SpvD prevents nuclear accumulation of p65 but not degradation of IĸBα.

<p>(A) SpvD does not inhibit activation of an AP-1-regulated promoter. HEK-293 cells were cotransfected with an AP-1-dependent luciferase reporter plasmid, pTK-Renilla luciferase and pRK5myc, pRK5myc-SpvC or pRK5myc-SpvD. Cells were then stimulated with PMA for 6 h. Firefly luciferase activity was normalized against Renilla luciferase activity. Results are expressed as fold induction compared to non-transfected (NT) cells. Values are expressed as mean ± SEM of 3 independent experiments and P-values were obtained using two-tailed unpaired Student's t-test (*** p < 0.005). (B-C) SpvD prevents nuclear translocation of p65. (B) Representative immunofluorescence fields of p65 localisation using anti-p65 (red) in <i>TLR4</i>^-/- BMMs infected for 10 h with indicated <i>Salmonella</i> strains (blue). Cell nuclei were stained with DRAQ5 (green). Scale bar, 8 μm. (C) Quantification above background levels of p65 intensity in the nucleus was analyzed by three-dimensional (3D) confocal microscopy. Background levels were determined using signals measured in uninfected cells. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05; ** p < 0.01). (D) SpvD inhibits activation of an NF-ĸB -regulated promoter. HEK-293 cells were co-transfected with an NF-ĸB -dependent luciferase reporter plasmid, pTK-Renilla luciferase and pRK5myc, pRK5myc-SpvC, pRK5myc-SpvD or pRK5myc-SpvD_K185A at the indicated amounts (in ng). The NF-ĸB pathway was activated with TNF-α and luciferase activity was measured after 8 h. Results are expressed as fold activation in relation to unstimulated and non-transfected (NT) cells. Values are expressed as mean ± SEM of at least 3 independent experiments and statistical significances were calculated using ANOVA followed by Bonferonni's multiple comparison test against NT cells (*p < 0.05; ** p < 0.01; *** p < 0.005). (E) Degradation of IĸBα is not affected by SpvD. HEK-293 cells transfected by pRK5myc-SpvD or pRK5myc-YopP were prepared at the indicated times after TNF-α stimulation and analysed by SDS-PAGE and immunoblotting with anti- IĸBα and anti-tubulin antibodies. Ratio of IĸBα normalised to unstimulated cells is indicated below immunoblots.</p

SpvD induces an intra-nuclear accumulation of importins.

<p>(A) HeLa cells were cotransfected with FLAG-KPNA1 or FLAG-KPNA3 and pRK5myc-SpvD or pRK5myc-SpvC. Samples were fixed, permeabilised in Triton X-100, labelled with anti-FLAG (red), anti-myc (blue) and anti-lamin (green) antibodies and analysed by confocal microscopy. Scale bar, 10 μm. White arrows indicate nuclear lamina-associated KPNAs. (B) Quantification of cells with nuclear lamina-associated KPNA1 or KPNA3 after transfection with pRK5myc-SpvD or pRK5myc-SpvC. Values are expressed as mean ± SEM of at least 4 independent experiments (*p < 0.05; *** p < 0.005). (C) HeLa cells were cotransfected with FLAG-KPNA1 and pRK5myc-SpvD. Samples were fixed, permeabilised in Triton X-100 or Saponin and labelled as in A. Scale bar, 8 um. (D) HeLa cells were cotransfected with FLAG-KPNA1 plasmids then infected for 14 h with <i>Salmonella</i> strains. Samples were fixed, permeabilised in Triton X-100 and labelled with anti-FLAG (red) and anti-<i>Salmonella</i> (blue) antibodies. Cell nuclei were stained with DRAQ5 (green). Scale bar, 10 μm. (E) Quantification of cells with nuclear lamina-associated KPNA1 after infection with <i>Salmonella</i> strains. Values are expressed as mean ± SEM of at least 4 independent experiments (** p < 0.01; *** p < 0.005). (F) HeLa cells were infected for 14 h with <i>Salmonella</i> strains. Nuclear and total cell extracts were analysed by SDS-PAGE and immunoblotting with anti-KPNA1, anti-histone H3 and anti-GAPDH antibodies. (G) Ratio of KPNA1 in the nucleus normalised to wild-type -infected cells. Values are expressed as mean ± SEM of 3 independent experiments. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05).</p

Effect of SpvD on virulence and cytokine expression <i>in vivo</i>.

<p>(A) Lack of SpvD causes virulence attenuation of <i>Salmonella</i> in the mouse model of systemic infection. C57BL/6 mice were inoculated by intraperitoneal injection (i.p.) with equal numbers (2.5 x 10⁴ CFU) of indicated bacteria. Bacteria were recovered from infected spleens 3 days post-inoculation. CI values were calculated as described in materials and methods. The scatter plot displays values obtained for individual mice and the mean is indicated (line) (*p < 0.05; *** p < 0.005). (B-D) C57BL/6 mice were inoculated by i.p. with indicated bacteria, RNAs from splenic macrophages were recovered at 2 days post-inoculation and <i>serpinB2</i> (B), <i>tnf</i>-α (C) and <i>il1-</i>β (D) mRNA levels were analysed by qRT-PCR. The transcript levels were normalized to the levels of <i>rsp9</i>, which were constant under all conditions used, and then expressed relative to those of wild-type-infected mice. Results are expressed as mean ± SEM of at least 3 independent experiments. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05). (E) Model depicting pathways targeted by SpvC and SpvD to overcome PAMP-induced defences in host cells. Immunoblots are representative of three independent experiments.</p

The SPI-2 T3SS reduces pro-inflammatory cytokine mRNA levels and protein secretion in <i>TLR4</i>^-/- BMM.

<p>Wild-type and <i>TLR4</i>^-/- BMMs were non-infected (NI), exposed to LPS (100 ng/ml) or infected with wild-type or Δ<i>ssaV</i> strains for 10 h and mRNA levels of <i>tnf</i>-<i>α</i> (A) and <i>il-1β</i> (B) were analysed. The transcript levels were normalized to the levels of <i>rsp9</i>, which were constant under all conditions used, and then expressed relative to those of NI wild-type BMMs. Results are expressed as mean ± SEM of at least 3 independent experiments. Levels of secreted TNF-α (C) and Il-1β (D) were quantified by ELISA in supernatants of non-infected (NI) <i>TLR4</i>^-/- BMMs, <i>TLR4</i>^-/- BMMs stimulated with LPS or <i>TLR4</i>^-/- BMMs infected with indicated strains of <i>S</i>. Typhimurium at 10 h or 24 h post-uptake respectively. Results presented are the means ± SEM of triplicates of one experiment. These results are representative of three independent experiments and significant differences were observed in each experiment. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05). Levels of secreted TNF-α at 10 h post-uptake (E) and Il-1β at 24 h post-uptake (F) were quantified by ELISA in supernatants of <i>TLR4</i>^-/- BMMs infected with indicated strains of <i>S</i>. Typhimurium. The cytokine levels were expressed relative to those of BMMs infected with wild-type bacteria. Results are expressed as mean ± SEM of at least 3 independent experiments. Statistical significances were calculated using ANOVA followed by Bonferonni's multiple comparison test against wild-type strain.</p