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Rabgdiα Is a Negative Regulator of Interferon- PNAS PLUS Γ–Inducible Gtpase-Dependent Cell-Autonomous Immunity to Toxoplasma Gondii

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Rabgdiα Is a Negative Regulator of Interferon- PNAS PLUS Γ–Inducible Gtpase-Dependent Cell-Autonomous Immunity to Toxoplasma Gondii RabGDIα is a negative regulator of interferon- PNAS PLUS γ–inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii Jun Ohshimaa,b,c, Miwa Sasaia,b, Jianfa Liud, Kazuo Yamashitae,JiSuMab, Youngae Leea, Hironori Bandoa, Jonathan C. Howardf, Shigeyuki Ebisuc, Mikako Hayashic, Kiyoshi Takedag,h, Daron M. Standleye, Eva-Maria Frickeli,j, and Masahiro Yamamotoa,b,1 aDepartment of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 5650871; bLaboratory of Immunoparasitology, World Premier International Research Center Immunology Frontier Research Center, Osaka University, Osaka, Japan 5650871; cDepartment of Restorative Dentistry and Endodontology, Graduate School of Dentistry, Osaka University, Osaka, Japan 5650871; dDepartment of Pathology and Pathogenic Biology, Medical College of Ningbo University, Ningbo City, China 315211; eLaboratory of System Immunology, World Premier International Research Center Immunology Frontier Research Center, Osaka University, Osaka, Japan 5650871; fInstitute for Genetics, University of Cologne, Cologne, Germany 50674; gLaboratory of Mucosal Immunology, World Premier International Research Center Immunology Frontier Research Center, Osaka University, Osaka, Japan 5650871; hDepartment of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Osaka, Japan 5650871; iDivision of Parasitology, Medical Research Council National Institute of Medical Research, London, United Kingdom SN2 1FL; and jThe Francis Crick Institute, London, United Kingdom NW7 1AA Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved July 8, 2015 (received for review May 21, 2015) IFN-γ orchestrates cell-autonomous host defense against various is an obligatory protozoan parasite that causes a life-threatening intracellular vacuolar pathogens. IFN-γ–inducible GTPases, such as toxoplasmosis in humans and animals (7). After the active in- p47 immunity-related GTPases (IRGs) and p65 guanylate-binding vasion of host cells, T. gondii forms a nonfusogenic cytoplasmic proteins (GBPs), are recruited to pathogen-containing vacuoles, membranous structure called the parasitophorous vacuole (PV), which is important for disruption of the vacuoles, culminating in in which the parasite efficiently proliferates (8, 9). In terms of the cell-autonomous clearance. Although the positive regulation cellular host defense against T. gondii, interleukin-12 (IL-12) is for the proper recruitment of IRGs and GBPs to the vacuoles has mainly produced from macrophages and dendritic cells, in which been elucidated, the suppressive mechanism is unclear. Here, we Toll-like receptors and the chemokine receptor CCR5 recognize α α show that Rab GDP dissociation inhibitor (RabGDI ), originally T. gondii-derived ligands. Also, IL-12 promotes development of identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ–producing Th1 cells (10–15). IFN-γ is critically required IFN-γ–inducible GTPases in cell-autonomous immunity to the intra- for suppression of T. gondii replication inside PVs and cell- cellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, autonomous clearance. Nitric oxide that is produced by inducible but not of RabGDIβ,impairedIFN-γ–dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibro- nitric oxide synthase (iNOS) in the infected cells mainly inhibits blasts enhanced the IFN-γ–induced clearance of T. gondii. Further- the replication (16, 17). On the other hand, T. gondii survival more, upon a high dose of infection by T. gondii, RabGDIα- within infected cells is suppressed by cooperative action between deficient mice exhibited a decreased parasite burden in the brain IRGs and GBPs (18). Indeed, various types of cells (such as and increased resistance in the chronic phase than did control macrophages, fibroblasts, and astrocytes) derived from mice mice. Among members of IRGs and GBPs important for the para- lacking IRGs [such as Irgm1 (also known as LRG-47), Irgm3 site clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in Significance RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions IFN-γ is a proinflammatory cytokine and stimulates induction α of RabGDI with Gbp2 through the lipid-binding pocket. Taken of ∼2,000 genes, including IFN-γ–inducible GTPases, such as im- α together, our results suggest that RabGDI inhibits host defense munity-related GTPases (IRGs) and guanylate-binding proteins – against T. gondii by negatively regulating the Gbp2 Irga6 axis of (GBPs), that are critically required for cell-autonomous host de- γ– IFN- dependent cell-autonomous immunity. fense against the vacuolar pathogen Toxoplasma gondii.Mech- anisms of how recruitment of these GTPases to the vacuoles is IFN-γ–inducible GTPase | cell-autonomous immunity | negative regulation | positively regulated have been gradually elucidated. However, Toxoplasma gondii the negative regulation remains unknown. