Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation

  • Nature Microbiology volume 2, Article number: 17066 (2017)
  • doi:10.1038/nmicrobiol.2017.66
  • Download Citation


Ubiquitination of invading Salmonella Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors such as p62/SQSTM1, NDP52/CALCOCO and optineurin (OPTN), which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. Besides this, the functional consequences and mechanistic regulation of differentially linked Ub chains at the host–Salmonella interface have remained unexplored. Here, we show, for the first time, that distinct Ub chains on cytosolic S. Typhimurium serve as a platform triggering further signalling cascades. By using single-molecule localization microscopy, we visualized the balance and nanoscale distribution pattern of linear (M1-linked) Ub chain formation at the surface of cytosolic S. Typhimurium. In addition, we identified the deubiquitinase OTULIN as central regulator of these M1-linked Ub chains on the bacterial coat. OTULIN depletion leads to enhanced formation of linear Ub chains, resulting in local recruitment of NEMO, activation of IKKα/IKKβ and ultimately NF-κB, which in turn promotes secretion of pro-inflammatory cytokines and restricts bacterial proliferation. Our results establish a role for the linear Ub coat around cytosolic S. Typhimurium as the local NF-κB signalling platform and provide insights into the function of OTULIN in NF-κB activation during bacterial pathogenesis.

  • Subscribe to Nature Microbiology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    & Self and nonself: how autophagy targets mitochondria and bacteria. Cell Host Microbe 15, 403–411 (2014).

  2. 2.

    & Autophagy in antimicrobial immunity. Mol. Cell 54, 224–233 (2014).

  3. 3.

    et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 6172–6176 (2008).

  4. 4.

    et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012).

  5. 5.

    et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

  6. 6.

    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).

  7. 7.

    et al. The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway. J. Immunol. 183, 5909–5916 (2009).

  8. 8.

    et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).

  9. 9.

    et al. OTULIN restricts Met1-linked ubiquitination to control innate immune signaling. Mol. Cell 50, 818–830 (2013).

  10. 10.

    et al. Molecular basis and regulation of OTULIN–LUBAC interaction. Mol. Cell 54, 335–348 (2014).

  11. 11.

    et al. Binding of OTULIN to the PUB domain of HOIP controls NF-κB signaling. Mol. Cell 54, 349–361 (2014).

  12. 12.

    et al. The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis. Nature 498, 318–324 (2013).

  13. 13.

    , & On the use of Ripley's K-function and its derivatives to analyze domain size. Biophys. J. 97, 1095–1103 (2009).

  14. 14.

    , , , & Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail. PLoS Pathogens 9, e1003198 (2013).

  15. 15.

    et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol. Cell 36, 831–844 (2009).

  16. 16.

    et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol. Cell 46, 746–758 (2012).

  17. 17.

    et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009).

  18. 18.

    & Regulation and function of IKK and IKK-related kinases. Sci. STKE 2006, re13 (2006).

  19. 19.

    et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell Biol. 11, 123–132 (2009).

  20. 20.

    et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).

  21. 21.

    et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).

  22. 22.

    et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011).

  23. 23.

    & Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).

  24. 24.

    , & Cargo recognition and trafficking in selective autophagy. Nat. Cell Biol. 16, 495–501 (2014).

  25. 25.

    et al. Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains. Proc. Natl Acad. Sci. USA 110, 15247–15252 (2013).

  26. 26.

    et al. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl Acad. Sci. USA 113, 4039–4044 (2016).

  27. 27.

    et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

  28. 28.

    et al. Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells. Mol. Cell 47, 797–809 (2012).

  29. 29.

    , , , & Receptor–ligand interactions: binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem 15, 671–676 (2014).

  30. 30.

    , & Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

  31. 31.

    , , & Correlative super-resolution imaging of RNA polymerase distribution and dynamics, bacterial membrane and chromosomal structure in Escherichia coli. Methods Appl. Fluoresc. 3, 014005 (2015).

  32. 32.

    et al. rapidSTORM: accurate, fast open-source software for localization microscopy. Nat. Methods 9, 1040–1041 (2012).

  33. 33.

    & Extracting quantitative information from single-molecule super-resolution imaging data with LAMA—localization microscopy analyzer. Sci. Rep. 6, 34486 (2016).

  34. 34.

    , , & Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

  35. 35.

    & ViSP: representing single-particle localizations in three dimensions. Nat. Methods 10, 689–690 (2013).

  36. 36.

    , , , & OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nat. Commun. 4, 2519 (2013).

