RabGDIα is a negative regulator of - 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 , 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 (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 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 and stimulates induction α of RabGDI with Gbp2 through the lipid-binding pocket. Taken of ∼2,000 , 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, , 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 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 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. In the absence γ– of essential -related proteins Atg3, Atg5, Atg7, and MEFs showed enhanced dose-dependent IFN- dependent re- duction of parasite numbers compared with WT cells (Fig. 1C). Atg16L1 and of another subfamily of IRGs called GMS-IRGs, β such as Irgm1 and Irgm3, the recruitment of IFN-γ–inducible In contrast, RabGDI -deficient MEFs were comparable with D GTPases and the killing of T. gondii are severely impaired (35–39). WT cells (Fig. 1 ). Taken together, these data demonstrate that RabGDIα, but not RabGDIβ, interacted with Gbp2 and pre- Thus, Atg3/Atg5/Atg7/Atg16L1 and Irgm1/Irgm3 are required for sumably plays a negative role in IFN-γ–dependent suppression proper targeting of GKS-IRGs and GBPs to T. gondii PVM and of T. gondii proliferation in MEFs. play positive roles in the cell-autonomous resistance to the path- ogen. On the other hand, the inhibitory mechanism for the IFN- γ– α γ– Enhanced IFN- Dependent Clearance of T. gondii by RabGDI Deficiency inducible GTPase-dependent immunity remains unclear. in Macrophages. Innate immune cells, such as macrophages, play a To explore the molecular mechanism to control the action of γ– γ– vital role in IFN- mediated cellular innate immunity against IFN- inducible GTPases, we have attempted to identify binding T. gondii in vivo (43). The expression levels of RabGDIα mRNA in partners of Gbp2 because a single deletion of Gbp2 in mice has macrophages as well as MEFs were unchanged after IFN-γ stimu- been shown to result in impaired in vitro and in vivo resistance to lation or T. gondii infection (Fig. S4). To analyze the physiological type II T. gondii (22). In the present study, we identify Rab GDP α α α role of RabGDI in macrophages, we generated mice harboring dissociation inhibitor (RabGDI ) as a Gbp2-interacting pro- loxP-flanked alleles of Gdi1 (Gdi1fl/fl) and deleted the floxed allele tein. We have an interest in this for two reasons: One is by Cre recombinase under control of the myeloid-specific lysozyme α because RabGDI has been shown to participate in the regula- LysM (LysM-Cre) gene (Fig. S2A). RabGDIα mRNA expression tion of Rab proteins, which, like GBPs, belong to another family was abolished in peritoneal macrophages from LysM-Cre Gdi1fl/fl of GTPases (40, 41), and the other is because we demonstrate mice (Fig. S2E). Rab proteins are involved in phagocytosis and α γ– that overexpression of RabGDI in cells impairs IFN- induced phagosome maturation (44). Therefore, we first tested whether reduction of T. gondii numbers. We have tested whether RabGDIα deficiency in macrophages affects the phagocytic activity α γ– RabGDI acts as a regulator of IFN- inducible GTPases under or the phagosomal acidification by analyzing the up- physiological conditions. Macrophages and fibroblasts from take of FITC-labeled or pH-sensitive dye-conjugated dextran, re- RabGDIα-deficient mice exhibit enhanced IFN-γ–dependent spectively (Fig. S5 A and B). RabGDIα-deficient macrophages clearance of T. gondii. Moreover, the enhanced clearance by normally incorporated FITC-labeled dextran (Fig. S5A). Further- RabGDIα deficiency is accompanied by increased recruitment of more, the phagosomal acidification in RabGDIα-deficient cells was Irga6 and Gbp2 to the parasite. Notably, Gbp2 is required for comparable with that in control cells (Fig. S5B), suggesting that Irga6 recruitment, which is suppressed by direct and specific RabGDIα is dispensable for the regulation of phagocytosis and interactions of RabGDIα with Gbp2 through a lipid-binding phagosomal maturation in macrophages. pocket. Furthermore, a high dose of type II T. gondii infection in Next, we assessed whether RabGDIα deficiency affected IFN- RabGDIα-deficient mice results in increased resistance, which is γ–mediated suppression of parasite replication or survival in characterized by a decreased parasite burden in the brain. Taken macrophages. Similar to MEFs, RabGDIα-deficient macro- together, our data indicate that RabGDIα plays a negative role phages exhibited an augmented IFN-γ–dependent reduction of in the Gbp2–Irga6 axis of IFN-γ–inducible GTPase-dependent parasite numbers that was time- or dose-dependent (Fig. 1 E and cell-autonomous resistance to T. gondii. F). Stimulation of macrophages by IFN-γ efficiently reduces T. gondii numbers in cells by suppression of parasite replication and Results survival. IFN-γ–dependent growth inhibition is mainly mediated by RabGDIα, but Not RabGDIβ, Associates with Gbp2. To elucidate the nitric oxide dependently on iNOS in the infected cells (16, 17), molecular mechanisms of Gbp2-dependent host defense against leading to reduction of the parasite number per vacuole. On the type II T. gondii, which is susceptible to Gbp2-dependent cell- other hand, T. gondii survival within infected cells is suppressed by autonomous immunity (22), we attempted to identify binding cooperative action between IRGs and GBPs, decrementing the partners of Gbp2. Immunoprecipitants of Flag-tagged Gbp2 in number of cells with parasites (17, 20, 21). The degree of parasite IFN-γ–stimulated macrophages infected with type II T. gondii infection and growth in macrophages from control or LysM-Cre were submitted for mass spectrometry analysis. We recovered Gdi1fl/fl mice by confocal microscopy showed that the percentage a fragment shared by RabGDIα and RabGDIβ that of T. gondii-infected cells was comparable between control or functioned as a Rab small GTPase (Rabs) inhibitor (Fig. S1A RabGDIα-deficient cells at 3 h postinfection (Fig. 1H). In contrast, and Table S1) (42). Gdi1 and Gdi2 genes encode RabGDIα and percentages for number of RabGDIα-deficient macrophages RabGDIβ, respectively. An immunoprecipitation assay to assess with parasites relative to total number of cells in visual fields at

E4582 | www.pnas.org/cgi/doi/10.1073/pnas.1510031112 Ohshima et al. Downloaded by guest on September 24, 2021 PNAS PLUS

