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LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB

Nature Microbiology volume 2, Article number: 17063 (2017) Cite this article

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Abstract

Cell-autonomous immunity relies on the ubiquitin coat surrounding cytosol-invading bacteria functioning as an ‘eat-me’ signal for xenophagy. The origin, composition and precise mode of action of the ubiquitin coat remain incompletely understood. Here, by studying Salmonella Typhimurium, we show that the E3 ligase LUBAC generates linear (M1-linked) polyubiquitin patches in the ubiquitin coat, which serve as antibacterial and pro-inflammatory signalling platforms. LUBAC is recruited via its subunit HOIP to bacterial surfaces that are no longer shielded by host membranes and are already displaying ubiquitin, suggesting that LUBAC amplifies and refashions the ubiquitin coat. LUBAC-synthesized polyubiquitin recruits Optineurin and Nemo for xenophagy and local activation of NF-κB, respectively, which independently restrict bacterial proliferation. In contrast, the professional cytosol-dwelling Shigella flexneri escapes from LUBAC-mediated restriction through the antagonizing effects of the effector E3 ligase IpaH1.4 on deposition of M1-linked polyubiquitin and subsequent recruitment of Nemo and Optineurin. We conclude that LUBAC-synthesized M1-linked ubiquitin transforms bacterial surfaces into signalling platforms for antibacterial immunity reminiscent of antiviral assemblies on mitochondria.

Mammalian cells maintain a sterile cytosol by deploying galectins to survey endomembrane damage and subsequently coat invading bacteria as well as damaged membranes with polyubiquitin1. The bacterial ubiquitin coat comprises multiple linkage types, synthesized by several E3 ligases such as LRSAM1, Parkin, Smurf1 and RNF166 (refs 2–5). Galectin-8 and the polyubiquitin coat provide ligands (‘eat-me’ signals) for NDP52, Optineurin and other cargo receptors that induce antibacterial autophagy (‘xenophagy’) and restrict bacterial proliferation1,6–10. Autophagy cargo receptors are not evenly dispersed around cytosol-invading bacteria7,9,11. Such a biased distribution of cargo receptors, sometimes referred to as ‘microdomains’, reflects the availability of their ligands, suggesting that recruitment signals and the substrate specificity of individual E3 ligases are crucial to successful xenophagy.

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Figure 1: LUBAC synthesizes M1-linked ubiquitin chains on cytosolic S. Typhimurium and restricts their proliferation.
Figure 2: HOIP senses and amplifies the ubiquitin coat of S. Typhimurium.
Figure 3: LUBAC recruits Optineurin and Nemo to S. Typhimurium.
Figure 4: LUBAC activates autophagy and NF-κB, which independently restrict cytosolic S. Typhimurium.

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Acknowledgements

This work was supported by the Medical Research Council (U105170648 to F.R. and U105192732 to D.K.) and the Wellcome Trust (WT104752MA) and Boehringer Ingelheim Fonds PhD fellowships (to C.P. and M.A.M.). The authors thank N. Mizushima for ATG5−/− MEFs, H. Walczack for Sharpincpdm MEFs, M. Gyrd-Hansen for RipK2−/− and HOIP−/− HCT116s and E. Werner for technical help.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Divison of Protein and Nucleic Acid Chemistry, Francis Crick Avenue, CB2 0QH, Cambridge, UK

    Jessica Noad, Alexander von der Malsburg, Claudio Pathe, Martin A. Michel, David Komander & Felix Randow

  2. Department of Medicine, University of Cambridge, Addenbrooke's Hospital, CB2 0QQ, Cambridge, UK

    Felix Randow

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Contributions

J.N. performed and analysed all experiments, except those in Fig. 2e (A.v.d.M., D.K.), Fig. 3h (C.P.), Fig. 3i (C.P., J.N.), Supplementary Fig. 1 (A.M., D.K.), Supplementary Fig. 2a (A.M.), Supplementary Fig. 6 (A.M.) and Supplementary Fig. 7 (A.M., M.A.M., D.K.). J.N. and F.R. wrote the manuscript.

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Correspondence to Felix Randow.

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Supplementary Information

Supplementary Figures 1–15. (PDF 10571 kb)

Supplementary Video 1

LUBAC is recruited after membrane damage and remains associated with daughter bacteria. Live imaging on a confocal spinning disk microscope of MEFs co-expressing mCherry:Galectin-8, GFP:HOIL-1, Flag:HOIP and Flag:Sharpin, infected with BFP-expressing S.Typhimurium and imaged every 2 min. Supplement to Fig. 2g. Scale bar, 7μm. (MOV 890 kb)

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Noad, J., von der Malsburg, A., Pathe, C. et al. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB. Nat Microbiol 2, 17063 (2017). https://doi.org/10.1038/nmicrobiol.2017.63

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