Figure 1.
Effect of EPEC infection on NF-κB activation.
A. Representative immunofluorescence fields showing p65 staining (green) in FAS-positive HeLa cells (red) infected with derivatives of EPEC E2348/69, stimulated with TNFα and stained for nucleic acid with DAPI (blue). Arrows indicate FAS-positive lesions. B. Quantification of p65 nuclear exclusion in cells infected with derivatives of EPEC E2348/69 and stimulated with TNFα or IL-1β. Results are expressed as the percentage of FAS-positive cells that exclude p65 from the nucleus and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 FAS-positive cells were counted per test. *significantly different to E2348/69 stimulated with TNFα **significantly different to E2348/69 stimulated with IL-1β (P<0.05, one way ANOVA).
Figure 2.
Effect of EPEC infection on IL-8 production and expression.
A. IL-8 production from Caco-2 cells infected with derivatives of EPEC E2348/69 for 4 h and stimulated with IL-1β for 24 h. Results are the mean ± SEM of 5 independent experiments carried out in triplicate. *ΔnleE significantly different to E2348/69 and ΔnleE (pNleE) (P<0.05, one way ANOVA) B. IL-8 production from Caco-2 cells infected with derivatives of EPEC E2348/69 for 4 h and left unstimulated. Results are the mean ± SEM of 5 independent experiments carried out in triplicate. *ΔnleE significantly different to ΔnleE (pNleE) (P<0.05, one way ANOVA) C. Reverse transcription qPCR analysis of IL8 expression in Caco-2 cells 4 h after infection and 3 h after IL-1β treatment. Results are expressed as log10 fold increase in mRNA expression over unstimulated control and are the mean ± SEM of 3 independent experiments carried out in triplicate. *ΔnleE significantly different to E2348/69 and ΔnleE (pNleE) (P<0.05, one way ANOVA).
Figure 3.
Effect of ectopically expressed NleE on NF-κB activation.
A. Representative immunofluorescence fields and quantification of p65 nuclear exclusion using anti-p65 (A), anti-c-Rel (B) or anti-p50 antibodies (C) (red) of HeLa cells transfected with pEGFP-C2 (GFP only) or pGFP-NleE (green), stimulated with TNFα for 30 min, and stained for nucleic acid (blue). Results are expressed as the percentage of GFP-positive cells that exclude p65, c-Rel or p50 and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 GFP-positive cells were counted per test. *significantly different to GFP only stimulated with TNFα, P = 0.0005 (unpaired, two-tailed t-test) D. Fold increase in NF-κB dependent luciferase activity in pEGFP-C2 (GFP) or pGFP-NleE transfected cells unstimulated (white bars) or stimulated with TNFα for 30 min (black bars). Results are the mean ± SEM of triplicate wells. *significantly different to GFP only stimulated with TNFα (P<0.05, one way ANOVA).
Figure 4.
Effect of NleE on nuclear translocation of STAT1 and STAT2.
A. Representative immunofluorescence fields and quantification of nuclear exclusion using antibodies to STAT1 (A) or STAT2 (B) (red) of HeLa cells transfected with pEGFP-C2 (GFP only) or pGFP-NleE (green), stimulated with IFNα for 30 min and stained for nucleic acid with DAPI (blue). Results are expressed as the percentage of GFP-positive cells that exclude STAT1 or STAT2 and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 GFP-positive cells were counted per test. C. Fold increase in STAT1/2 dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP), pGFP-NleE or pGFP-OspZ. Results are the mean ± SEM of 3 independent experiments performed in triplicate. Differences between GFP and GFP-NleE or GFP-OspZ were not significant (P>0.05, one way ANOVA).
Figure 5.
Effect of NleE and OspZ homologues on NF-κB activation.
A. Fold increase in NF-κB dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP) or pGFP-NleE/OspZ cloned from C. rodentium (GFP-NleECR), EHEC O157:H7 EDL933 (GFP-NleEO157), EPEC E2348/69 (GFP-NleEEPEC), S. flexneri 2a (GFP-OspZSF2a), S. boydii (GFP-OspZSB) and S. flexneri 6 (GFP-OspZSF6) and left unstimulated (white bars) or stimulated with TNFα for 30 min (black bars). Results are the mean ± SEM of 3 independent experiments performed in triplicate. *significantly different to GFP-NleEEPEC (P<0.05, one way ANOVA) B. Representative immunofluorescence fields using anti-p65 antibodies (red) of HeLa cells transfected with derivatives of pGFP-NleE and GFP-OspZ (green) labelled as in Fig. 5A and stimulated with TNFα for 30 min. C. Fold increase in NF-κB dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP) or pGFP-NleE, pGFP-NleE1-188 or pGFP-NleE188-224 and stimulated with TNFα for 30 min where indicated. Results are the mean ± SEM of 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with TNFα (P<0.05, one way ANOVA).
Figure 6.
Effect of NleE deletions and mutations on NF-κB activation.