Fig. 1. Enhanced IFN-γ–dependent T. gondii clearance in RabGDIα-deficient cells. (A) MEFs stably transfected with empty or Flag-tagged RabGDIα expression plasmids were untreated or treated with IFN-γ and lysed. The lysates were immunoprecipitated with anti-Flag and detected with the indicated Abs by Western blot. (B) T. gondii numbers at 36 h postinfection in control and MEFs overexpressing RabGDIα (Left)orRabGDIβ (Right) untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated control or cells overexpressing RabGDIα or RabGDIβ relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” (C and D) MEFs lacking RabGDIα (C)orRabGDIβ (D), and WT cells were untreated or treated with the indicated concentrations of IFN-γ. Untreated or IFN-γ–treated cells were infected with ME49 T. gondii expressing luciferase [multiplicity of infection (moi) = 1] and harvested at 36 h postinfection. The luciferase units (LUs) were assayed with the lysates. Error bars represent means ± SD of triplicates. (E) Control and RabGDIα conditional KO (cKO) macrophages stimulated with 10 ng/mL IFN-γ were infected with ME49 T. gondii expressing luciferase (moi = 0.5) and harvested at the indicated points postinfection. The luciferase units (LU) were assayed with the lysates. Indicated values are means ± SD of triplicates. (F) Control and RabGDIα cKO macrophages were untreated or treated with the indicated concentrations of IFN-γ. Untreated or IFN-γ–treated cells were infected with ME49 T. gondii expressing luciferase (moi = 0.5) and harvested at 36 h postinfection. The LUs were assayed with the lysates. Indicated values are means ± SD of triplicates. (G) IFN-γ–stimulated control and RabGDIα cKO peritoneal macrophages were infected with ME49 T. gondii (moi = 0.5), fixed at 36 h postinfection, and stained with rabbit anti-T. gondii (green) or rat anti-CD11b (red). (Scale bars: 20 μm.) (H) The percentage (number of cells with parasites relative to total number of cells) of control and RabGDIα cKO macrophages containing at least one parasite at the indicated points postinfection. Indicated values are means ± SD of triplicates. (I) The number of T. gondii parasites per vacuole in control or RabGDIα cKO macrophages at 36 h postinfection. Indicated values are means ± SD of triplicates. N.S., not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of three (A and C–F)andtwo(B and G) independent experiments. Data in H and I are pooled from two independent experiments in which almost 200 cells and 100 vacuoles were counted, respectively.

later time points were significantly lower than those of controls Thus, loss of RabGDIα in macrophages and MEFs resulted in (Fig. 1 G and H). Parasite replication in control or RabGDIα-deficient the augmented IFN-γ–dependent suppression of T. gondii sur- macrophages was determined by counting parasite numbers in vival whereas the inhibition of replication and cell death was PVs and demonstrated that T. gondii numbers in PVs of unaffected. RabGDIα-deficient cells were similar to controls (Fig. 1 G and I). In addition, cell death in response to IFN-γ has been shown Increased in Vivo Resistance to T. gondii by Myeloid-Specific Ablation to restrict parasite growth (45). However, RabGDIα-deficient of RabGDIα. Tumor necrosis factor-α (TNF-α) plays a critical role

macrophages showed comparable cell death in the parasite in- in the in vivo resistance to T. gondii and is shown to strongly INFLAMMATION fection in comparison with control macrophages (Fig. S5C). enhance anti-T. gondii activity in macrophages mainly by the IMMUNOLOGY AND

Ohshima et al. PNAS | Published online August 3, 2015 | E4583 Downloaded by guest on September 24, 2021 synergistic production of nitric oxide (NO) (46). Both control and mouse survival were assessed (Fig. 2 B–E). In vivo imaging and RabGDIα-deficient macrophages produced comparable analysis indicated reduced abdominal parasite signal in LysM-Cre levels of nitrite ion (NO2-) in costimulation of IFN-γ and TNF-α Gdi1fl/fl mice compared with control mice at 7 or 9 dpi (Fig. 2 B and (Fig. S6A). Furthermore, RabGDIα-deficient macrophages still C). Although control and LysM-Cre Gdi1fl/fl mice showed an initially exhibited augmented IFN-γ–inducible reduction of T. gondii numbers comparable mortality up to 16 dpi and serum levels of proin- even in the presence of TNF-α (Fig. 2A). Therefore, we tested flammatory , such as IFN-γ and IL-12 p40, in the acute whether increased T. gondii clearance activity in RabGDIα-deficient phase (Fig. S6B), LysM-Cre Gdi1fl/fl mice were less susceptible in the macrophages results in in vivo resistance to the parasite. Control or chronic phase of the infection (Fig. 2D). In terms of parasite burdens LysM-Cre Gdi1fl/fl mice were intraperitoneally infected with a high in organs, high numbers in spleens at 8 dpi in both control and LysM- doseoftypeIIT. gondii, to which all control mice succumbed around Cre Gdi1fl/fl mice, albeit slightly lower in spleens of LysM-Cre Gdi1fl/fl 30 d postinfection (dpi), and parasite spread and burden in organs mice, were observed. In sharp contrast, the parasite numbers in

Fig. 2. Myeloid-specific RabGDIα-ablated mice are resistant to T. gondii infection. (A) Control and RabGDIα cKO macrophages were untreated or treated with 10 ng/mL IFN-γ and/or TNF-α for 24 h, and followed by the infection of the luciferase expressing T. gondii (moi = 0.5). T. gondii numbers at 36 h postinfection were analyzed by luciferase assay. Indicated values are means ± SD of triplicates. (B)Control(Gdi1flox/flox)orRabGDIα conditional KO (cKO; LyzM-Cre Gdi1flox/flox) mice (n = 4 per each group) were intraperitoneally infected with 5 × 103 ME49 T. gondii-expressing luciferase, and the progress of infection was assessed by bioluminescence imaging at day 7 postinfection. The color scales indicate photon emission during a 60-s exposure. (C) Total photon emission analysis from mice (n = 4 per group) in B at indicated days postinfection. Abdominal photon emission was assessed during a 60-s exposure. The flux (photons/s/cm2/sr) was de- termined as a measure of parasite burden. The statistical significance was determined by Mann–Whitney U test. (D)Control(n = 16) or RabGDIα cKO (n = 19) mice were infected with 5 × 103 T. gondii, and the survival rates were monitored for 60 d. The statistical significance was determined by log-rank test. (E) Quantification of parasites in indicated tissues from mice at day 8 or 20 postinfection by the standard curve (in Fig. S6C). The statistical significance was determined by Mann– Whitney U test. N.S., not significant; *P < 0.05, **P < 0.01. Data are representative of two independent experiments (A–C and E). Data in D are pooled from three independent experiments.