  37. 37.

    , & Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner. J. Cell Biol. 163, 315–326 (2003).

  38. 38.

    , & Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain-specific sensors. Nat. Protoc. 8, 1449–1458 (2013).

Download references


The authors thank D. Bumann for providing S. Typhimurium strains SFH4, SL1344 and DsRed expressing SL1344 and E. Laplantine for providing the NEMO-GFP plasmid. The authors also thank D. Hoeller, A. Stolz and K. Koch for critical reading and commenting on the manuscript. This work was supported by the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177; I.D., M.H., F.F.), by the DFG-funded Cluster of Excellence ‘Macromolecular Complexes’ (EXC115; I.D., M.H., F.F.), by the DFG-funded SPP 1580 programme ‘Intracellular Compartments as Places of Pathogen–Host-Interactions’ (I.D.) and by the LOEWE programme ‘Ubiquitin Networks’ (Ub-Net; I.D.) and the LOEWE Center for Gene and Cell Therapy Frankfurt (CGT; I.D.), both funded by the State of Hesse/Germany. L.H. is funded by an EMBO long-term postdoctoral fellowship.

Author information

Author notes

    • Sjoerd J. L. van Wijk
    •  & Franziska Fricke

    These authors contributed equally to this work.


  1. Institute of Biochemistry II, Goethe University – Medical Faculty, University Hospital Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

    • Sjoerd J. L. van Wijk
    • , Lina Herhaus
    • , Paolo Grumati
    • , Manuel Kaulich
    •  & Ivan Dikic
  2. Institute for Experimental Cancer Research in Pediatrics, Goethe University, Komturstrasse 3a, 60528 Frankfurt am Main, Germany

    • Sjoerd J. L. van Wijk
    •  & Simone Fulda
  3. German Cancer Consortium (DKTK), Heidelberg, Germany

    • Sjoerd J. L. van Wijk
    • , Florian R. Greten
    •  & Simone Fulda
  4. German Cancer Research Centre (DKFZ), Heidelberg, Germany

    • Sjoerd J. L. van Wijk
    • , Florian R. Greten
    •  & Simone Fulda
  5. Institute of Physical and Theoretical Chemistry, Goethe University, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany

    • Franziska Fricke
    •  & Mike Heilemann
  6. Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt am Main, Germany

    • Jalaj Gupta
    •  & Florian R. Greten
  7. Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany

    • Katharina Hötte
    • , Francesco Pampaloni
    •  & Ivan Dikic
  8. Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan

    • Yu-shin Sou
    •  & Masaaki Komatsu


  1. Search for Sjoerd J. L. van Wijk in:

  2. Search for Franziska Fricke in:

  3. Search for Lina Herhaus in:

  4. Search for Jalaj Gupta in:

  5. Search for Katharina Hötte in:

  6. Search for Francesco Pampaloni in:

  7. Search for Paolo Grumati in:

  8. Search for Manuel Kaulich in:

  9. Search for Yu-shin Sou in:

  10. Search for Masaaki Komatsu in:

  11. Search for Florian R. Greten in:

  12. Search for Simone Fulda in:

  13. Search for Mike Heilemann in:

  14. Search for Ivan Dikic in:


S.J.L.v.W., M.H. and I.D. conceived the study and interpreted all the results. S.J.L.v.W. performed immunofluorescence, dSTORM sample preparation, bacterial infections and colony-formation assays. F.F. performed dSTORM imaging, acquirement and data analysis. L.H. performed luciferase assays and qRT–PCR analysis. M.Ka. supported the generation of OTULIN CRISPR/Cas9 cells. K.H. performed whole-mount infected organoid immunofluorescence experiments. K.H. and F.P. performed live-cell Salmonella infection experiments and data analysis. J.G., P.G. and F.R.G. performed organoid culture experiments, Y.-s.S. and M.Ko made Otulinf/f;CAG-Cre-ER+/– animals. S.F. provided support with reagents and expertise. S.J.L.v.W., M.H. and I.D. wrote the manuscript. All authors discussed and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Mike Heilemann or Ivan Dikic.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1-12.


  1. 1.

    Supplementary Video 1

    Live-cell imaging of doxycycline (Dox)-induced HeLa

  2. 2.

    Supplementary Video 2

    Live-cell imaging of doxycycline (Dox)-induced HeLa

  3. 3.

    Supplementary Video 3

    Confocal microscope z-stack scan of S. Typhimurium

  4. 4.

    Supplementary Video 4

    Confocal microscope z-stack scan of S. Typhimurium