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

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 responses1,2,3. 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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Figure 1: NleB1 binds death domain proteins and inhibits caspase-8 activation.
Figure 2: Enzymatic activity of NleB1.
Figure 3: Inhibition of FasL-induced DISC formation and cell death by EPEC.
Figure 4: Infection of mice deficient for FAS signalling with C. rodentium.

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Acknowledgements

We gratefully acknowledge P. Bouillet for the gift of Bid−/− mice and T. Cumming for assistance with animal work. This work was supported by the Australian National Health and Medical Research Council (Program Grant no.606788 to E.L.H., Project Grants no.637332, no.1009145 to A.S., no.1009145 to L.O.R., Australia Fellowship to A.S.), the Wellcome Trust to G.F., the Juvenile Diabetes Foundation; the Leukaemia and the Lymphoma Society (New York; SCOR grant no.7413) to A.S. E.L.H was supported by an Australian Research Council Future Fellowship. J.S.P., M.K., T.W., C.G. and P.R. were supported by Australian Postgraduate Awards. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.

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J.S.P., C.G., S.Y.O., C.L.K., M.K., K.S.R., T.W.F.L., P.R., C.V.O. and A.B. designed and performed the experiments. V.F.C. and O.M. generated reagents. C.S.A., N.A.W., G.I. and A.I.W. performed mass spectrometry analyses. L.A.O., A.Z., S.M., U.N., A.M., A.S., J.S., G.F., J.S.P. and E.L.H. contributed to experimental design. J.S.P., A.S., J.S., G.F. and E.L.H. wrote the manuscript.

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Correspondence to Elizabeth L. Hartland.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 NleB1 binds FADD, TRADD and RIPK1 and blocks extrinsic apoptosis.

a, Co-immunoprecipitation of bacterially delivered NleB1–2HA with FADD–Flag, TRADD–Flag and RIPK1–Flag in HeLa cells. Lane 1, HeLa cells infected with EPEC ΔnleB1 (pNleB1) (negative control). Lane 2, HeLa cells infected with EPEC ΔnleB1 (pNleB1–2HA). Actin, loading control. Representative immunoblot from at least three independent experiments. b, GFP-Trap pull-down of FADD–Flag or FADDΔDD–Flag. NleB1 from EPEC E2348/69, EHEC O157:H7 EDL933 and C. rodentium ICC169 fused with EGFP were used as indicated. Actin, loading control. Representative immunoblot from at least three independent experiments. c, HeLa cells were infected with derivatives of EPEC as indicated for 4 h and left unstimulated (dark grey bars) or stimulated with TNF for 8 h (light grey bars). Results are the mean ± s.e.m. of at least three independent experiments carried out in duplicate. The * indicates significantly greater than uninfected, unstimulated cells, P < 0.0001, one-way ANOVA with Dunnett’s multiple comparison test. d, Apoptosis was induced by FasL and HeLa cells were stained with antibodies to cleaved caspase-8. Arrows indicate cells with cleaved caspase-8. Scale bar, 5 μm. Representative images from at least three independent experiments. e, Quantification of cleaved caspase-8 or cleaved caspase-3 by immunofluorescence microscopy in HeLa cells expressing EGFP or EGFP–NleB1. UT, untransfected; Results are the mean ± s.e.m. of percentage of cells with cleaved caspase-8 or cleaved caspase-3 from at least three independent experiments counting 200 cells in triplicate. *P < 0.0001, unpaired, two-tailed t-test. f, N-acetylglucosylation of FADD–Flag detected using a monoclonal antibody to GlcNAc of immunoprecipitated lysate from HEK293T cells. Actin, loading control. Representative immunoblot from at least three independent experiments.

Extended Data Figure 2 Mass spectra of peptides identifying FADD Arg 117 GlcNAc modification.

Fragment ions of overlapping tryptic missed cleaved peptides confirm Arg 117 of FADD as the modification site.

