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


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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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.

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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.

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Authors and Affiliations



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.

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Correspondence to Mike Heilemann or Ivan Dikic.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-12. (PDF 1672 kb)

Supplementary Video 1

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

Supplementary Video 2

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

Supplementary Video 3

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

Supplementary Video 4

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

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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).

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