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Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection


Ubiquitylation is a widespread post-translational protein modification in eukaryotes and marks bacteria that invade the cytosol as cargo for antibacterial autophagy1,2,3. The identity of the ubiquitylated substrate on bacteria is unknown. Here we show that the ubiquitin coat on Salmonella that invade the cytosol is formed through the ubiquitylation of a non-proteinaceous substrate, the lipid A moiety of bacterial lipopolysaccharide (LPS), by the E3 ubiquitin ligase ring finger protein 213 (RNF213). RNF213 is a risk factor for moyamoya disease4,5, which is a progressive stenosis of the supraclinoid internal carotid artery that causes stroke (especially in children)6,7. RNF213 restricts the proliferation of cytosolic Salmonella and is essential for the generation of the bacterial ubiquitin coat, both directly (through the ubiquitylation of LPS) and indirectly (through the recruitment of LUBAC, which is a downstream E3 ligase that adds M1-linked ubiquitin chains onto pre-existing ubiquitin coats8). In cells that lack RNF213, bacteria do not attract ubiquitin-dependent autophagy receptors or induce antibacterial autophagy. The ubiquitylation of LPS on Salmonella that invade the cytosol requires the dynein-like core of RNF213, but not its RING domain. Instead, ubiquitylation of LPS relies on an RZ finger in the E3 shell. We conclude that ubiquitylation extends beyond protein substrates and that ubiquitylation of LPS triggers cell-autonomous immunity, and we postulate that non-proteinaceous substances other than LPS may also become ubiquitylated.

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Fig. 1: Ubiquitylation of LPS.
Fig. 2: RNF213 is required for ubiquitylation of LPS.
Fig. 3: Ubiquitylation of LPS by RNF213 is a RING-independent, RZ-finger-mediated reaction.
Fig. 4: RNF213 provides cell-autonomous immunity.

Data availability

All data are included in the Article and its Supplementary Information. Gel source images are provided in Supplementary Fig. 1. Materials can be obtained from the corresponding authors upon request. Source data are provided with this paper.


  1. Huang, J. & Brumell, J. H. Bacteria–autophagy interplay: a battle for survival. Nat. Rev. Microbiol. 12, 101–114 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Perrin, A. J., Jiang, X., Birmingham, C. L., So, N. S. Y. & Brumell, J. H. Recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system. Curr. Biol. 14, 806–811 (2004).

    CAS  PubMed  Google Scholar 

  3. Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kamada, F. et al. A genome-wide association study identifies RNF213 as the first moyamoya disease gene. J. Hum. Genet. 56, 34–40 (2011).

    CAS  PubMed  Google Scholar 

  5. Liu, W. et al. Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development. PLoS ONE 6, e22542 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Scott, R. M. & Smith, E. R. Moyamoya disease and moyamoya syndrome. N. Engl. J. Med. 360, 1226–1237 (2009).

    CAS  PubMed  Google Scholar 

  7. Kuroda, S. & Houkin, K. Moyamoya disease: current concepts and future perspectives. Lancet Neurol. 7, 1056–1066 (2008).

    PubMed  Google Scholar 

  8. Noad, J. et al. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB. Nat. Microbiol. 2, 17063 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Schaible, U. E. & Haas, A. Intracellular Niches of Microbe: A Pathogens Guide through the Host Cell (Wiley Blackwell, 2009).

  10. Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T. & Brumell, J. H. Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J. Biol. Chem. 281, 11374–11383 (2006).

    CAS  PubMed  Google Scholar 

  11. Nakagawa, I. et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040 (2004).

    ADS  CAS  PubMed  Google Scholar 

  12. Thurston, T. L. M., Wandel, M. P., von Muhlinen, N., Foeglein, A. & Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–418 (2012).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Thurston, T. L. M., Ryzhakov, G., Bloor, S., von Muhlinen, N. & Randow, F. The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat. Immunol. 10, 1215–1221 (2009).

