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.