Extended Data Figure 3 Mass spectra obtained using electron transfer dissociation fragmentation on the Orbitrap Elite mass spectrometer.

a, Red and blue spectra represent the c and z series ions that matches the peptide sequence DWKRLAR-HexNAc-ELKVSEAKM (addition of 203.0793 Da) produced from an AspN endoproteinase digest of FADD incubated with NleB1. Insert box shows the precursor ion present as a 4+ charge isotope series with a delta mass of 0.15 p.p.m. between the measured and theoretical mass of the modified peptide. Xcorr score of 6.26 was obtained using the Sequest HT (V1.3) search engine. b, Total ion current of the AspN digest. c, Extracted ion chromatogram of mass 541.5449 belonging to the HexNAc modified peptide DWKRLARELKVSEAKM using isolation mass tolerance of 10 p.p.m. d, ETD fragmentation spectra of the 541.55 precursor ion. Insert table shows the theoretical masses associated with the respective fragmentation ions.

Extended Data Figure 4 GlcNAc modification of FADD by GST–NleB1.

a, Intact protein mass spectrometry of FADD with an R117A substitution incubated with GST–NleB1 and UDP-GlcNAc reveals no GlcNAc modification. b, Intact protein mass spectrometry of FADD with S122A substitution incubated with GST–NleB1 and UDP-GlcNAc reveals a single GlcNAc modification. c, In vitro assay for NleB1 GlcNAc modification of mutated forms of FADD as indicated. Representative immunoblot from at least three independent experiments.

Extended Data Figure 5 Comparison of FasL and TNF-mediated caspase-8 processing and analysis of NleB2.

a, GFP-Trap pull-down of FADD–Flag, TRADD–Flag or RIPK1–Flag with EGFP–NleB2 in HEK293T cells. Actin, loading control. Representative immunoblot from at least three independent experiments. bd, Co-immunoprecipitation of bacterially delivered NleB2–2HA with FADD–Flag, TRADD–Flag and RIPK1–Flag in HeLa cells using HA antibodies. Lane 1, HeLa cells infected with EPEC ΔnleB2 (pNleB2) (negative control). Lane 2, HeLa cells infected with EPEC ΔnleB2 (pNleB2–2HA). Actin, loading control. Representative immunoblots from at least three independent experiments. e, Unprocessed and cleaved caspase-8 in HeLa cells infected for 3 h with derivatives of EPEC and left untreated or treated with Fcγ–FasL or TNF for 1 h, as indicated. Representative immunoblot from at least three independent experiments. f, Unprocessed and cleaved caspase-8 in HeLa cells infected for 45 min with derivatives of EPEC and left untreated or treated with TNF and cyclohexamide (CHX) for 1 h as indicated. Actin, loading control. Representative immunoblot from at least three independent experiments. g, Adherence of EPEC to HeLa cells. HeLa cells were infected with derivatives of EPEC for 3 h followed by treatment with FasL for 1 h. Number of recovered bacteria and the inoculum are shown for comparison. Mean ± s.e.m. are indicated. Data are from three independent experiments performed in triplicate.

Extended Data Figure 6 Infection of C57BL/6 and Faslpr/lpr mice with C. rodentium.

a, Immunofluorescence staining of cleaved caspase-8 in different colonic sections from C57BL/6 mice infected with wild type C. rodentium and an nleB mutant as indicated, using antibodies to C. rodentium O-antigen (anti-O152, green) and cleaved caspase-8 (red). Intestinal tissue was visualized with Hoechst staining for DNA (blue). Arrows indicate cleaved caspase-8 positive cells sloughed into the gut lumen. Scale bar, 100 μm. 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. b, Time course of infection of C57BL/6 and Faslpr/lpr mutant mice with C. rodentium. Each data point represents log10 c.f.u. per 100 mg faeces per individual animal on the indicated days. Mean ± s.e.m. are indicated, dotted line represents detection limit. C. rodentium, mice infected with wild type C. rodentium; ΔnleB mice infected with C. rodentium nleB mutant. *P < 0.05, **P < 0.01, ***P < 0.001. P values from Mann–Whitney U-test. Dotted line represents the detection limit.