E4584 | www.pnas.org/cgi/doi/10.1073/pnas.1510031112 Ohshima et al. Downloaded by guest on September 24, 2021 spleens from both groups were dramatically reduced at 20 dpi strated that myeloid cell-specific deletion of RabGDIα leads to a PNAS PLUS (Fig. 2E and Fig. S6C). When other organs were tested at 20 dpi, lower parasitic burden in the brain and confers increased in vivo we found that brains of control mice contained strikingly higher resistance to T. gondii, which is because of neither IL-12 p40 nor parasite numbers than those from LysM-Cre Gdi1fl/fl mice (Fig. 2E). IFN-γ production defects. However, no significant differences in the parasite burdens in lungs, kidneys, and livers were measured in LysM-Cre Gdi1fl/fl mice Increased Loading of Irga6 onto T. gondii PVs by RabGDIα Deficiency. compared with control mice (Fig. 2E). Thus, these results demon- IFN-γ–induced cellular clearance of T. gondii is dependent on

Fig. 3. Increased accumulation of Irga6 and Gbp2 in RabGDIα-deficient cells. (A) Control and RabGDIα cKO macrophages were untreated or treated with IFN-γ. Untreated or IFN-γ–treated cells were infected with RH (type I) or ME49 (type II) T. gondii expressing luciferase (moi = 0.5) and harvested at 36 h postinfection. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated control or RabGDIα-deficient cells relative to those in the unstimulated re- spective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (B and C)IFN-γ–stimulated control and RabGDIα-deficient macrophages were infected with ME49 T. gondii (moi = 0.5), fixed at 3 h postinfection, and incubated with rabbit anti-T. gondii (green) and mouse anti-Irga6 (red in B) or goat anti-Irgb6 (red in C). (Scale bars: 10 μm.) Arrows indicate colocalization of Irga6 or Irgb6 with T. gondii (Upper). The percentage of parasites positive for Irga6 or Irgb6 staining at the indicated points postinfection in IFN-γ–stimulated control and RabGDIα-deficient macrophages (Lower). Indicated values are means ± SD of triplicates. (D) The percentage of WT or RabGDIα-deficient MEFs positive for indicated Gbps staining at 1 h postinfection. Indicated values are means ± SD of triplicates. (E)IFN-γ–stimulated WT or RabGDIα-deficient MEFs were infected with T. gondii (moi = 4), fixed at 1 h postinfection, and incubated with rabbit anti-Gbp2 (red), and goat anti-T. gondii (green). (Scale bars: 10 μm.) Arrows indicate colocalization of endogenous Gbp2 with T. gondii.(F) The percentage of WT or RabGDIα- deficient MEFs positive for endogenous Gbp2 costaining with T. gondii at the indicated time points postinfection. Indicated values are means ± SD of triplicates. (G and H) Proximity-ligation (PL) assay of both endogenous Irga6 and Gbp2 in IFN-γ–stimulated or unstimulated (G), and WT or RabGDIα-deficient (H) MEFs. Cells were treated with IFN-γ, infected with ME49 T. gondii (moi = 4), and fixed at 3 h postinfection: [red, PL-positive (PL+) signals; green, T. gondii; blue, nuclei]. Arrows indicate colocalization of PL signal with T. gondii.(I) The percentage of parasites positive for PL signals (in H) at 3 h postinfection. Indicated values are means ± SD of triplicates.

N.D., not detected; N.S., not significant; **P < 0.01, ***P < 0.001. Data are representative of two (A, B, Upper, C, Upper, D, E, G,andH) independent experiments. INFLAMMATION Data in B, Lower, C, Lower, D, F,andI are pooled from three independent experiments, in which almost 150 cells at each time point were counted. IMMUNOLOGY AND

