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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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.
The authors declare no competing interests.
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.
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Extended data figures and tables
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.
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.
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. c–o, Representative confocal micrographs for Fig. 4d–o. c–e, Wild-type or Rnf213-knockout MEFs stably expressing the indicated GFP-RNF213 alleles infected with mCherry-expressing S. Typhimurium, fixed 3 h after infection. f–o, 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 (c–f, h–n) 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 (c–n). 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 (c–o).
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.
Source gel data for the cropped immunoblots shown in Figs 1–3 and Extended Data Figs 1–3.
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). https://doi.org/10.1038/s41586-021-03566-4