Extended Data Figure 7 Histological analysis of intestinal sections at day 10 from FAS-deficient mice infected with C. rodentium.

a, Bacterial load in the colon of mice infected with C. rodentium (CR). Each data point represents log10 c.f.u. per 100 mg colon per individual animal on day 10 post-infection. C57BL/6 CR, wild-type mice infected with wild-type C. rodentium; C57BL/6 ΔnleB, wild-type mice infected with C. rodentium nleB mutant; Faslpr/lpr CR, Faslpr/lprmice infected with wild-type C. rodentium, dotted line is the detection limit. P = 0.0002, Mann–Whitney U-test. Mean ± s.e.m. are indicated. b, Resected colon weights (between rectum and caecum) of individual animals on day 10 post-infection. C57BL/6 CR, wild-type mice infected with wild-type C. rodentium; C57BL/6 ΔnleB, wild-type mice infected with C. rodentium nleB mutant; Faslpr/lpr CR, Faslpr/lpr mice infected with wild type C. rodentium. P = 0.0164, Mann–Whitney U-test. Mean ± s.e.m. are indicated. c, Mean ± s.e.m. crypt height in μm in haematoxylin and eosin stained sections from C57BL/6 and Faslpr/lprmice infected with wild type C. rodentium. UI, uninfected. Data are from 4 sections of colon measured at least 50 μm apart per animal from at least 3 individual mice per group. d, Mean ± s.e.m. tissue damage score in colon sections for individual mice infected with wild type C. rodentium. Scoring system is described in the Methods. UI, uninfected. P < 0.05, Mann–Whitney U-test. e, Haematoxylin and eosin stained sections of colon from C57BL/6 (wild-type) or Faslpr/lprmice uninfected or infected with wild type C. rodentium (day 10 post-infection). UI, uninfected. Scale bar, 100 μm. Mucosal inflammation (asterisk), neutrophil invasion of the muscularis mucosa (arrow), erosion of the epithelium (arrowhead). Representative images from two sections of colon at least 100 μm apart (transverse or longitudinal) from at least three individual mice per group.

Extended Data Figure 8 Diarrhoea score and histological analysis of intestinal sections at day 12 from C57BL/6 (wild-type), Faslpr/lpr, Fasgld/gld and Bid−/− mice infected with C. rodentium.

a, Bacterial load in the faeces of different mouse strains infected with wild-type C. rodentium as indicated. Each data point represents log10 c.f.u. per 100 mg colon per individual animal. Dotted line is detection limit. Mean ± s.e.m. are indicated. b, Diarrhoea score at day 12 post-infection of different strains of mice infected with C. rodentium. Scoring system is described in the Methods. Mean ± s.e.m. are indicated. P values from one-way ANOVA. c, Mean ± s.e.m. crypt height in μm in haematoxylin and eosin stained sections from different mouse strains infected with wild-type C. rodentium. UI, uninfected. Data are from 4 sections of colon measured at least 50 μm apart per animal from at least three individual mice per group. d, Haematoxylin and eosin stained sections of colon from different mouse strains infected with wild type C. rodentium. Scale bar, 100 μm. Representative images from two sections of colon at least 100 μm apart (transverse or longitudinal) from at least three individual mice per group.

Extended Data Figure 9 Bacterial load in the faeces of Faslpr/lprmice infected with wild-type C. rodentium, an espI mutant and an espF mutant.

a, Bacterial load in the faeces of mice during infection with derivatives of C. rodentium. Each data point represents log10 c.f.u. per 100 mg faeces or colon as indicated per individual animal on days 4, 8, 10 and 12 post-infection. Mean ± s.e.m. are indicated, dotted line represents detection limit. b, Diarrhoea score at day 4, 8, 10 and 12 post-infection. Scoring system is described in the Methods. Mean ± s.e.m. are indicated. P values from one-way ANOVA. c, d, Bacterial load in the faeces of mice infected with derivatives of C. rodentium. Each data point represents log10 c.f.u. per 100 mg faeces per individual animal on day 8 post-infection. Mean ± s.e.m. are indicated, dotted line represents detection limit. P values from Mann–Whitney U-test.

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Supplementary Data

This file contains Supplementary Table 1, which shows matched fragment ion of all peptides. (XLSX 48 kb)

Supplementary Data

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Pearson, J., Giogha, C., Ong, S. et al. A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature 501, 247–251 (2013). https://doi.org/10.1038/nature12524

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