Ohshima et al. PNAS | Published online August 3, 2015 | E4585 Downloaded by guest on September 24, 2021 IRGs (17). The virulent type I T. gondii parasite is resistant to assay. No PL signals were observed in unstimulated cells (Fig. 3G). the IRG-dependent clearance due to ROP18, a parasite-secreted In sharp contrast, strong PL signals between endogenous Gbp2 kinase that acts as a virulence effector molecule (33, 34). Con- and Irga6 were observed in IFN-γ–stimulated cells (Fig. 3G). sistently, IFN-γ–stimulated reduction of type I T. gondii was not Moreover, markedly higher numbers of T. gondii with PL signals as efficient as that of the avirulent IRG-susceptible type II strain between Gbp2 and Irga6 were also detected in RabGDIα- (Fig. 3A). Moreover, although type II parasite numbers were deficient cells than in WT cells (Fig. 3 H and I), which is consistent significantly more decreased in RabGDIα-deficient macrophages with more frequent accumulation of Irga6 and Gbp2 in RabGDIα- after IFN-γ stimulation, those of type I parasite were unchanged deficient cells (Fig. 3 B and E). Together, these results indicated between control and RabGDIα-deficient macrophages (Fig. 3A), that Irga6 and Gbp2 were located in close proximity to allow a suggesting that the phenotype caused by the RabGDIα deficiency physical association with the parasites. may be associated with IRGs. Analysis of the recruitment of two different IRG family members, Irga6 and Irgb6, to T. gondii in Gbp2 Plays a Positive Role in Irga6 Recruitment to T. gondii. To assess IFN-γ–stimulated macrophages by confocal microscopy indicated the relationship between Irga6 and Gbp2 directly, we created a significantly higher percentage of Irga6 recruitment to parasites MEFs derived from embryos of Gbp2-deficient mice, generated by in RabGDIα-deficient macrophages at 3 or 6 h postinfection CRISPR/Cas9-mediated genome editing (Fig. S9A). Although compared with controls (Fig. 3B and Fig. S7A). In contrast, the Irga6 proteins were similarly induced upon IFN-γ stimulation, rates of Irgb6-positive parasites were comparable between con- Gbp2 proteins were not detected in Gbp2-deficient MEFs (Fig. trol and RabGDIα-deficient macrophages (Fig. 3C and Fig. S9B). Compared with WT cells, Gbp2-deficient cells had a de- S7B). Furthermore, RabGDIα-deficient MEFs also showed in- fective suppression of T. gondii proliferation in response to IFN-γ creased Irga6 loading onto parasites compared with WT cells (Fig. 4A). In addition, the percentages of Irga6-positive parasites (Fig. S7 C and D), indicating that RabGDIα deficiency leads to a in Gbp2-deficient MEFs were significantly lower than in WT more frequent recruitment of Irga6, but not Irgb6, to T. gondii. cells (Fig. 4B), which is opposite to that observed in RabGDIα- Furthermore, the negative effect of RabGDIα on Irga6 loading deficient cells (Fig. 3B and Fig. S7D). On the other hand, the was not determined by protein levels because Irga6 proteins percentages of Irgb6-positive T. gondii were comparable between were similarly induced and expressed in WT and RabGDIα- WT and Gbp2-deficient MEFs (Fig. 4C). Thus, Gbp2 positively deficient cells (Fig. S7E). Moreover, we did not detect an asso- regulated the recruitment of Irga6 to PVs of T. gondii. ciation between RabGDIα and Irga6 by coprecipitation (Fig. S7F), suggesting that the higher rates of Irga6 loading to Prenylation of Gbp2 Is Required for Its Positive Function on IFN- T. gondii might be indirectly caused by the RabGDIα deficiency. γ–Dependent Clearance and Irga6 Recruitment. GBPs, including Gbp2, comprise two structural domains, an N-terminal globular Gbp2 Is Specifically More Recruited onto T. gondii PVs in RabGDIα- nucleotide-binding domain, where the GTP-binding motif is lo- Deficient Cells. In the first place, we identified RabGDIα as a cated, and a C-terminal helical effector domain. Gbp2 contains a Gbp2-interacting protein (Fig. S1A). To test whether RabGDIα C-terminal isoprenylated Caax motif (22, 48, 49). To assess the role specifically interacted with Gbp2 alone or with other GBP family of GTP-binding status and Gbp2 prenylation in anti-T. gondii re- members, we performed an immunoprecipitation assay. Flag- sponses, we generated K51A and C586S mutants, where either tagged RabGDIα coprecipitated with endogenous Gbp2 but not lysine 51 or cysteine 586 was substituted to alanine or serine, re- Gbp1 (Fig. 1A). Next, to compare the recruitment of individual spectively (Fig. 4D). K51A causes the defective binding of human GBPs in WT or RabGDIα-deficient MEFs, Flag- or HA-tagged GBP1 with nucleotides, including GTP and GDP (50). The C586S GBPs (such as Gbp1, Gbp2, Gbp3, Gbp5, and Gbp7) were stably mutation abolishes prenylation by geranylgeranyltransferase I (51, expressed in WT or RabGDIα-deficient MEFs (Fig. S8 A and B). 52). We introduced these mutations into Gbp2-deficient MEFs and The percentages of accumulation of Gbp1, Gbp3, Gbp5, and tested the IFN-γ–dependent killing of T. gondii and Irga6 loading Gbp7 on T. gondii were comparable between WT and RabGDIα- (Fig. 4E). Gbp2-deficient MEFs expressing WT Gbp2, but neither deficient cells (Fig. 3D and Fig. S8C). In sharp contrast, the rate K51A nor C586S, restored the IFN-γ–dependent reduction of par- of loading of HA-tagged Gbp2 alone in RabGDIα-deficient asite numbers and recruitment of Irga6 and Gbp2 (Fig. 4 F–I). These MEFswassignificantlygreaterthaninWTcells(Fig.3D and Fig. data indicate that both nucleotide binding and prenylation of Gbp2 S8C). Furthermore, endogenous Gbp2 proteins in RabGDIα- areessentialfortheIFN-γ–dependent cellular immunity. deficient cells were more recruited to T. gondii than in WT cells (Fig. 3 E and F), indicating that specific association of RabGDIα The Lipid-Binding Pocket in RabGDIα Is Required for Its Negative with Gbp2, but not with other GBPs, may account for enhanced Function. RabGDI functions as a negative regulator of Rabs by Gbp2 recruitment observed in RabGDIα deficiency. sequestering Rabs in an inactive GDP-bound form in the cyto- plasm (53). The extraction of prenylated Rabs from membranes Gbp2 and Irga6 on T. gondii Are Colocalized in the Very Proximity. by RabGDI is achieved by the direct interaction of Rabs with These results suggested that, among IRGs and GBPs, the be- RabGDIα through the Rab-binding platform and the lipid-binding havior of Irga6 and Gbp2 were similar in RabGDIα-deficient pocket, into which the prenyl group of Rabs is inserted (54, 55). cells. We previously demonstrated that GBPchr3 regulated Irgb6 The significance of the two domains of RabGDIα was originally and Irgb10 loading to T. gondii (21). Furthermore, a previous characterized in the context of Rabs; however, their function in study showed that Gbp1 was important for Irgb6 recruitment on the context of IFN-γ–induced cell-autonomous immunity is un- T. gondii-containing vacuoles (20). Thus, a molecular link be- known. Therefore, we analyzed the localization of interactions tween IRGs and GBPs has been suggested; however, a specific between RabGDIα and Gbp2 by PL assay. In contrast to in- association between Irga6 and Gbp2 is unclear. Thus, we tested teractions between Irga6 and Gbp2, which were strongly detected whether Irga6 and Gbp2 were colocalized on T. gondii-containing on parasites (Fig. 3 G and H), IFN-γ–dependent PL signals for the vacuoles in IFN-γ–stimulated MEFs. Confocal microscopy and association of Flag-tagged RabGDIα with endogenous Gbp2 were intensity profiling of fluorescent signals showed that both endog- not detected on parasites, but only in the cytoplasm (Fig. 5A). In enous Irga6 and Gbp2 proteins were detected at similar sites (Fig. addition, PL signals between endogenous Irga6 and Flag-tagged S8 D and E). Although we examined the association of Irga6 and RabGDIα were not detected in IFN-γ–stimulated cells (Fig. 5B), Gbp2, we failed to detect an interaction by coprecipitation anal- suggesting that RabGDIα and Irga6 are not localized in the ysis. To test whether Irga6 and Gbp2 proteins were in close proximity. Next, we characterized the Rab-binding platform of proximity (<16 nm) (47), we performed a proximity ligation (PL) RabGDIα in IFN-γ–mediated immunity. Consistent with previous

E4586 | www.pnas.org/cgi/doi/10.1073/pnas.1510031112 Ohshima et al. Downloaded by guest on September 24, 2021 PNAS PLUS

