title: EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.
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.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Jaclyn S
authors.initials: JS
authors.lastname: Pearson
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Cristina
authors.initials: C
authors.lastname: Giogha
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.|Department of Molecular Infection Biology, Helmholtz-Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Mühlen
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Ueli
authors.initials: U
authors.lastname: Nachbur
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, New South Wales 2006, Australia.
authors.forename: Chi L L
authors.initials: CL
authors.lastname: Pham
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Hildebrand
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Clare V
authors.initials: CV
authors.lastname: Oates
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Tania Wong Fok
authors.initials: TW
authors.lastname: Lung
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.|Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute and Centre for Systems Genomics, University of Melbourne, Victoria 3010, Australia.
authors.forename: Danielle
authors.initials: D
authors.lastname: Ingle
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Laura F
authors.initials: LF
authors.lastname: Dagley
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Bankovacki
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Petrie
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, SW7 2AZ, UK.|Centre for Experimental Medicine, Queen's University Belfast, BT9 7BL, UK.
authors.forename: Gunnar N
authors.initials: GN
authors.lastname: Schroeder
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, SW7 2AZ, UK.
authors.forename: Valerie F
authors.initials: VF
authors.lastname: Crepin
authors.affiliation: MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, SW7 2AZ, UK.
authors.forename: Gad
authors.initials: G
authors.lastname: Frankel
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Seth L
authors.initials: SL
authors.lastname: Masters
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Vince
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, New South Wales 2006, Australia.
authors.forename: Margaret
authors.identifier: 0000-0002-0150-3203
authors.initials: M
authors.lastname: Sunde
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Webb
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Melbourne, Australia.|Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: John
authors.initials: J
authors.lastname: Silke
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
mesh_terms: D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D044082:Citrobacter rodentium / Q000472:pathogenicity; D057056:Cysteine Proteases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054308:Enteropathogenic Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009336:Necrosis; D010766:Phosphorylation; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000069296:Type III Secretion Systems
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C000624428:EspL protein, E coli; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000069296:Type III Secretion Systems; C506249:RIPK1 protein, human; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; C485615:Ripk3 protein, mouse; D057056:Cysteine Proteases