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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Super-resolution imaging (dSTORM) of Ub signals at the surface of cytosolic S. Typhimurium.
Figure 2: OTULIN regulates linear Ub at the surface of cytosolic S. Typhimurium.
Figure 3: OTULIN, by controlling linear Ub levels, regulates NF-κB activation from the surface of cytosolic S. Typhimurium and mediates bacterial clearance.
Figure 4: OTULIN regulates NF-κB activation by controlling M1 Ub formation in the bacterial Ub coat upon S. Typhimurium infection in primary intestinal crypt organoids.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Gomes, L. C. & Dikic, I. Autophagy in antimicrobial immunity. Mol. Cell 54, 224–233 (2014).

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    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  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Muranyi, W., Malkusch, S., Muller, B., Heilemann, M. & Krausslich, H. G. 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).

    Article  Google Scholar 

  15. 15

    Haas, T. L. 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).

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

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

    Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

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

    CAS  Article  Google Scholar 

  26. 26

    Richter, B. 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).

    CAS  Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    van Wijk, S. J. 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).

    CAS  Article  Google Scholar 

  29. 29

    Dietz, M. S., Fricke, F., Kruger, C. L., Niemann, H. H. & Heilemann, M. Receptor–ligand interactions: binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem 15, 671–676 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Spahn, C., Cella-Zannacchi, F., Endesfelder, U. & Heilemann, M. Correlative super-resolution imaging of RNA polymerase distribution and dynamics, bacterial membrane and chromosomal structure in Escherichia coli. Methods Appl. Fluoresc. 3, 014005 (2015).

    Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

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

    CAS  Article  Google Scholar 

  34. 34

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

    Herhaus, L., Al-Salihi, M., Macartney, T., Weidlich, S. & Sapkota, G. P. OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nat. Commun. 4, 2519 (2013).

    Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

    van Wijk, S. J., Fiskin, E. & Dikic, I. Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain-specific sensors. Nat. Protoc. 8, 1449–1458 (2013).

    Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding authors

Correspondence to Mike Heilemann or Ivan Dikic.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-12. (PDF 1672 kb)

41564_2017_BFnmicrobiol201766_MOESM18_ESM.avi

Live-cell imaging of doxycycline (Dox)-induced HeLa (AVI 542 kb)

Supplementary Video 1

Live-cell imaging of doxycycline (Dox)-induced HeLa (AVI 542 kb)

41564_2017_BFnmicrobiol201766_MOESM19_ESM.avi

Live-cell imaging of doxycycline (Dox)-induced HeLa (AVI 1435 kb)

Supplementary Video 2

Live-cell imaging of doxycycline (Dox)-induced HeLa (AVI 1435 kb)

41564_2017_BFnmicrobiol201766_MOESM20_ESM.avi

Confocal microscope z-stack scan of S. Typhimurium (AVI 23104 kb)

Supplementary Video 3

Confocal microscope z-stack scan of S. Typhimurium (AVI 23104 kb)

41564_2017_BFnmicrobiol201766_MOESM21_ESM.avi

Confocal microscope z-stack scan of S. Typhimurium (AVI 11683 kb)

Supplementary Video 4

Confocal microscope z-stack scan of S. Typhimurium (AVI 11683 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

van Wijk, S., Fricke, F., Herhaus, L. et al. Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation. Nat Microbiol 2, 17066 (2017). https://doi.org/10.1038/nmicrobiol.2017.66

Download citation

Further reading