Fig. 4. Gbp2 positively regulates Irga6 recruitment to T. gondii through prenylation. (A) T. gondii numbers at 36 h postinfection in WT and Gbp2-deficient MEFs untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated WT or Gbp2-deficient cells relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (B and C) The percentage of parasites positive for Irga6 (B) or Irgb6 (C) staining at the indicated points postinfection in IFN-γ–stimulated WT and Gbp2-deficient MEFs. Indicated values are means ± SD of triplicates. (D) Gbp2 mutants possessing amino acid substitutions in the GTPase domain or Caax motif. (E) Lysates of Gbp2-deficient MEFs stably transfected with empty or indicated HA-tagged Gbp2 expression plasmids were detected with indicated Abs by Western blot. (F and G) IFN-γ–stimulated Gbp2-deficient MEFs stably transfected with empty or indicated HA-tagged Gbp2 expression plasmids were infected with ME49 T. gondii (moi = 4), fixed at 1 h postinfection, and incubated with rabbit anti-T. gondii (green), mouse anti-HA (red), and DAPI (blue). (Scale bars, 10 μm.) Arrows indicate colocalization of Gbp2 with T. gondii. Percentage of parasites positive for Gbp2 staining is shown G. Indicated values are means ± SD of triplicates. (H) T. gondii numbers at 36 h postinfection in Gbp2-deficient MEFs stably transfected with empty (control) or indicated HA-tagged Gbp2 expression plasmids untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated control or indicated Gbp2 variants expressing cells relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (I) The percentage of parasites positive for Irga6 staining in Gbp2-deficient MEFs expressing in- dicated Gbp2 variants at 3 h postinfection. Indicated values are means ± SD of triplicates. N.D., not detected; N.S., not significant; **P < 0.01, ***P < 0.001. Data are representative of two (E and F) or three (A and H) independent experiments. Data in B, C, G, and I are pooled from two independent experiments, in which almost 150 cells at each time point were counted.

reports (54, 56), RabGDIα Y39V, R218A/Y219A, and E233S/ sponses might be independent of Rabs. To verify the importance of E240A mutants nearly abolished the interaction with HA-tagged the lipid-binding pocket of RabGDIα (Fig. 5F), we generated an Rab10 (Fig. 5C). However, when these Rab-binding platform M132I mutant of RabGDIα andtestedanassociationwithGbp2. mutants of RabGDIα were stably expressed in RabGDIα-deficient Notably, the Flag-tagged M132I mutant of RabGDIα failed to MEFs, the RabGDIα mutants coprecipitated with endogenous coprecipitate with endogenous Gbp2 (Fig. 5G). Moreover, in- Gbp2 and restored the suppressive activity of RabGDIα in the troduction of the RabGDIα M132I mutant in RabGDIα-deficient γ– γ–

IFN- induced reduction of parasite numbers (Fig. 5 D and E). MEFs did not restore suppression of IFN- mediated killing activity INFLAMMATION Thus, the negative effect of RabGDIα on Gbp2-mediated re- of T. gondii and reduced Irga6 loading onto PVs (Fig. 5 H and I). IMMUNOLOGY AND

Ohshima et al. PNAS | Published online August 3, 2015 | E4587 Downloaded by guest on September 24, 2021 Fig. 5. RabGDIα suppresses Gbp2-dependent responses through the lipid-binding pocket. (A and B) PL assay of Flag-tagged RabGDIα and endogenous Gbp2 (A) or Irga6 (B)inIFN-γ–stimulated or unstimulated MEFs. Cells were infected with ME49 T. gondii (moi = 4), fixed at 3 h postinfection; red, PL-positive (PL+) signals; green, T. gondii;blue,nuclei.(C) Lysates of 293T cells transiently cotransfected with indicated Flag-tagged RabGDIα vectors and/or HA-tagged Rab10 were immunoprecipitated with anti-Flag and detected using the indicated Abs by Western blot. (D)RabGDIα-deficient MEFs expressing indicated Flag-tagged RabGDIα were untreated or treated with IFN-γ and lysed. The lysates were immunoprecipitated with anti-Flag and detected using with the indicated Abs by Western blot. (E and H) T. gondii numbers at 36 h postinfection in RabGDIα-deficient MEFs stably transfected with empty (control) or indicated Flag-tagged RabGDIα expression plasmids untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated control or indicated RabGDIα variants-expressing cells relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (F) In silico profiling of the molecular surface of RabGDIα, colored by electrostatic potential, with red (or blue) representing the most negative (or positive) values, and by hydrophobicity, with yellow representing the most hydrophobic. The lipid-binding pocket is framed by a dashed line,anditsclose-upviewisshownRight. The approximate location of the methionine residue 132 is indicated by an arrow. (G) Lysates of RabGDIα-deficient MEFs stably transfected with empty or indicated Flag-tagged RabGDIα expression plasmids were detected with indicated Abs by Western blot. (I) The percentage of parasites positive for Irga6 staining in RabGDIα-deficient MEFs expressing indicated RabGDIα variants at 3 h postinfection. Indicated values are means ± SD of triplicates. N.S., not significant; *P < 0.05, **P < 0.01. Data are representative of two (A–D and G)orthree(E and H) independent experiments and two in- dependent calculations (F). Data in I are pooled from two independent experiments, in which almost 150 cells at each time point were counted.

Taken together, these results indicate that lipid-binding activity, IFN-γ–inducible GTPases. RabGDIα-deficient cells exhibited in- but not the Rab-binding function, of RabGDIα is critical creased IFN-γ–mediated Gbp2/Irga6-depedent in vitro clearance for IFN-γ–induced Gbp2-dependent cell-autonomous immunity of T. gondii. Furthermore, mice specifically lacking RabGDIα in against T. gondii. myeloid cell linage showed increased resistance to a high dose of type II T. gondii infection due to a decreased parasite burden in Discussion the brain, which is an opposite phenotype of Gbp2-deficient mice In the present study, we have found that RabGDIα plays a sup- showing increased parasite numbers in the brain and decreased pressive role in cell-autonomous host defense against T. gondii by resistance in the chronic phase (22).

