Abstract

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.

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Acknowledgements

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.

Affiliations

  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

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Contributions

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

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    Supplementary Information

    Supplementary Figures 1-12.

Videos

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    Supplementary Video 1

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

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    Supplementary Video 2

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

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    Supplementary Video 3

    Confocal microscope z-stack scan of S. Typhimurium

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    Supplementary Video 4

    Confocal microscope z-stack scan of S. Typhimurium

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DOI

https://doi.org/10.1038/nmicrobiol.2017.66