the indicated mGBP2 constructs. Cells were pretreated overnight with IFN-γ and infected with T. gondii ME49 for the indicated time points. T. gondii were stained with anti-SAG1 and anti–mouse-Cy3. The amount of T. gondii PVs colocalizing with GFP was enumerated (n = 2).
Fig. 4.
Fig. 4.
The C-terminal domain of mGBP2 is required to inhibit T. gondii proliferation. (A) mGBP2−/− MEFs transduced with the indicated constructs were pretreated o/n with IFN-γ and infected with T. gondii ME49 for 32 h. T. gondii were stained with anti-SAG1 mAb and anti–mouse-Cy3 (red). The amount of intracellular T. gondii rosettes and the amount of PVs containing a single parasite were enumerated microscopically, and the ratio was built (n = 3). *P < 0.05. (B) Cells described in A were analyzed microscopically. Rosettes containing PVs show no colocalization with mGBP2 constructs. One representative experiment out of three is shown.
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