Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 18 Dec 2018 17:03:12 Enterohaemorrhagic, but not enteropathogenic, Escherichia coli infection of epithelial cells disrupts signalling responses to tumour necrosis factor- alpha Me´ lanie G. Gareau, 1 Nathan K. Ho, 1,2 Dirk Brenner, 3 Andrew J. Sousa, 1 Lionel LeBourhis, 4 Tak W. Mak, 3 Stephen E. Girardin, 2 Dana J. Philpott 4 and Philip M. Sherman 1,2 Correspondence Philip M. Sherman philip.sherman@sickkids.ca Received 4 May 2011 Revised 30 June 2011 Accepted 21 July 2011 1 Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada 2 Department of Laboratory Medicine and Pathobiology University of Toronto, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, ON M5S 1A8, Canada 3 Campbell Family Cancer Research Institute, Princess Margaret Hospital, 620 University Avenue, Toronto, ON M5G 2C1, Canada 4 Department of Immunology, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, ON M5S 1A8, Canada Enterohaemorrhagic Escherichia coli (EHEC), serotype O157 : H7 is a non-invasive, pathogenic bacterium that employs a type III secretion system (T3SS) to inject effector proteins into infected cells. In this study, we demonstrate that EHEC blocks tumour necrosis factor-alpha (TNFa)- induced NF-kB signalling in infected epithelial cells. HEK293T and INT407 epithelial cells were challenged with EHEC prior to stimulation with TNFa. Using complementary techniques, stimulation with TNFa caused activation of NF-kB, as determined by luciferase reporter assay (increase in gene expression), Western blotting (phosphorylation of IkBa), immunofluorescence (p65 nuclear translocation) and immunoassay (CXCL-8 secretion), and each was blocked by EHEC O157 : H7 infection. In contrast, subversion of host cell signalling was not observed following exposure to either enteropathogenic E. coli, strain E2348/69 (O127 : H6) or the laboratory E. coli strain HB101. Heat-killed EHEC had no effect on NF-kB activation by TNFa. Inhibition was mediated, at least in part, by Shiga toxins and by the O157 plasmid, but not by the T3SS or flagellin, as demonstrated by using isogenic mutant strains. These findings indicate the potential for developing novel therapeutic targets to interrupt the infectious process. INTRODUCTION Disease burden associated with attaching and effacing enteric pathogens is high, with outbreaks occurring yearly around the world (Croxen & Finlay, 2010; Jay et al., 2007). Enterohaemorrhagic Escherichia coli (EHEC), serotype O157 : H7 asymptomatically colonizes the gut of ruminants (La Ragione et al., 2009), but is a zoonotic infection linked to both food- and water-borne outbreaks of diarrhoea and haemorrhagic colitis in humans. EHEC O157 : H7 infection is the primary cause of diarrhoea-associated haemolytic– uraemic syndrome, primarily afflicting children and the elderly (Tarr et al., 2005). The related organism, enter- opathogenic E. coli (EPEC) primarily causes non-bloody protracted diarrhoea in children living in developing countries (Kaper et al., 2004). It is now well appreciated that enteric pathogens exploit host inflammatory responses to promote gut colonization (Pe´dron & Sansonetti, 2008). EHEC O157 : H7 causes rearrangement of the infected cell cytoskeleton, leading to the development of attaching and effacing lesions on infected epithelial cells within 4–6 h (Sherman et al., 2010). This non-invasive pathogen uses a type III secretion system (T3SS), encoded on the locus of enterocyte effacement (LEE) pathogenicity island, as a molecular syringe to inject multiple effector proteins that alter host cell signal transduction cascades. Both EHEC O157 : H7 and non- O157 : H7 Shiga toxin (Stx)-producing E. coli (STEC) Abbreviations: EHEC, enterohaemorrhagic E. coli; EPEC, enteropatho- genic E. coli; IKK, IkB kinase; IL, interleukin; LEE, locus of enterocyte effacement; STEC, Stx-producing E. coli; Stx, Shiga-like toxin; T3SS, type III secretion system; TNFa, tumour necrosis factor-alpha. Microbiology (2011), 157, 2963–2973 DOI 10.1099/mic.0.051094-0 051094 G 2011 SGM Printed in Great Britain 2963