E4588 | www.pnas.org/cgi/doi/10.1073/pnas.1510031112 Ohshima et al. Downloaded by guest on September 24, 2021 The function of RabGDIα, as well as yeast RabGDI, has been the Irga6 accumulation in GBPchr3-deleted cells was almost com- PNAS PLUS well-characterized for the regulation of Rabs, which play im- parable with that in Gbp2-deficient cells, indicating that IFN- portant roles in membrane vesicular trafficking. Rabs cycle be- γ–induced Irga6 recruitment to the parasite may be controlled in tween a GDP-bound inactive state and a GTP-bound active state a Gbp2 (GBPchr3)-dependent and -independent manner. Al- (57). Inactive Rabs are activated by an exchange of GDP for though recruitment of all IRGs, comprising 23 members in mice, GTP on donor membranes that generate a carrier vesicle (58). was not tested in Gbp1-, Gbp2-, or GBPchr3-deficient cells, these Active Rabs on carrier vesicles facilitate docking and fusion with results suggest that individual GBPs may differentially regulate target membranes, culminating in the inactivation of Rabs by the recruitment and/or retention of IRGs. We did not detect an hydrolysis of GTP to GDP. RabGDI associates with inactive interaction between Gbp2 and Irga6 by coprecipitation; however, Rabs on target membranes through the Rab binding platform, both proteins were localized in close proximity as demonstrated which contains a prenyl group of Rabs in the lipid-binding by PL assay. The detailed molecular mechanism by which GBPs pocket. This association between Rabs and RabGDI allows the associate with IRGs and positively regulate the recruitment and/or retrieval of Rabs from target membranes to the cytosolic pool for retention should be analyzed in the future. recycling for the next round of vesicular transport (59). In the GMS-IRGs (Irgm1, Irgm2, and Irgm3) are shown to prevent context of IFN-γ–induced responses, the Rab-binding platform activation of GKS-IRGs, including Irga6 and Irgb6, by limiting of RabGDIα was dispensable for the negative function, in- the GTP nucleotide exchange from GDP in the detailed biochemical dicating that RabGDIα might function in a Rab-independent analysis (27). Together with our current data, cell-autonomous im- manner. In contrast, the lipid-binding pocket of RabGDIα was munity dependent on IRGs or GBPs may possess differential in- indispensable for the suppressive effect. In addition, prenylation hibitory circuits by GMS-IRGs and RabGDIα, respectively. It is of Gbp2 in the C-terminal CTIL motif was essential for both the of note that mice lacking Irgm1 or Irgm3 showed high suscep- recruitment to PV membranes of T. gondii and the IFN- tibility to T. gondii, probably due to dysregulation of GKS-IRGs; γ–dependent killing of parasites (22). Similarly, prenylation of Rabs in stark contrast, RabGDIα-deficient mice displayed resistance was critical for membrane anchoring and function (54). In terms of to the pathogen. RabGDIα and Gbp2 localization, PL signals indicative of direct in- In conclusion, we have identified RabGDIα as a negative regu- teractions between both proteins were detected mainly in the cyto- latorintheIrga6-Gbp2axisofIFN-γ–inducible GTPase-dependent plasm. Thus, by analogy with the relationship between RabGDIα cell-autonomous immunity to T. gondii. By close analysis of each and Rabs in membrane vesicular trafficking, RabGDIα might re- step of IFN-γ–inducible GTPase-dependent T. gondii PV disrup- trieve Gbp2 from target PV membranes to the cytoplasm, resulting tion, other positive and negative regulators for this cell-autonomous in termination of Gbp2-dependent Irga6 loading on PV membranes. immune response might be further identified in the future. Compared with the two isoforms of RabGDI in mammals, yeast has only one copy of the GDI gene that encodes yeast Materials and Methods RabGDI, and its disruption results in lethality (57). Although Mice, Cells, and Parasites. All animal experiments were conducted with the conventional RabGDIα-deficient mice are viable and healthy approval of the Animal Research Committee of the Research Institute for except for impaired cognitive functions (60), we failed to obtain Microbial Diseases in Osaka University. Detailed information for mice, cells, postnatal RabGDIβ-deficient mice due to unknown reasons. In and T. gondii are provided in SI Materials and Methods. this regard, the RabGDIβ deficiency could not be compensated α Reagents. Detailed information for antibodies and other reagents is provided for by RabGDI in vivo. In addition, the differential roles of in SI Materials and Methods. RabGDIα and RabGDIβ in the regulation of Rabs as well as embryonic development are still unclear. Both RabGDIα and β Immunofluorescence. Detailed methods for indirect immunofluorescence are RabGDI bind and solubilize similar members of GDP-formed provided in SI Materials and Methods. inactive Rabs in vitro (61). Of note, we demonstrated that RabGDIα, but not RabGDIβ, specifically associated with Gbp2 Generation of Conditional Gdi1-Deficient Mice, Conventional Gdi2-Deficient and suppressed Gbp2-dependent cell-autonomous immune re- Mice, and Gbp2-Deficient MEFs. Detailed protocols for the generation of sponses. Currently, the specific function of RabGDIβ in IFN- these knockout mice and MEFs are provided in SI Materials and Methods. γ–induced responses is unknown. RabGDIβ might associate with Gbp family members other than Gbp2, or be totally unrelated Statistical Analysis. The unpaired Student’s t test or ANOVA plus post hoc Tukey to this immune response. In terms of phagocytosis, RabGDIβ was used to determine the statistical significance of the in vitro experimental – rather than RabGDIα may mainly regulate the activity and the data. Animal experiments in vivo were evaluated with the Mann Whitney U test or log-rank test. All data were analyzed with Prism software (GraphPad). acidification. Genes encoding murine GBPs consist of 13 family members (11 ACKNOWLEDGMENTS. We thank M. Enomoto for secretarial and technical active members and 2 pseudo genes), are organized in two clusters assistance. We thank Dr. Akiko Iwasaki for suggestions and discussion of this on 3 and 5 in mice, and share a high degree of ho- study. We also thank Dr. Dominique Soldati-Favre for providing us with the mology (62). We previously generated mice lacking GBPchr3 and anti-T. gondii antibody. This work was supported by a Grant-in-Aid for Sci- chr3 entific Research on Innovative Areas (Homeostatic regulation by various demonstrated that GBP participated in the recruitment and/or types of cell death, 15H01377; and Matoryoshka-type evolution, 26117713) retention of IRGs to T. gondii or Chlamydia trachomatis (21, 39). A from the Ministry of Education, Culture, Sports, Science and Technology; The subsequent study revealed that Gbp1 plays a role in the recruitment Inoue Research Award; the Takeda Science Foundation; the Naito Founda- and/or retention of Irgb6, but not of Irga6, to T. gondii (20). Here, tion; the Daiichi-Sankyo Foundation of Life Science; and the Sumitomo Foun- dation and Research Foundation for Microbial Diseases of Osaka University. we found that Gbp2 positively regulated the recruitment of Irga6, E.-M.F. is supported by a Wellcome Trust Career Development Fellowship but not Irgb6, to the parasite. Gbp2-deficient cells displayed almost and by Medical Research Council Grant MC_UP_1202/12. J.O. is the recipient 50% loss of Irga6 loading to T. gondii. Furthermore, the defect in of a scholarship from the Iwadare Scholarship Foundation.