A. Fold increase in NF-κB dependent luciferase activity in cells transfected with pEGFP-C2 or derivatives of pGFP-NleE as labelled and left unstimulated or stimulated with TNFα for 30 min where indicated. Results are the mean ± SEM of at least 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with TNFα (P<0.05, one way ANOVA) B. Fold increase in NF-κB dependent luciferase activity in cells transfected with pEGFP-C2, pGFP-OspZ6 or pGFP-OspZΔIDSYMK and left unstimulated (white bars) or stimulated with TNFα for 30 min (black bars). Results are the mean ± SEM of at least 3 independent experiments performed in triplicate. *significantly different to GFP-OspZ stimulated with TNFα (P<0.05, one way ANOVA) C. Alignment of the C-terminal region of NleE and OspZ from A/E pathogens and Shigella as well as the NleE6A variant. The IDSY(M/I)K motif is shaded and the Jpred prediction for an amino acid contributing to secondary structure is given as an accuracy score between 0 and 9, where 9 represents the most reliable prediction [16]. D. Fold increase in NF-κB dependent luciferase activity in cells transfected with pEGFP-C2 or derivatives of pGFP-NleE as labelled and left unstimulated (white bars) or stimulated with TNFα for 30 min (black bars). Results are the mean ± SEM at least 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with TNFα (P<0.05, one way ANOVA). E. Fold increase in NF-κB dependent luciferase activity in cells transfected with pEGFP-C2 or derivatives of pGFP-NleE and pGFP-OspZ as labelled and left unstimulated (white bars) or stimulated with TNFα for 30 min (black bars). Results are the mean ± SEM at least 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with TNFα (P<0.05, one way ANOVA).
Figure 7.
Inhibition of NF-κB activation by NleB.
A. Fold increase in NF-κB dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP only) or pGFP-NleE, pGFP-NleH1, pGFP-NleH2, pGFP-NleD or pGFP-NleB and stimulated with TNFα. Results are the mean ± SEM at least 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with TNFα (P<0.05, one way ANOVA) B. Representative immunofluorescence fields and quantification of nuclear exclusion using antibodies to p65 (red) of HeLa cells transfected with pGFP-C2 or pGFP-NleB (green) and stimulated with TNFα for 30 min. Results are expressed as the percentage of GFP-positive cells that exclude p65 and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 GFP-positive cells were counted per test. *significantly different to GFP only stimulated with TNFα P<0.0005 (unpaired, two-tailed t-test). C. Fold increase in NF-κB dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP), pGFP-NleE or pGFP-NleB and stimulated with IL-1β. Results are the mean ± SEM at least 3 independent experiments performed in triplicate. *significantly different to GFP-NleE stimulated with IL-1β (P<0.05, one wayANOVA) D. Fold increase in AP-1 dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP only), pGFP-NleE or pGFP-NleB and stimulated with PMA. Results are the mean ± SEM at least 3 independent experiments performed in triplicate. There was no significant difference between any tests (P>0.05, one way ANOVA).
Figure 8.
Effect of bacterially injected NleB and NleE on NF-κB activation.
A. Representative immunofluorescence fields showing p65 staining (green) in FAS-positive HeLa cells (red) infected with derivatives of EPEC E2348/69, stimulated with TNFα or IL-1β as indicated and stained for nucleic acid with DAPI (blue). Arrows indicate FAS-positive lesions. B. Quantification of p65 nuclear exclusion in cells infected with derivatives of EPEC E2348/69 and stimulated with TNFα or IL-1β as indicated. Results are expressed as the percentage of FAS-positive cells that exclude p65 from the nucleus and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 FAS-positive cells were counted per test. *significantly different to E2348/69 stimulated with TNFα or IL-1β **significantly different to E2348/69 left unstimulated (P<0.05, one way ANOVA).
Figure 9.
Effect of NleB and NleE on IκB degradation.
A. Time course of IκB degradation in HeLa cells transfected with pEGFP-C2, pGFP-NleE, pGFP-OspZ and pGFP-NleB. Cells were stimulated with TNFα or IL-1β for the times indicated and IκB was detected by immunoblot using anti-IκB antibodies. UT, untransfected. Un, unstimulated B. IκB degradation in HeLa cells transfected with GFP-NleE and GFP-OspZ variants and GFP-NleB and stimulated with TNFα or IL-1β for 30 min. IκB was detected by immunoblot using anti-IκB antibodies. p65 was used as a loading control (LC) and detected using anti-p65 antibodies. A representative immunoblot is shown. UT, untransfected. Un, unstimulated C. Infection of HeLa cells with derivatives of EPEC E2348/69 for 90 min followed by stimulation with TNFα or IL-1β for 30 min. Cells were collected for immunoblotting with anti-IκB antibodies. p65 was used as a loading control (LC) and detected using anti-p65 antibodies. A representative immunoblot is shown. UI, unininfected. Un, unstimulated D. Proposed model of the inhibition of NF-κB signaling by EPEC. Components of both the TNFα and IL-1β pathways are labelled and the predicted points at which NleE and NleB act on the pathways are shown as blocked arrows. The dashed line represents IκB degradation and shaded (P) represents phosphorylation.
Table 1.
Bacterial strains and plasmids used in this study.