Abstract
Vertebrate immunity to infection enlists a newly identified family of 47-kilodalton immunity-related GTPases (IRGs). One IRG in particular, Irgm1, is essential for macrophage host defense against phagosomal pathogens, including Mycobacterium tuberculosis (Mtb). Here we show that Irgm1 targets the mycobacterial phagosome through lipid-mediated interactions with phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) and PtdIns(3,4,5)P3. An isolated Irgm1 amphipathic helix conferred lipid binding in vitro and in vivo. Substitutions in this region blocked phagosome recruitment and failed to complement the antimicrobial defect in Irgm1−/− macrophages. Removal of PtdIns(3,4,5)P3 or inhibition of class I phosphatidylinositol-3-OH kinase (PI(3)K) mimicked this effect in wild-type cells. Cooperation between Irgm1 and PI(3)K further facilitated the engagement of Irgm1 with its fusogenic effectors at the site of infection, thereby ensuring pathogen-directed responses during innate immunity.
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Acknowledgements
We thank L. Cantley (Harvard Medical School), P. De Camilli (Yale University School of Medicine), J. Galan (Yale University School of Medicine), P. Lengyel (Yale University School of Medicine), R. Lin (Stony Brook University), J. Lucocq (University of Dundee), T. Meyer (Stanford University), C. Roy (Yale University School of Medicine), B. Vanhaesebroeck (University of London) and G. Warren (Vienna Biocenter) for antibodies, plasmids or cDNAs used in this study; P. Cresswell (Yale University School of Medicine) for the yeast two-hybrid mouse embryonic fibroblast library; G. Taylor (Duke University Medical Center) for Irgm1−/− mice, Y. Lu (University of Iowa) for BCG-GFP; and A. Shenoy for help with Irgm1 crystallographic modeling. Supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (R01 AI068041-01A1), the Burroughs-Wellcome Fund (1007845), Edward R. Mallinckrodt Foundation (R06152), the Searle Foundation (05-F-114), the Cancer Research Institute, the W.W. Winchester Foundation (J.D.M.) and the Japanese Society for the Promotion of Science (T.M.).
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S.T., T.M., H.-P.C., M.P. and J.D.M. did the experimental design, data analysis and interpretation; S.T., H.-P.C. and T.M. did molecular and biochemical analyses (affinity purification, gel filtration, yeast-two hybrid, in vitro assays); S.T., T.M., H.-P.C. and J.D.M. did scanning confocal microscopy; M.P. and J.D.M. did transmission electron microscopy of immunolabeled cryosections; J.D.M. wrote the manuscript; and all authors discussed the results and commented on the manuscript.
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Supplementary Figures 1–9 and Supplementary Tables 1–2 (PDF 6130 kb)
Supplementary Movie 1
Live imaging of Irgm1 (RFP-Irgm1, pseudocolored green) translocation to sites of M. bovis BCG internalization (white arrows) in IFN-γ-activated RAW264.7 macrophages. EGFP-BCG, pseudocolored red. (AVI 11535 kb)
Supplementary Movie 2
Triple-labeled live imaging of Irgm1 translocation to PtdIns(3,4,5)P3-pseudopods internalizing M. bovis BCG in IFN-γ-activated RAW264.7 cells (white arrow). CFP-Irgm1 (pseudocolored green); YFP-GRP1-PH (pseudocolored red); Cy5-BCG (blue). (AVI 951 kb)
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Tiwari, S., Choi, HP., Matsuzawa, T. et al. Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P2 and PtdIns(3,4,5)P3 promotes immunity to mycobacteria. Nat Immunol 10, 907–917 (2009). https://doi.org/10.1038/ni.1759
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DOI: https://doi.org/10.1038/ni.1759
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