The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-κB p65

et al. (2010) The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-kB p65. PLoS Pathog 6(5): e1000898. doi:10.1371/journal.ppat.1000898 The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-kB p65 Hayley J. Newton 0 Jaclyn S. Pearson 0 Luminita Badea 0 Michelle Kelly 0 Mark Lucas 0 Gavan 0 Holloway 0 Kylie M. Wagstaff 0 Michelle A. Dunstone 0 Joan Sloan 0 James C. Whisstock 0 James B. Kaper 0 Roy M. Robins-Browne 0 David A. Jans 0 Gad Frankel 0 Alan D. Phillips 0 Barbara S. Coulson 0 Elizabeth L. Hartland 0 Guy Tran Van Nhieu, Institut Pasteur, France 0 1 Department of Microbiology and Immunology, University of Melbourne , Parkville, Victoria , Australia , 2 Centre for Pediatric Gastroenterology, UCL Medical School, Royal Free Campus , London , United Kingdom , 3 Department of Biochemistry and Molecular Biology, Monash University , Clayton, Victoria , Australia , 4 Department of Microbiology, Monash University , Clayton, Victoria , Australia , 5 ARC Centre for Structural and Functional Microbial Genomics, Monash University , Clayton, Victoria , Australia , 6 Centre for Vaccine Development and Department of Microbiology and Immunology, University of Maryland School of Medicine , Baltimore , Maryland, United States of America, 7 Centre for Molecular Microbiology and Infection, Division of Cell and Molecular Biology, Imperial College London , London , United Kingdom Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-kB, to the host cell nucleus. NF-kB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-kB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-kB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-kB activation. Whereas NleE inhibited both TNFa and IL-1b stimulated p65 nuclear translocation and IkB degradation, NleB inhibited the TNFa pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-kB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators. - Funding: This work was supported by grants from the Australian Research Council (ARC) and the Australian National Health and Medical Research Council (NHMRC). ELH is an ARC Future Fellow. JSP and MK are recipients of Australian Postgraduate Awards. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Many bacterial pathogens have the ability to inject virulence effector proteins into the host cell using a type III secretion system (T3SS). The effector proteins perform a variety of functions that allow the pathogen to persist in the host and cause disease [1]. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) deliver T3SS effector proteins to the intestinal epithelium that mediate attaching and effacing lesion (A/E) lesion formation. A/E lesions are characterized by intimate bacterial attachment, effacement of the brush border microvilli and actin pedestal formation [2]. T3SS effectors from other pathogens such as Salmonella and Shigella have various roles in invasion, intracellular survival and the inhibition of innate immune responses through targeting host inflammatory signaling pathways [1]. Many of the T3SS effectors belong to conserved protein families that are found in a range of bacterial pathogens of plants and animals. For example, the OspF family of T3SS effectors from Shigella, Salmonella and Pseudomonas exhibit phosphothreonine lyase activity and induce irreversible dephosphorylation of mitogen-activated protein kinases (MAPKs) in the host cell nucleus [3,4,5]. In Shigella, this leads to gene-specific repression of a subset of NF-kB regulated genes, including IL8 [3]. Given the remarkable specificity of their biochemical function, the discovery of the mechanism of action of T3SS effectors remains an important step towards understanding the pathogenesis of many bacterial infections. Bacterial intestinal pathogens have evolved distinct ways of colonizing the gut and causing disease. Enteropathogenic E. coli (EPEC) and its close relative enterohemorrhagic E. coli O157:H7 (EHEC) are extracellular pathogens that cause a characteristic lesion on the intestinal mucosa known as an attaching and effacing lesion. In contrast, Shigella is an intracellular pathogen that invades the intestinal mucosa and spreads from cell to cell. Both pathogens utilize a bacterial type III secretion system that injects virulence effector proteins into the host cell upon contact. We have discovered that an effector shared by EPEC/EHEC and Shigella, known as NleE or OspZ, as well as another EPEC/EHEC effector, NleB, inhibit the host cell inflammatory response by preventing translocation of the immune regulator NF-kB to the cell nucleus. Thus, although EPEC/EHEC and Shigella have evolved different colonization strategies, they share a common virulence determinant that suppresses the inflammatory response of the host, and both pathogens mediate a multi-effector attack on NF-kB signaling. The activation of gene expression during inflammation is tightly regulated by transcription factors such as NF-kB. The NF-kB/Rel family comprises five members that share an N-terminal Rel homology domain that mediates DNA binding, dimerization and nuclear translocation [6]. The p65, c-Rel and RelB NF-kB subunits have an additional C-terminal transactivation domain, which strongly activates transcription from NF-kB-binding sites in target genes. The p50 and p52 subunits lack the transactivation domain but still bind to NF-kB consensus sites and act as transcriptional repressors [6]. The most abundant form of NF-kB in mammalian (...truncated)