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EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation

Nature Microbiology volume 2, Article number: 16258 (2017) Cite this article

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An Erratum to this article was published on 13 February 2017

Abstract

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling1–3. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis4,5. Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

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Figure 1: EspL is a T3SS cysteine protease that degrades RIPK1 and RIPK3.
Figure 2: Distribution of EspL in Gram-negative pathogens and substrate specificity.
Figure 3: EspL inhibits TNF-induced necroptosis.
Figure 4: EspL activity inhibits TLR3/4-mediated signalling and contributes to in vivo persistence.

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Acknowledgements

The authors thank E. Mocarski (Emory University) for the gift of Flag-ZBP1/DAI and Flag-M45 and G. Belz (Walter and Eliza Hall Institute) for animal ethics assistance. The authors thank S. Young (Walter and Eliza Hall Institute) for technical assistance. This work was supported by the Australian National Health and Medical Research Council (program grant ID606788 to E.L.H., project grants APP1057888 to J.S., APP1051210 to J.V. and APP1057905 to J.M.M. and J.S., fellowships APP1090108 to J.S.P., APP1052598 to J.V. and APP1105754 to J.M.M.) and the Australian Research Council (Future Fellowship FT130100166 to U.N., Discovery Project DP150104227 to M.S.). C.G. and D.I. were supported by Australian Postgraduate Awards. T.W.F.K. was supported by a University of Melbourne International Research Scholarship (MIRS). G.N.S. is funded by the Medical Research Council, UK. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.

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Author notes

  1. Sabrina Mühlen & Gunnar N. Schroeder

    Present address: † Present addresses: Department of Molecular Infection Biology, Helmholtz-Centre for Infection Research, 38124 Braunschweig, Germany (S.M.); Centre for Experimental Medicine, Queen's University Belfast, BT9 7BL, UK (G.N.S.),

  2. Jaclyn S. Pearson and Cristina Giogha: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, 3000, Australia

    Jaclyn S. Pearson, Cristina Giogha, Sabrina Mühlen, Ying Zhang, Clare V. Oates, Tania Wong Fok Lung, Danielle Ingle & Elizabeth L. Hartland

  2. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia

    Ueli Nachbur, Joanne M. Hildebrand, Laura F. Dagley, Aleksandra Bankovacki, Emma J. Petrie, Seth L. Masters, James Vince, James M. Murphy, Andrew I. Webb & John Silke

  3. Department of Medical Biology, University of Melbourne, 3010, Victoria, Australia

    Ueli Nachbur, Joanne M. Hildebrand, Laura F. Dagley, Aleksandra Bankovacki, Emma J. Petrie, Seth L. Masters, James Vince, James M. Murphy, Andrew I. Webb & John Silke

  4. Discipline of Pharmacology, School of Medical Sciences, University of Sydney, 2006, New South Wales, Australia

    Chi L. L. Pham & Margaret Sunde

  5. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute and Centre for Systems Genomics, University of Melbourne, Victoria 3010, Australia

    Danielle Ingle

  6. MRC Centre for Molecular Bacteriology and Infection Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, SW7 2AZ, UK

    Gunnar N. Schroeder, Valerie F. Crepin & Gad Frankel

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Contributions

J.S.P., C.G., S.M., U.N., C.L.L.P., Y.Z., J.M.H., T.W.F.L., D.I., A.B., E.J.P., J.V. and M.S. designed and performed the experiments. S.L.M., J.M.M., G.N.S., C.V.O., V.F.C. and G.F. contributed reagents and expertise. L.F.D. and A.I.W. performed mass spectrometry analyses. J.S.P., C.G., J.S. and E.L.H. designed the experiments and wrote the manuscript.

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Correspondence to Elizabeth L. Hartland.

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Supplementary Figures 1–10, Supplementary Table 1. (PDF 47035 kb)

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Pearson, J., Giogha, C., Mühlen, S. et al. EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation. Nat Microbiol 2, 16258 (2017). https://doi.org/10.1038/nmicrobiol.2016.258

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