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A bacterial effector deubiquitinase specifically hydrolyses linear ubiquitin chains to inhibit host inflammatory signalling

Nature Microbiology volume 4, pages 1282–1293 (2019)Cite this article

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Abstract

Linear ubiquitin (Ub) chains regulate many cellular processes, including NF-κB immune signalling. Pathogenic bacteria have evolved to secrete effector proteins that harbour deubiquitinase activity into host cells to disrupt host ubiquitination signalling. All previously identified effector deubiquitinases hydrolyse isopeptide-linked polyubiquitin (polyUb). It has been a long-standing question whether bacterial pathogens have evolved an effector deubiquitinase to directly cleave linear Ub chains. In this study, we performed extensive screening of bacterial pathogens and found that Legionella pneumophila—the causative agent of human Legionnaire’s disease—encodes an effector protein, RavD, which harbours deubiquitinase activity exquisitely specific for linear Ub chains. RavD hydrolyses linear Ub chains but not any type of isopeptide-linked polyUb. The crystal structure of RavD with linear diubiquitin reveals that RavD adopts a papain-like fold with a Cys–His–Ser catalytic triad. The Ub-binding surface and specific interacting residues in RavD determine its specificity for Met1 linkages. RavD prevents the accumulation of linear Ub chains on Legionella-containing vacuoles established by the pathogen in host cells to inhibit the NF-κB pathway during infection. This study identified a unique linear Ub chain-specific effector deubiquitinase and indicates its potential application as a tool to dissect linear polyUb-mediated signalling in mammalian cells.

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Fig. 1: A deubiquitinase activity screen of bacterial pathogens revealed that L. pneumophila can hydrolyse linear Ub chains.
Fig. 2: The Legionella effector RavD hydrolyses linear Ub chains.
Fig. 3: RavD is a linear Ub chain-specific deubiquitinase.
Fig. 4: Complex structure of RavDlc with linear diUb.
Fig. 5: RavD can inhibit linear Ub chain-mediated NF-κB signalling.
Fig. 6: RavD prevents the accumulation of linear Ub chains on LCVs and inhibits IκBα degradation during infection.

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Data availability

Coordinates and structure factors for RavD and the RavD-linear diUb complex have been deposited in the Protein Data Bank under accession codes 6NII and 6NJD, respectively. All data generated during this study are included in this published article and its Supplementary Information.

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Acknowledgements

We thank the staff at the beamline BL18U1 of SSRF and the National Center for Protein Science Shanghai for assistance with diffraction data collection, F. Shao, L. Wang, V. Dixit and Z.-Q. Luo for providing bacterial strains and antibodies, and the staff at the core facilities of the Life Sciences Institute Zhejiang University for assistance with SIM. This work was supported by grants from MOST (2017YFA0503900 to Y.Zhu), NSFC (81530068 to Y.Zhu and 81501717 to Y.Zhou), the National High-level Talents Special Support Plan, the leading scientist program of Zhejiang High-level Talents Special Support Plan and the Fundamental Research Funds for the Central Universities.

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Author notes

  1. These authors contributed equally: Muyang Wan, Xiaofei Wang.

Authors and Affiliations

  1. MOE Key Laboratory of Biosystems Homeostasis & Protection, Innovation Center for Cell Signalling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Muyang Wan, Xiaofei Wang, Chunfeng Huang, Dandan Xu, Zhao Wang & Yongqun Zhu

  2. Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, China

    Zhao Wang & Yan Zhou

  3. Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China

    Yongqun Zhu

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Contributions

Y.Zhu designed the study. M.W. performed the biochemical, cell and infection assays with assistance from Y.Zhou, X.W., C.H., D.X. and Z.W. X.W. determined the structures and generated the mutant strains. M.W., X.W., Y.Zhou and Y.Zhu analysed the data and wrote the manuscript.

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Correspondence to Yongqun Zhu.

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Supplementary Figures 1–19, Supplementary Table 1 and uncropped blots

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Wan, M., Wang, X., Huang, C. et al. A bacterial effector deubiquitinase specifically hydrolyses linear ubiquitin chains to inhibit host inflammatory signalling. Nat Microbiol 4, 1282–1293 (2019). https://doi.org/10.1038/s41564-019-0454-1

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