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. 2013 Sep 12;501(7466):247-51.
doi: 10.1038/nature12524.

A type III effector antagonizes death receptor signalling during bacterial gut infection

Jaclyn S Pearson  1 , Cristina Giogha, Sze Ying Ong, Catherine L Kennedy, Michelle Kelly, Keith S Robinson, Tania Wong Fok Lung, Ashley Mansell, Patrice Riedmaier, Clare V L Oates, Ali Zaid, Sabrina Mühlen, Valerie F Crepin, Olivier Marches, Ching-Seng Ang, Nicholas A Williamson, Lorraine A O'Reilly, Aleksandra Bankovacki, Ueli Nachbur, Giuseppe Infusini, Andrew I Webb, John Silke, Andreas Strasser, Gad Frankel, Elizabeth L Hartland
Affiliations

A type III effector antagonizes death receptor signalling during bacterial gut infection

Jaclyn S Pearson et al. Nature. .

Abstract

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

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Figures

Figure 1
Figure 1. NleB1 binds death domain proteins and inhibits caspase-8 activation
a, Yeast two-hybrid analysis of protein-protein interactions in S. cerevisiae PJ69-4A. Results are mean ± SEM β-galactosidase activity from at least three independent experiments performed in triplicate. b, Growth of S. cerevisiae PJ69-4A on medium to select for protein-protein interactions (left panel) or plasmid maintenance (right panel). Representative images from at least three independent experiments c, Yeast two-hybrid analysis of protein interactions in S. cerevisiae PJ69-4A. Results are mean ± SEM β-galactosidase activity from at least three independent experiments performed in triplicate. d, GFP-Trap® of GFP-NleB1 and detection of FADD-FLAG, TRADD-FLAG and RIPK1-FLAG in HEK293T cells. Actin; loading control. Representative immunoblot from at least three independent experiments e, MTT reduction in HeLa cells expressing GFP, GFP-NleB1 or GFP-NleB2. UT, untransfected. Results are the mean ± SEM of absorbance at 540 nm from three independent experiments performed in triplicate. *P < 0.0001, unpaired, two-tailed t-test. f, Cleaved caspase-8 in HeLa cells expressing GFP or GFP-NleB1. p43/41 and p18 are products of processed caspase-8. UT, untransfected. Representative immunoblot from at least three independent experiments.
Figure 2
Figure 2. Enzymatic activity of NleB1
a, In vitro assay for NleB1 GlcNAc modification of FADD using recombinant proteins and 1 mM UDP-GlcNAc. Representative immunoblot from at least three independent experiments b, Intact protein mass spectrometry of FADD incubated with GST-NleB1 and UDP-GlcNAc. c, High resolution CID spectrum of the peptide corresponding to FADD115-125. *Diagnostic fragment ions that carry the GlcNAc modification. d, Cleaved caspase-8 in FasL-treated HeLa cells expressing GFP, GFP-NleB1 or GFP-NleB1AAA. UT, untransfected. Actin; loading control. Representative immunoblot from at least three independent experiments e, Cleaved caspase-8 in HeLa cells infected with derivatives of EPEC and treated with FasL. Representative immunoblot from at least three independent experiments f, Quantification of cleaved caspase-8 by immunofluorescence microscopy of HeLa cells infected with derivatives of EPEC and treated with FasL. Results are mean ± SEM percentage cells with cleaved caspase-8 from two independent experiments counting ~200 cells in triplicate. *P < 0.0001 compared to uninfected, unstimulated control, one-way ANOVA g, Immunofluorescence staining for detection of cleaved caspase-8 induced by FasL in HeLa cells infected with derivatives of EPEC. Scale bar 10 μm. Representative images from at least three independent experiments
Figure 3
Figure 3. Inhibition of FasL-induced DISC formation and cell death by EPEC
a, Cleaved caspase-8 in HeLa cells infected with derivatives of EPEC. Actin; loading control. Representative immunoblot from at least three independent experiments b, Cell death visualised by PI staining in HeLa cells infected with derivatives of EPEC and treated with FasL. Scale bar 20 μm. Representative images from at least three independent experiments c, Quantification of PI staining and microscopic analysis in HeLa cells infected with derivatives of EPEC and treated with FasL. Results are mean ± SEM percentage cells with PI staining from two independent experiments counting ~200 cells in triplicate. *P < 0.0001 compared to E2348/69 infected cells, one-way ANOVA. d, Cleaved and full-length caspase-8 in HeLa cells infected with derivatives of EPEC and treated with FasL. Actin; loading control. Representative immunoblot from at least three independent experiments e, DISC components induced by FasL coupled to Fcδ and precipitated with protein G beads. UN, untreated and uninfected; UI, uninfected. Actin; loading control. Representative immunoblot from at least three independent experiments.
Figure 4
Figure 4. Infection of mice deficient for FAS signalling with CR
a, Immunofluorescence staining and quantification of cleaved caspase-8 in colonic sections from C57BL/6 mice infected with CR or a CR nleB mutant. Representative images from at least three separate sections of colon at least 100 μm apart (transverse or longitudinal), per animal from five individual mice per group. Results are mean ± SEM percentage fluorescence intensity of cleaved caspase-8 staining relative to Hoechst staining of five fields with CR infection from three mice per group. *P = 0.0001, unpaired two tailed t-test. b, Bacterial load in the faeces during acute infection with CR. Each data point represents log10CFU/100 mg faeces per individual animal on days 4, 8 and 12 after infection. Mean ± SEM are indicated, dotted line represents detection limit. P values from Mann Whitney U. c, Diarrhoea score at day 4, 8 and 12 post-infection. Scoring system is described in the Supplementary Methods. Mean ± SEM are indicated. P values from one-way ANOVA. d, Immunofluorescence staining and quantification of CR penetration into intestinal crypts in colonic sections from C57BL/6, Faslpr/lpr and Fasgld/gld mice. Representative images from at least two separate sections of colon at least 100 μm apart (transverse or longitudinal), per animal from five individual mice per group. Results are the mean ± SEM maximum distance of CR staining from the epithelial surface (in μm) of five independent sections with at least three measurements per section. P = 0.0001, unpaired two tailed t-test. e, Bacterial load in the faeces during the resolving phase of CR infection. Each data point represents log10CFU/100 mg faeces per individual animal on days 12, 16, 21, 23 and 25 post-infection. Mean ± SEM are indicated, dotted line represents detection limit. P values from Mann Whitney U.
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References

    1. Wong AR, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80:1420–1438. - PubMed
    1. Srikanth CV, Mercado-Lubo R, Hallstrom K, McCormick BA. Salmonella effector proteins and host-cell responses. Cell Mol Life Sci. 2011;68:3687–3697. - PMC - PubMed
    1. Ashida H, et al. Shigella deploy multiple countermeasures against host innate immune responses. Curr Opin Microbiol. 2011;14:16–23. - PubMed
    1. Park HH, et al. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu Rev Immunol. 2007;25:561–586. - PMC - PubMed
    1. Strasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in the immune system. Immunity. 2009;30:180–192. - PMC - PubMed

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