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα) acts as a suppressor γ– α FN-γ is an important T-helper 1 (Th1) cytokine that inhibits of IFN- inducible GTPases, such as Gbp2 and Irga6. RabGDI γ– the survival and growth of a wide range of intracellular patho- deficiency resulted in enhanced IFN- mediated T. gondii clear- I α gens, such as viruses, bacteria, and parasites (1). Stimulation of ance in vitro and in vivo. Furthermore, RabGDI inhibited the act innate immune cells, such as macrophages, by IFN-γ up-regulates of Gbp2 and Irga6 through the lipid-binding pocket. Thus, our almost 2,000 effector genes encoding various IFN-γ–inducible current study demonstrates a negative regulatory mechanism for γ– proteins, including immunity-related GTPases such as the MX IFN- inducible GTPase-dependent cell-autonomous immunity. proteins, p47 immunity-related GTPases (IRGs), and p65 guany- Author contributions: J.O., M.S., S.E., M.H., K.T., D.M.S., E.-M.F., and M.Y. designed re- late-binding proteins (GBPs) (2). MX proteins and GBPs have search; J.O., M.S., K.Y., J.S.M., Y.L., H.B., and E.-M.F. performed research; J.S.M. and J.C.H. been shown to restrict replication of viruses (3). Moreover, IRGs contributed new reagents/analytic tools; J.L., K.Y., E.-M.F., and M.Y. analyzed data; and and GBPs play roles in host defense against vacuole-forming D.M.S., E.-M.F., and M.Y. wrote the paper. bacteria, including Salmonella, Chlamydia, Mycobacteria,andLis- The authors declare no conflict of interest. teria, by induction of antibacterial responses involving autophagic This article is a PNAS Direct Submission. – effectors, inflammasomes, and phagocytic oxidases (4 6). 1To whom correspondence should be addressed. Email: [email protected]. Not only viruses and bacteria but also the vacuolar parasite This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND Toxoplasma gondii is targeted by IFN-γ–inducible GTPases. T. gondii 1073/pnas.1510031112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1510031112 PNAS | Published online August 3, 2015 | E4581–E4590 Downloaded by guest on September 24, 2021 (IGTP), and Irga6 (IIGP1)] or GBPs [such as Gbp1, Gbp2, and a whether Gbp2 associated with RabGDIα and/or RabGDIβ dem- cluster of GBPs on murine chromosome 3 (GBPchr3;Gbp1, onstrated that Flag-tagged RabGDIα, but neither RabGDIβ nor Gbp2, Gbp3, Gbp5, and Gbp7)] were defective for IFN- Gbp1, coprecipitation with endogenous Gbp2 was dependent on γ–mediated intracellular killing of T. gondii (19–25). After the IFN-γ (Fig. 1A and Fig. S1 B and C). formation of PVs, GBPs and a subfamily of IRG members called GKS-IRGs [such as Irga6, Irgb6 (TGTP), and Irgb10] are shown RabGDIα, but Not RabGDIβ,PlaysaRoleinIFN-γ–Dependent Responses to accumulate on PV membrane (PVM) and oligomerize de- to T. gondii. Next we examined the effect of overexpression of α β α pendently on GTP binding to destroy PV membrane integrity and RabGDI or RabGDI in anti-T. gondii response. RabGDI β γ– structure (26, 27), resulting in cell-autonomous clearance by in- or RabGDI overexpression in IFN- mediated suppression of tracellular digestive pathways (20, 21, 28). The IFN-γ–mediated T. gondii proliferation in mouse embryonic fibroblasts (MEFs) α clearance by these GTPases is T. gondii strain-specific. Most showed that retroviral ectopic expression of RabGDI , but not β γ– T. gondii in North America and Europe belong to type I, type II, of RabGDI , significantly impaired IFN- dependent reduction and type III (29). Virulent type I strain inactivates IFN- of T. gondii numbers (Fig. 1B and Fig. S1 D and E). To assess the γ– physiological roles of RabGDIα and RabGDIβ, we generated inducible GTPases by effectors, such as ROP18 and ROP5, α β during the parasite infection (30). On the other hand, avirulent type MEFs derived from embryos lacking RabGDI - or RabGDI - II and type III strains are susceptible to IFN-γ–dependent clearance deficient mice by conventional ES cell-based gene targeting (Fig. – S2 A–D) or CRISPR/Cas9-mediated genome editing (Fig. S3), due to polymorphisms or reduced expression of the effectors (31 34). γ– The regulatory mechanism of how IFN-γ–induced GTPases respectively, and tested the IFN- dependent inhibition of T. gondii proliferation (Fig. 1 C and D). RabGDIα-deficient are recruited to PVs has gradually been elucidated.
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