    CAS  PubMed  Google Scholar 

  14. Wild, P. et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dupont, N. et al. Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy. Cell Host Microbe 6, 137–149 (2009).

    CAS  PubMed  Google Scholar 

  16. Huett, A. et al. The LRR and RING domain protein LRSAM1 is an E3 ligase crucial for ubiquitin-dependent autophagy of intracellular Salmonella Typhimurium. Cell Host Microbe 12, 778–790 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Manzanillo, P. S. et al. The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature 501, 512–516 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. van Wijk, S. J. L. et al. Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation. Nat. Microbiol. 2, 17066 (2017).

    PubMed  Google Scholar 

  19. Franco, L. H. et al. The ubiquitin ligase Smurf1 functions in selective autophagy of Mycobacterium tuberculosis and anti-tuberculous host defense. Cell Host Microbe 21, 59–72 (2017).

    CAS  PubMed  Google Scholar 

  20. Fiskin, E., Bionda, T., Dikic, I. & Behrends, C. Global analysis of host and bacterial ubiquitinome in response to Salmonella Typhimurium infection. Mol. Cell 62, 967–981 (2016).

    CAS  PubMed  Google Scholar 

  21. Engström, P. et al. Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence. Nat. Microbiol. 4, 2538–2551 (2019).

    PubMed  PubMed Central  Google Scholar 

  22. Raetz, C. R. H. & Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 (2002).

    CAS  PubMed  Google Scholar 

  23. Whitfield, C. & Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014).

    CAS  PubMed  Google Scholar 

  24. Ahel, J. et al. Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism. eLife 9, e56185 (2020).

    PubMed  PubMed Central  Google Scholar 

  25. Piccolis, M. et al. Probing the global cellular responses to lipotoxicity caused by saturated fatty acids. Mol. Cell 74, 32–44.e8 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Sugihara, M. et al. The AAA+ ATPase/ubiquitin ligase mysterin stabilizes cytoplasmic lipid droplets. J. Cell Biol. 218, 949–960 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Banh, R. S. et al. PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia. Nat. Cell Biol. 18, 803–813 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Morito, D. et al. Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state. Sci. Rep. 4, 4442 (2014).

    PubMed  PubMed Central  Google Scholar 

  29. Wang, Y. et al. Mitochondria-localised ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS. Nat. Cell Biol. 21, 1346–1356 (2019).

    CAS  PubMed  Google Scholar 

  30. Liu, W., Hitomi, T., Kobayashi, H., Harada, K. H. & Koizumi, A. Distribution of moyamoya disease susceptibility polymorphism p.R4810K in RNF213 in East and Southeast Asian populations. Neurol. Med. Chir. (Tokyo) 52, 299–303 (2012).

    Google Scholar 

  31. Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014).

    ADS  CAS  PubMed  Google Scholar 

  32. Wandel, M. P. et al. Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat. Immunol. 21, 880–891 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Santos, J. C. et al. Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat. Commun. 11, 3276 (2020).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Randow, F. & Sale, J. E. Retroviral transduction of DT40. Subcell. Biochem. 40, 383–386 (2006).

    PubMed  Google Scholar 

  35. Glover, J. D. et al. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration. PLoS ONE 8, e77222 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ogawa, M. et al. A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens. Cell Host Microbe 9, 376–389 (2011).

    CAS  PubMed  Google Scholar 

  37. Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc. Natl Acad. Sci. USA 108, 1531–1536 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. Perkins, D. N., Pappin, D. J. C., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    CAS  PubMed  Google Scholar 

  40. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    CAS  PubMed  Google Scholar 

  41. Johnson, L. S., Eddy, S. R. & Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).

    PubMed  PubMed Central  Google Scholar 

  42. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Liu, Y., Schmidt, B. & Maskell, D. L. MSAProbs: multiple sequence alignment based on pair hidden Markov models and partition function posterior probabilities. Bioinformatics 26, 1958–1964 (2010).

