Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

Staying alive: inhibition of cell death by enteropathogenic E. coli (#147)

Jaclyn S Pearson 1 , Sabrina Muehlen 1 , Cristina Giogha 1 , Tania Wong 1 , Joanne Hildebrand 2 , Chi Le Lan Pham 3 , Emma Petrie 2 , Ueli Nachbur 2 , Laura Dagley 4 , Margie Sunde 3 , James Murphy 2 , Andrew Webb 4 , John Silke 2 , Elizabeth L Hartland 1
  1. Department of Infectious Diseases, Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia
  2. Division of Cell Signaling and Cell Death, The Walter and Eliza Hall Institute for Medical Research, Parkville, Melbourne, Victoria, Australia
  3. Department of Pharmacology, University of Sydney, Sydney, New South Wales, Australia
  4. Proteomics Laboratory, Division of Systems Biology and Personalised Medicine, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

Enteropathogenic E. coli (EPEC) is a gastrointestinal pathogen that adheres intimately to the apical surface of intestinal enterocytes. EPEC utilises a type III secretion system (T3SS) to directly translocate a diverse repertoire of virulence (effector) proteins directly into host cells. The effector proteins subvert a multitude of host cellular processes including innate immune signaling and apoptosis in order to persist in the host. The host adaptor proteins RIPK1, RIPK3, TRIF and ZBP1/DAI, all contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM), and play a key role in cell death and inflammatory signaling. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis. Here we report that EPEC uses the T3SS effector EspL to directly cleave and inactivate RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This required a previously unrecognised tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaved within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL led to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of TNF, LPS or poly (I:C)-induced necroptosis and inflammatory signaling. Furthermore, EPEC infection inhibited TNF-induced phosphorylation and plasma membrane localization of MLKL, a key mediator of necroptosis. In vivo, Ripk3-/- but not Mlkl-/- mice exhibited increased pathology upon infection with the EPEC-like mouse pathogen Citrobacter rodentium compared to wild type C57BL/6 mice. The activity of EspL defines a new family of T3SS cysteine protease effectors found in a range of bacteria and reveals a new mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signaling pathways.