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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Sun, X., Yin, J., Starovasnik, M. A., Fairbrother, W. J. & Dixit, V. M. Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J. Biol. Chem. 277, 9505–9511 (2002).
Kaiser, W. J. & Offermann, M. K. Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif. J. Immunol. 174, 4942–4952 (2005).
Rebsamen, M. et al. DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-κB. EMBO Rep. 10, 916–922 (2009).
Li, J. et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 150, 339–350 (2012).
Pasparakis, M. & Vandenabeele, P. Necroptosis and its role in inflammation. Nature 517, 311–320 (2015).
Micheau, O. & Tschopp, J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003).
Holler, N. et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495 (2000).
Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123 (2009).
He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009).
Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).
Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).
Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat. Cell Biol. 16, 55–65 (2014).
Hildebrand, J. M. et al. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc. Natl Acad. Sci. USA 111, 15072–15077 (2014).
Kaiser, W. J. et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J. Biol. Chem. 288, 31268–31279 (2013).
Upton, J. W., Kaiser, W. J. & Mocarski, E. S. DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe 11, 290–297 (2012).
Lawlor, K. E. et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat. Commun. 6, 6282 (2015).
Giogha, C., Lung, T. W., Pearson, J. S. & Hartland, E. L. Inhibition of death receptor signaling by bacterial gut pathogens. Cytokine Growth Factor Rev. 25, 235–243 (2014).
Pearson, J. S. et al. A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature 501, 247–251 (2013).
Li, S. et al. Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains. Nature 501, 242–246 (2013).
Zhang, L. et al. Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation. Nature 481, 204–208 (2012).
Charpentier, X. & Oswald, E. Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter. J. Bacteriol. 186, 5486–5495 (2004).
Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).
Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).
Lin, Y., Devin, A., Rodriguez, Y. & Liu, Z. G. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13, 2514–2526 (1999).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).
Shao, F., Merritt, P. M., Bao, Z., Innes, R. W. & Dixon, J. E. A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109, 575–588 (2002).
Humphries, F., Yang, S., Wang, B. & Moynagh, P. N. RIP kinases: key decision makers in cell death and innate immunity. Cell Death Differ. 22, 225–236 (2015).
Vince, J. E. et al. IAP antagonists target cIAP1 to induce TNFα-dependent apoptosis. Cell 131, 682–693 (2007).
Wickham, M. E. et al. Bacterial genetic determinants of non-O157 STEC outbreaks and hemolytic-uremic syndrome after infection. J. Infect. Dis. 194, 819–827 (2006).
Conzen, S. D. & Cole, C. N. The three transforming regions of SV40T antigen are required for immortalization of primary mouse embryo fibroblasts. Oncogene 11, 2295–2302 (1995).
Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9, 847–856 (2008).
Tanzer, M. C. et al. Evolutionary divergence of the necroptosis effector MLKL. Cell Death Differ. 23, 1185–1197 (2016).
Catanzariti, A. M., Soboleva, T. A., Jans, D. A., Board, P. G. & Baker, R. T. An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci. 13, 1331–1339 (2004).
Galan, J. E., Ginocchio, C. & Costeas, P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of invA to members of a new protein family. J. Bacteriol. 174, 4338–4349 (1992).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
McKenzie, G. J. & Craig, N. L. Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiol. 6, 39 (2006).
Huang, K. F., Chiou, S. H., Ko, T. P., Yuann, J. M. & Wang, A. H. The 1.35 Å structure of cadmium-substituted TM-3, a snake-venom metalloproteinase from Taiwan habu: elucidation of a TNFα-converting enzyme-like active-site structure with a distorted octahedral geometry of cadmium. Acta Crystallogr. D 58, 1118–1128 (2002).
Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999).
Coppolino, M. G. et al. Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis. J. Biol. Chem. 276, 4772–4780 (2001).
Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
Huson, D. H. & Scornavacca, C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst. Biol. 61, 1061–1067 (2012).
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.
The authors declare no competing financial interests.
About this article
Cite this article
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
Annual Review of Immunology (2021)
Science Signaling (2021)
Cell Death & Disease (2021)
Seminars in Cell & Developmental Biology (2021)
Seminars in Cell & Developmental Biology (2021)