1. Boehm U, Klamp T, Groot M, Howard JC (1997) Cellular responses to interferon- 5. Deretic V (2006) Autophagy as an immune defense mechanism. Curr Opin Immunol gamma. Annu Rev Immunol 15:749–795. 18(4):375–382. 2. MacMicking JD (2012) Interferon-inducible effector mechanisms in cell-autonomous 6. Shenoy AR, et al. (2012) GBP5 promotes NLRP3 assembly and immu- immunity. Nat Rev Immunol 12(5):367–382. nity in mammals. Science 336(6080):481–485. 3. Sadler AJ, Williams BR (2008) Interferon-inducible antiviral effectors. Nat Rev 7. Montoya JG, Remington JS (2008) Management of Toxoplasma gondii infection Immunol 8(7):559–568. during pregnancy. Clin Infect Dis 47(4):554–566.

4. Kim BH, Shenoy AR, Kumar P, Bradfield CJ, MacMicking JD (2012) IFN-inducible 8. Boothroyd JC (2009) Toxoplasma gondii: 25 years and 25 major advances for the field. INFLAMMATION IMMUNOLOGY AND GTPases in host cell defense. Cell Host Microbe 12(4):432–444. Int J Parasitol 39(8):935–946.

Ohshima et al. PNAS | Published online August 3, 2015 | E4589 Downloaded by guest on September 24, 2021 9. Sibley LD (2011) Invasion and intracellular survival by protozoan parasites. Immunol 38. Zhao Z, et al. (2008) Autophagosome-independent essential function for the auto- Rev 240(1):72–91. phagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe 10. Yarovinsky F, Sher A (2006) Toll-like receptor recognition of Toxoplasma gondii. Int J 4(5):458–469. Parasitol 36(3):255–259. 39. Haldar AK, et al. (2013) IRG and GBP host resistance factors target aberrant, “non- 11. Hunter CA, Remington JS (1995) The role of IL12 in toxoplasmosis. Res Immunol 146(7-8): self” vacuoles characterized by the missing of “self” IRGM proteins. PLoS Pathog 9(6): 546–552. e1003414. 12. Koblansky AA, et al. (2013) Recognition of profilin by Toll-like receptor 12 is critical 40. Matsui Y, et al. (1990) Molecular cloning and characterization of a novel type of for host resistance to Toxoplasma gondii. Immunity 38(1):119–130. regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol Cell 13. Andrade WA, et al. (2013) Combined action of nucleic acid-sensing Toll-like receptors Biol 10(8):4116–4122. and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice. Cell 41. Sasaki T, et al. (1990) Purification and characterization from bovine brain of a Host Microbe 13(1):42–53. protein that inhibits the dissociation of GDP from and the subsequent binding of GTP – 14. Aliberti J, et al. (2000) CCR5 provides a signal for microbial induced production of to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem 265(4):2333 2337. IL-12 by CD8 alpha+ dendritic cells. Nat Immunol 1(1):83–87. 42. Nishimura N, Nakamura H, Takai Y, Sano K (1994) Molecular cloning and character- 15. Aliberti J, et al. (2003) Molecular mimicry of a CCR5 binding-domain in the microbial ization of two rab GDI species from rat brain: Brain-specific and ubiquitous types. – activation of dendritic cells. Nat Immunol 4(5):485–490. J Biol Chem 269(19):14191 14198. 16. Langermans JA, et al. (1992) IFN-gamma-induced L-arginine-dependent toxoplasmastatic 43. Suzuki Y, Orellana MA, Schreiber RD, Remington JS (1988) Interferon-gamma: The major mediator of resistance against Toxoplasma gondii. Science 240(4851):516–518. activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis 44. Fairn GD, Grinstein S (2012) How nascent phagosomes mature to become phag- factor-alpha. J Immunol 148(2):568–574. olysosomes. Trends Immunol 33(8):397–405. 17. Zhao Y, et al. (2009) Virulent Toxoplasma gondii evade immunity-related GTPase- 45. Zhao YO, Khaminets A, Hunn JP, Howard JC (2009) Disruption of the Toxoplasma mediated parasite vacuole disruption within primed macrophages. J Immunol 182(6): gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases 3775–3781. (IRG proteins) triggers necrotic cell death. PLoS Pathog 5(2):e1000288. 18. Hunter CA, Sibley LD (2012) Modulation of innate immunity by Toxoplasma gondii 46. Sibley LD, Adams LB, Fukutomi Y, Krahenbuhl JL (1991) Tumor necrosis factor-alpha virulence effectors. Nat Rev Microbiol 10(11):766–778. triggers antitoxoplasmal activity of IFN-gamma primed macrophages. J Immunol 19. Taylor GA, Feng CG, Sher A (2004) p47 GTPases: Regulators of immunity to in- 147(7):2340–2345. tracellular pathogens. Nat Rev Immunol 4(2):100–109. 47. Weibrecht I, et al. (2010) Proximity ligation assays: A recent addition to the proteo- 20. Selleck EM, et al. (2013) Guanylate-binding protein 1 (Gbp1) contributes to cell- mics toolbox. Expert Rev Proteomics 7(3):401–409. autonomous immunity against Toxoplasma gondii. PLoS Pathog 9(4):e1003320. 48. Virreira Winter S, et al. (2011) Determinants of GBP recruitment to Toxoplasma gondii γ 21. Yamamoto M, et al. (2012) A cluster of interferon- -inducible p65 GTPases plays a vacuoles and the parasitic factors that control it. PLoS One 6(9):e24434. – critical role in host defense against Toxoplasma gondii. Immunity 37(2):302 313. 49. Prakash B, Praefcke GJ, Renault L, Wittinghofer A, Herrmann C (2000) Structure of 22. Degrandi D, et al. (2013) Murine guanylate binding protein 2 (mGBP2) controls human guanylate-binding protein 1 representing a unique class of GTP-binding – Toxoplasma gondii replication. Proc Natl Acad Sci USA 110(1):294 299. proteins. Nature 403(6769):567–571. γ 23. Liesenfeld O, et al. (2011) The IFN- -inducible GTPase, Irga6, protects mice against 50. Praefcke GJ, et al. (2004) Identification of residues in the human guanylate-binding Toxoplasma gondii but not against Plasmodium berghei and some other intracellular protein 1 critical for nucleotide binding and cooperative GTP hydrolysis. J Mol Biol pathogens. PLoS One 6(6):e20568. 