    CAS  PubMed  Google Scholar 

  44. Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank F. Begum, S. Peak Chew and M. Shekel of the LMB mass spectrometry facility for analysing samples; D. Morito for providing RNF213 cDNA; and C. Gladkova and A. von der Malsburg for advice. This work was supported by a PhD fellowship from the Boehringer Ingelheim Trust to V.D., and by grants from the MRC (U105170648) and the Wellcome Trust (WT104752MA) to F.R.

Author information

Authors and Affiliations



E.W. performed and analysed experiments that led to the discovery of LPS ubiquitylation. E.G.O. identified and characterized RNF213, with contributions from A.C.C. (super-resolution microscopy, and complementation and analysis of RNF213-knockout MEFs), K.B.B. (restriction of Salmonella by RNF213), C.P. (generation of Salmonella knockouts and analysis of RNF213-knockout MEFs), V.D. (validation of RNF213-knockout cells) and B.S. (bioinformatic analysis). E.G.O. and F.R. designed the study and wrote the manuscript.

Corresponding authors

Correspondence to Elsje G. Otten or Felix Randow.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Zhijian (James) Chen, J. Wade Harper and Samuel Miller for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 LPS structure in S. Typhimurium and S. Minnesota.

a, c, The composition of lipid A, inner core, outer core and O-antigen from S. Typhimurium (a) and rough variants of S. Minnesota (c). The S. Typhimurium mutants that are deficient in specific steps of LPS biosynthesis, and truncated LPS species from S. Minnesota, are indicated in red. Substoichiometric modifications introducing amino groups and the enzymes that are responsible are indicated in blue. ArnC (the undecaprenyl-phosphate 4-deoxy-4-formamido-l-arabinose transferase) functions upstream of ArnT and is required for the ultimate incorporation of L4AraN into LPS. EptA, EptB and CptA incorporate phosphoethanolamine into LPS. b, Immunoblot analysis of the indicated S. Typhimurium strains, extracted from HeLa cells. Blots were probed with the indicated antibodies. DnaK, loading controls for bacterial lysates. Δ4, ΔeptA ΔarnC ΔcptA ΔeptB in ΔrfaL background. Representative of three biological repeats. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 RNF213 is required for ubiquitylation of LPS.

a, In vitro ubiquitylation of S. Typhimurium Δrfc extracted from infected HeLa cells. The reaction comprised Flag–ubiquitin, E1 enzyme (UBE1), E2 enzyme (UBCH5C) and fractionated HeLa cell lysate, as indicated. In the chromatograms depicted below each blot, light grey indicates fractions with little or no LPS-ubiquitylating activity, whereas blue indicates fractions with LPS-ubiquitylating activity used for further fractionation or mass spectrometry. b, Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and wild-type HeLa and RNF213-knockout HeLa cells c, d, Immunoblot analysis of S. Typhimurium Δrfc (c, d left) or wild type (d right) extracted from HeLa cells transfected with the indicated siRNAs (c) or extracted from wild-type or Rnf213-knockout MEFs (d). Representative of three biological repeats. Blots were probed with the indicated antibodies. Actin and GroEL or DnaK, loading controls for mammalian and bacterial lysates, respectively. Asterisk, non-specific band. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3 Ubiquitylation of LPS by RNF213 is a RING-independent, RZ-finger-mediated reaction.

