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Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense

Nature Immunology volume 18, pages 899–910 (2017)Cite this article

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Abstract

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

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Figure 1: GABARAPs, but not LC3 proteins, are indispensable for the full IFN-γ-induced response to T. gondii infection.
Figure 2: GABARAPs are required for recruitment of interferon-inducible GTPases and effectors to T. gondii and S. typhimurium PCVs.
Figure 3: LC3-, FIP200-, Atg9a- or Atg14-dependent canonical autophagy is not involved in GBP localization.
Figure 4: The C-terminal lipidation of Gate-16 is essential for IFN-γ-induced clearance of T. gondii.
Figure 5: Arf1-dependent generation of GBP-containing vesicles is required for the IFN-γ-induced response.
Figure 6: The ubiquitin-like domain of Gate-16 interacts with Arf1.
Figure 7: Gate-16 is essential for acute resistance to T. gondii in vivo.
Figure 8: Myeloid-cell-specific Gate-16 deficiency results in high susceptibility to T. gondii infection.

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Acknowledgements

We thank M. Enomoto (Osaka University) for secretarial and technical assistance, D. Soldati-Favre (University of Geneva), J.C. Howard (Instituto Gulbenkian de Ciência) and E.M. Frickel (Francis Crick Institute) for providing antibodies, M. Komatsu (Niigata University) for Atg7-deficient MEFs and H.W. Virgin (Washington University at St. Louis) for helpful discussion. Supported by the Research Program on Emerging and Re-emerging Infectious Diseases (grant no. 17fk0108120h0001) from the Agency for Medical Research and Development (AMED) (M.Y.), the Grant-in-Aid for Scientific Research on Innovative Areas (“Homeostatic regulation by various types of cell death”, grant no. 17H0557 (M.Y.), and “Matoryoshka-type evolution”, grant no. 26117713 (M.Y.)) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.Y.), a Cooperative Research Grant of the Institute for Enzyme Research (M.Y.), the Joint Usage/Research Center of Tokushima University (M.Y.), the Takeda Science Foundation (M.Y.), the Ohyama Health Foundation (M.Y.), the Heiwa Nakajima Foundation (M.Y.), the Cell Science Research Foundation (M.Y.),the Research Foundation for Microbial Diseases of Osaka University (M.Y.) and the Naito Foundation (M.S.).

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Authors and Affiliations

  1. Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Miwa Sasai, Naoya Sakaguchi, Ji Su Ma, Hironori Bando & Masahiro Yamamoto

  2. Laboratory of Immunoparasitology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Miwa Sasai, Naoya Sakaguchi, Youngae Lee & Masahiro Yamamoto

  3. Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan

    Shuhei Nakamura, Tsuyoshi Kawabata & Tamotsu Yoshimori

  4. Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

    Shuhei Nakamura, Tsuyoshi Kawabata & Tamotsu Yoshimori

  5. Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Tatsuya Saitoh & Shizuo Akira

  6. Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Tatsuya Saitoh & Shizuo Akira

  7. Division of Inflammation Biology, Institute for Enzyme Research, Tokushima University, Tokushima, Japan

    Tatsuya Saitoh

  8. Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

    Akiko Iwasaki

  9. Department of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Daron M Standley

  10. Laboratory of System Immunology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Daron M Standley

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Contributions

M.S., N.S., H.B., Y.L., J.S.M. and M.Y. performed experiments; D.M.S. performed in silico analysis; M.S., S.N., T.K., T.Y., T.S., S.A., A.I. and M.Y. designed the experiments and analyzed the data; T.S. and S.A. provided critical materials; and M.S., A.I. and M.Y. wrote the manuscript.

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Correspondence to Masahiro Yamamoto.

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Integrated supplementary information

Supplementary Figure 1 Generation of mice or MEFs lacking mouse Atg8 family members or FIP200 by CRISPR-mediated genome editing.

(a) Unrooted phylogenic tree of murine Atg8 family members. (b, c, d, e, f, i) Schematic of the gRNA-targeting sites in the (b) Map1lc3a (LC3a), (c) Map1lc3b (LC3b), (d) Gabarap, (e) Gabarapl1, or (f) Gabarapl2 (Gate-16), (i) Rb1cc1 (FIP200) genes. Boxed and underlined regions are the gRNA target or PAM sequences, respectively. (g, j) Immunoblot analysis for the expression of indicated proteins and Actin in GABARAP TKO cells transfected with Flag-tagged empty or indicated vectors (g) or in wild-type and FIP200 KO MEFs (j). Data is one of the representative data in three independent experiments. (h) 293T cells were stimulated with 10 ng/ml hIFN-γ for 24 hrs. Lysates of unstimulated or stimulated cells were immunoprecipitated by indicated antibodies, and subjected to immunoblot to detect indicated proteins. Data was the representative of three independent experiments.

Supplementary Figure 2 Normal recruitment of interferon-inducible GTPases and effectors to T. gondii PCVs in MEFs lacking FIP200, Atg9a or Atg14.