344(1):257–269. 24. Taylor GA, et al. (2000) Pathogen-specific loss of host resistance in mice lacking the 51. Sinensky M, Lutz RJ (1992) The prenylation of proteins. BioEssays 14(1):25–31. IFN-gamma-inducible gene IGTP. Proc Natl Acad Sci USA 97(2):751–755. 52. Stickney JT, Buss JE (2000) Murine guanylate-binding protein: Incomplete ger- 25. Melzer T, Duffy A, Weiss LM, Halonen SK (2008) The gamma interferon (IFN-gamma)- anylgeranyl isoprenoid modification of an interferon-gamma-inducible guanosine inducible GTP-binding protein IGTP is necessary for toxoplasma vacuolar disruption triphosphate-binding protein. Mol Biol Cell 11(7):2191–2200. and induces parasite egression in IFN-gamma-stimulated astrocytes. Infect Immun 53. Alory C, Balch WE (2001) Organization of the Rab-GDI/CHM superfamily: The func- 76(11):4883–4894. tional basis for choroideremia disease. Traffic 2(8):532–543. 26. Papic N, Hunn JP, Pawlowski N, Zerrahn J, Howard JC (2008) Inactive and active states 54. Rak A, et al. (2003) Structure of Rab GDP-dissociation inhibitor in complex with of the interferon-inducible resistance GTPase, Irga6, in vivo. J Biol Chem 283(46): prenylated YPT1 GTPase. Science 302(5645):646–650. 32143–32151. 55. Ignatev A, Kravchenko S, Rak A, Goody RS, Pylypenko O (2008) A structural model of 27. Hunn JP, et al. (2008) Regulatory interactions between IRG resistance GTPases in the the GDP dissociation inhibitor rab membrane extraction mechanism. J Biol Chem cellular response to Toxoplasma gondii. EMBO J 27(19):2495–2509. 283(26):18377–18384. 28. Ling YM, et al. (2006) Vacuolar and plasma membrane stripping and autophagic 56. Schalk I, et al. (1996) Structure and mutational analysis of Rab GDP-dissociation in- elimination of Toxoplasma gondii in primed effector macrophages. J Exp Med 203(9): hibitor. Nature 381(6577):42–48. 2063–2071. 57. Garrett MD, Zahner JE, Cheney CM, Novick PJ (1994) GDI1 encodes a GDP dissociation 29. Howe DK, Sibley LD (1995) Toxoplasma gondii comprises three clonal lineages: Cor- inhibitor that plays an essential role in the yeast secretory pathway. EMBO J 13(7): – relation of parasite genotype with human disease. J Infect Dis 172(6):1561–1566. 1718 1728. 30. Niedelman W, et al. (2012) The rhoptry proteins ROP18 and ROP5 mediate Toxo- 58. Nuoffer C, Balch WE (1994) GTPases: Multifunctional molecular switches regulating – plasma gondii evasion of the murine, but not the human, interferon-gamma re- vesicular traffic. Annu Rev Biochem 63:949 990. 59. Ullrich O, Horiuchi H, Bucci C, Zerial M (1994) Membrane association of Rab5 medi- sponse. PLoS Pathog 8(6):e1002784. ated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 31. Behnke MS, et al. (2011) Virulence differences in Toxoplasma mediated by amplifi- 368(6467):157–160. cation of a family of polymorphic pseudokinases. Proc Natl Acad Sci USA 108(23): 60. D’Adamo P, et al. (2002) Deletion of the mental retardation gene Gdi1 impairs associative 9631–9636. memory and alters social behavior in mice. Hum Mol Genet 11(21):2567–2580. 32. Reese ML, Zeiner GM, Saeij JP, Boothroyd JC, Boyle JP (2011) Polymorphic family of 61. Shisheva A, Chinni SR, DeMarco C (1999) General role of GDP dissociation inhibitor 2 injected pseudokinases is paramount in Toxoplasma virulence. Proc Natl Acad Sci USA in membrane release of Rab proteins: Modulations of its functional interactions by in 108(23):9625–9630. vitro and in vivo structural modifications. Biochemistry 38(36):11711–11721. 33. Steinfeldt T, et al. (2010) Phosphorylation of mouse immunity-related GTPase (IRG) 62. Kresse A, et al. (2008) Analyses of murine GBP homology clusters based on in silico, in resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol vitro and in vivo studies. BMC Genomics 9:158. 8(12):e1000576. 63. Yamamoto M, et al. (2011) ATF6beta is a host cellular target of the Toxoplasma 34. Fentress SJ, et al. (2010) Phosphorylation of immunity-related GTPases by a Toxo- gondii virulence factor ROP18. J Exp Med 208(7):1533–1546. plasma gondii-secreted kinase promotes macrophage survival and virulence. Cell Host 64. Wang H, et al. (2013) One-step generation of mice carrying mutations in multiple – Microbe 8(6):484 495. genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. 35. Choi J, et al. (2014) The parasitophorous vacuole membrane of Toxoplasma gondii is 65. Ma JS, et al. (2014) Selective and strain-specific NFAT4 activation by the Toxoplasma targeted for disruption by ubiquitin-like conjugation systems of autophagy. Immunity gondii polymorphic dense granule protein GRA6. J Exp Med 211(10):2013–2032. 40(6):924–935. 66. Soding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein ho- 36. Haldar AK, Piro AS, Pilla DM, Yamamoto M, Coers J (2014) The E2-like conjugation mology detection and structure prediction. Nucleic Acids Res 33(Web Server issue): enzyme Atg3 promotes binding of IRG and Gbp proteins to Chlamydia- and Toxo- W244–W248. plasma-containing vacuoles and host resistance. PLoS One 9(1):e86684. 67. Liang S, Zheng D, Zhang C, Standley DM (2011) Fast and accurate prediction of 37. Ohshima J, et al. (2014) Role of mouse and human autophagy proteins in IFN-γ-in- protein side-chain conformations. Bioinformatics 27(20):2913–2914. duced cell-autonomous responses against Toxoplasma gondii. J Immunol 192(7): 68. Kinoshita K, Nakamura H (2004) eF-site and PDBjViewer: Database and viewer for 3328–3335. protein functional sites. Bioinformatics 20(8):1329–1330.

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