a, RNF213 domain structure (Protein Data Bank 6TAX) (left) with domain colours corresponding to Fig. 3a, and zoom-in on the E3 module (middle, right) indicating N-terminal (N), middle (M) and C-terminal (C) lobes of the E3 shell domain. Red arrows point at the position of the unresolved RZ finger. b, Coomassie-stained gel of purified RNF213. c, d, Left panels show immunoblot analysis of S. Typhimurium Δrfc extracted from HeLa cells and subjected to in vitro ubiquitylation using HeLa, Sf9 or Sf9 expressing human RNF213 lysates (c) or purified RNF213 (d) corresponding to Fig. 3c, d. c, Right, Coomassie gel and immunoblot analysis of lysates used in Fig. 3c. d, Right, immunoblot analysis of in vitro ubiquitylation reaction supernatants, separated under non-reducing and reducing (+βme) conditions, corresponding to Fig. 3d. In c, d, blots were probed with indicated antibodies. e, Alignment and conservation scores of RZ fingers in RNF213 and ZNFX1 from the indicated species. Colour and height denote the degree of conservation (from Jalview) (top). RZ finger alignment (bottom). In b, d, representative shown of three biological repeats. In c, n = 1. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4 RNF213 provides cell-autonomous immunity.

a, CFU of S. Typhimurium at 1 h after infection, extracted from wild-type and Rnf213-knockout MEFs. Bacteria were counted by serial dilution of cell lysate on LB agar plates. Data are mean ± s.e.m. of three experiments. b, Percentage of cytosolic S. Typhimurium positive for Flag–GFP–RNF213 at 3 h after infection in Rnf213-knockout MEFs stably expressing the indicated GFP-RNF213 alleles. co, Representative confocal micrographs for Fig. 4d–o. ce, Wild-type or Rnf213-knockout MEFs stably expressing the indicated GFP-RNF213 alleles infected with mCherry-expressing S. Typhimurium, fixed 3 h after infection. fo, Wild-type and Rnf213-knockout MEFs (f, g, n), Rnf213-knockout MEFs stably expressing GFP–RNF213H4509A (g, o) or wild-type and Rnf213-knockout MEFs stably expressing GFP–HOIP(1–438) or GFP–HOIP(1–438, T360A) (h), GFP–Nemo (j), GFP–optineurin(F178S) (k), GFP–NDP52 (l), GFP–p62 (m), infected with mCherry-expressing (cf, hn) or BFP-expressing (g, o) S. Typhimurium, fixed 3 h after infection and stained for FK2 (ubiquitin) (f, g), M1-linked linear ubiquitin chains (i) or LC3 (n, o). In g, o, for better visibility of bacteria, the Hoechst and BFP channel has been depicted in white in the zoomed sections. Scale bar, 10 μm (cn). Statistical significance was assessed by two-tailed unpaired Student’s t-test (a) or one-way analysis of variance (b). ns, not significant. Data are mean ± s.e.m. of three independent experiments (a, b) and micrographs are representative of three biological repeats (co).

Source data

Extended Data Fig. 5 Model of RNF213-mediated ubiquitylation of LPS during bacterial infection.

Damage of Salmonella-containing vacuoles releases S. Typhimurium into the host cytosol, where RNF213 associates with the bacterial surface and ubiquitylates LPS, resulting in LUBAC recruitment and the deposition of M1-linked ubiquitin chains linked to an unidentified substrate. Recruitment of the autophagy cargo receptors NDP52 and p62 requires RNF213 but not LUBAC, while that of Optn and the IKK subunit NEMO relies on the activity of both RNF213 and LUBAC. Yellow insert, the structure of the Gram-negative cell envelope. PG, peptidoglycan. Red insert, lipid A, the minimal substrate for RNF213-mediated ubiquitylation of LPS. Ubiquitylation of lipid A is predicted to target its hydroxy or phosphate groups. The C6′ OH function is not available as a potential ubiquitylation site when the core is present.

Supplementary information

Supplementary Figure 1

Source gel data for the cropped immunoblots shown in Figs 1–3 and Extended Data Figs 1–3.

Reporting Summary

Peer Review File

Video 1

The spread of RNF213 across the surface of S. Typhimurium. Instant structured illumination microscopy (iSIM) of RNF213 KO MEFs expressing FLAG-GFP-RNF213 infected with mCherry-expressing S. Typhimurium. Time p.i. as indicated. Scale bar, 1 μm.

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Otten, E.G., Werner, E., Crespillo-Casado, A. et al. Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection. Nature 594, 111–116 (2021).

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