(a) Immunostaining of T. gondii-infected IFN-γ-stimulated wild-type and indicated KO MEFs with antibodies against indicated antibodies (red), T. gondii (green), and DAPI (blue). Scale bars, 10 μm. Three independent experiments were performed. (b) Wild-type and indicated KO MEFs were treated with 10 ng/ml IFN-γ or not for 24 hours and infected with C. koseri (moi = 10) for 2 hours. The cells were fixed and stained with antibodies against indicated antibodies GBP1-5 (red), GBP2 (green), and DAPI (blue). Representative confocal microscope images of the recruitment of GBP1-5 or GBP2 on DAPI (indicative of bacteria) in WT or indicated KO MEFs. Scale bars, 10 μm. Images are from one of the representative data in three independent experiments. (Right) Results of quantification analysis for GBP1-5- or GBP2- and DAPI positive bacteria are shown. Indicated values were means ± s.d (three independent experiments per group). The “C. koseri” was not colocalized with DAPI. Although we easily detected DAPI staining of bacterial DNA inside of GABARAP TKO and Atg3 KO cells, we failed to detect such a pattern of DAPI staining at all in wild-type cells. Instead, WT cells contained corn-shaped accumulation of GBPs, which is very similar to the shape of C. koseri-containing vacuole and hardly detected in GABARAP TKO and Atg3 KO cells. Given the dramatic reduction of the C. koseri number after IFN-γ stimulated wild-type cells, we regarded the corn- shaped accumulation of GBPs as remnants of the bacteria. (c) Indicated MEFs treated with 10 ng/ml IFN-γ for 24 hr were infected with C. koseri (moi = 50) for 18-20 hours. Bacterial numbers are shown. Indicated values were absolute bacterial number ± s.d. of two independent experiments.

Supplementary Figure 3 HA-tagged Gate-16 is rarely localized on T. gondii PCVs.

(a) Immunostaining of IFN-γ-stimulated GABARAP TKO MEFs transfected with HA-tagged Gate-16 vectors with anti-GBP1 (green), anti-HA (red), anti- T. gondii (magenta) and DAPI (blue). Images are from one of the representative of three independent experiments. Indicated values were means ± s.d. (three biological replicates per group, in which almost 100 parasites were counted). ***P < 0.001 (two-tailed Student’s t-test).
(b, c) Immunostaining of T. gondii-infected IFN-γ-stimulated wild GABARAP TKO MEFs (b) or Gate-16 KO MEFs (c) transfected with empty or indicated HA-tagged Gate-16 vectors with anti- T. gondii (green), indicated antibodies (red) and DAPI (blue). Images are from one of three independent experiments. Indicated values were means ± s.d. of three independent experiments, in which almost 150 parasites were counted. ***P < 0.001 (two-tailed Student’s t- test).

Supplementary Figure 4 GBP aggregation localizes to the Golgi or ER.

Immunostaining of IFN-γ-stimulated GABARAP TKO MEFs transfected with HA-tagged Gate-16(G116A) with indicated antibodies (green), anti-GBP1-5 (red) and DAPI (blue). Images are from a representative data in three independent experiments.

Supplementary Figure 5 In silico structural analysis of the Gate-16(25-LC3-29) mutant.

(a) Indicated values were means ± s.d. of GBP1 dot numbers per cell (Fig. 6i), in which three cells (Empty), three cells (Gate-16(WT)), or three cells (Gate-16(25-LC3-29)) calculated by ImageJ. **P < 0.01 (two-tailed Student’s t-test). Three independent experiments were performed.
(b) Ribbon model of Gate-16 (WT) and Gate-16 (25-LC-29). Amino acids at residue 25-29 are shown as spheres. (c) Alignment of amino acids sequence of murine Atg8 family members around residue 25-29. Light blue and pink portions indicate the key five amino acids in GABARAPs and LC3s, respectively.

Supplementary Figure 6 Loss of acute resistance to Type III T. gondii infection in Gate-16-deficient mice.

(a) Wild-type (n = 9) or indicated KO mice (Gate-16 KO: n = 9 and IFNγR KO: n = 9) were infected with 1 ×104 CTG T. gondii, and the survival rates were monitored for 15 days. (b) Calculus equation of luciferase unit to parasite number. The luciferase units were measured at indicated parasite numbers, and used to generate a standard curve.
(c) Prior to infection (upper) or at 4 d.p.i. (lower), spleens were collected and indicated surface markers of splenocytes from wild-type (left) or Gate-16 KO (right) mice were analysed by flow cytometry. Composition of CD19/CD3 and CD8/CD4 populations were reflected on each panel. Data was representative of three independent experiments. (d) Immunoblot analysis for the expression of indicated proteins and Actin in MLNs from wild-type and Gate-16 KO mice infected with T. gondii at 7 d.p.i. Data is one of the representative data in three independent experiments.

Supplementary Figure 7 Recruitment of inflammatory monocytes and neutrophils in control or LysM-Cre;Gate-16flox/flox mice infected with T. gondii.

(a) Peritoneal macrophages from indicated mice were stimulated with 100 ng/ml LPS for 24 hours. Indicated cytokine concentrations in the supernatants were determined by ELISA. Indicated values were means ± s.d. (three independent experiments per group). (b) LPS-induced upregulation of surface markers in peritoneal macrophages from indicated mice. The cells were stimulated with 100 ng/ml LPS for 24 hours. The indicated markers were tested by flow cytometory. Data were representative of three independent experiments.
(c) Unstimulated or IFN-γ-stimulated wild-type and Gate-16 KO bone marrow-derived macrophages were infected with T. gondii (moi = 0.5) for 24 hours. Concentrations of NO2- or the indicated cytokines in the culture supernatants were measured by Griess method or ELISA, respectively. Indicated values were means ± s.e.m (two independent experiments per group). (d) Prior to infection (upper) or at 5 d.p.i. (lower), peritoneal cells were collected and indicated surface markers and YFP (T. gondii) of peritoneal cells from control (left) or LysM-Cre Gate-16fl/fl (right) mice were analysed by flow cytometry. Composition of CD11b/YFP, CD11b/Ly6C and CD11b/Ly6G populations were reflected on each panel. Data was representative of three independent experiments.

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Sasai, M., Sakaguchi, N., Ma, J. et al. Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense. Nat Immunol 18, 899–910 (2017). https://doi.org/10.1038/ni.3767

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