IRG proteins load onto PVM in a cooperative manner.

<p>MEFs were induced with IFNγ for 24 h and then infected with <i>E. cuniculi</i> spores for 12 h (<b>A</b>) or 24 h (<b>B, C</b>). Fixed cells were stained for meronts (mouse mAB 6G2, red) as well as for endogenous Irga6 (rabbit pAS165/3, green) and Irgb6 (goat pAB A20, far-red pseudocolored in magenta). Nuclei were labeled with DAPI. Representative images from 4 independent experiments are shown; white box indicates enlarged area shown below; scale bar: 10 µm. (<b>D</b>) Quantification of cooperative loading after 24 h; Irgb6-single, Irga6-single or Irgb6/Irga6-double (both) positive meronts are shown as % of total 6G2-positive meronts; 100 vacuoles were counted in each independent experiment (Exp. 1–3).</p

IFNγ suppressive effect on <i>E. cuniculi</i> growth is impaired in GMS-IRG knock-out cells.

<p>(<b>A</b>) <i>Wildtype (wt)</i> or <i>Irgm1/Irgm3</i> knock-out (KO) MEFs were induced with 200 U/ml IFNγ for 24 h and then infected with <i>E. cuniculi</i> spores for 24 h or left untreated. Cells were fixed and stained for meronts using 6G2 mAB (red) and host nuclei with DAPI (pseudocolored in cyan). Representative fluorescence microscopic images are shown. (B) Quantification of A, representative of two independent experiments. (C/D) Transformed <i>wildtype</i> or transformed IRG knock-out MEFs were induced with IFNγ for 24 h and then infected with <i>E. cuniculi</i> spores or left untreated. Cells were harvested after 2 days (in D) and 5 days (in E) post-infection. Cell lysates were separated by SDS-PAGE and Western blots were cut into three regions and simultaneously probed for anti-meront mAB 6G2, anti-Calnexin pAB, which served as loading control and anti-Irgb6 (mAB B34) or anti-Irga6 (mAB 10E7 for <i>Irgm1</i> KO and <i>Irgm3</i> KO MEFs both at 5 d post infection; 165/3 pAS for <i>Irgm1/Irgm3</i>KO MEFs at 5 d post infection) as IFNγ-induction control. The black arrows highlight a 6G2-positive protein band indicating <i>E. cuniculi</i> growth despite presence of IFNγ, which is inhibited in <i>wt</i> cells (grew arrows). The asterisk marks an unknown <i>E. cuniculi</i>-derived protein that is detected by the Calnexin antibody. The white asterisk marks unspecific bands. The four samples of one cell line per time point were analyzed together by one single SDS-PAGE and Western Blot, except for the <i>Irgm1</i>KO MEFs at 2 d post infection. The data represents at least three independent experiments.</p

Tryptophan supplementation cannot reverse the IFNγ-mediated <i>E. cuniculi</i> restriction.

<p>C57/BL/6 MEFs (<b>A</b>) or mouse enterocytic CMT-93 cells (<b>B</b>) were treated with IFNγ for 24 h or left uninduced. Indicated doses of L-Tryptophan (W) were added to the medium 30 minutes prior infection with <i>E. cuniculi</i> spores. Cell lysates were prepared 5 days post infection and separated by one SDS-PAGE. Western blots were cut into three regions probed simultaneously with for anti-meront mAB 6G2, anti-Calnexin pAB as loading control and anti-Irga6 (10E7 mAB) as IFNγ-induction control. The data are representative of three independent experiments.</p

IFNγ restricts <i>E. cuniculi</i> growth in mouse embryonic fibroblasts.

<p>(<b>A</b>) Mouse embryonic fibroblasts (MEFs) from C57BL/6 mice were induced with IFNγ for 24 h or left uninduced before infection with <i>E. cuniculi</i> spores. Cells were fixed at the indicated time points and the number of meronts (stained with anti-meront mAb 6G2) per 500 host nuclei (stained with DAPI) was counted. The inhibition in the IFNγ-treated sample compared to the uninduced control sample is presented as mean +/− standard deviation (SD) of 3–7 replicates per time point from at least 2 individual experiments. Significant differences (of 0.5 h, 1 h and 2–3 h compared to 24–26 h) were calculated with a two tailed T-test. (<b>B</b>) MEFs were induced with IFNγ or left uninduced, infected with <i>E. cuniculi</i> spores for 24 h and stained as in A. Single meronts and meronts that divided once (double meront) were counted per 500 host nuclei and shown as percent of total vacuoles of uninduced controls. Numbers indicate the counted number of single or double meronts per 500 host cells. Data from three independent experiments (Exp. 1–3) is presented. (<b>C</b>) MEFs were stimulated with IFNγ and/or infected with <i>E. cuniculi</i> spores for 2 or 5 days or left untreated. Cell lysates were separated by SDS-PAGE and Western Blots were cut into three regions to probe for anti-meront mAB 6G2 as well as anti-spore wall protein 1 pAS SWP1. Calnexin staining served as loading control and Irgb6 staining (mAB B34) to proof IFNγ-induction. The asterisk marks an unknown <i>E. cuniculi</i>-derived protein that is detected by the Calnexin antibody. These Western Blots emerged from one single SDS-PAGE, the 45–70 kDa region was first probed with mouse mAB B34, stripped, and then probed for anti-SWP1 rabbit pAS. Experiments for both time points were performed at least three times.</p

Scheme of the IRG resistance system and its target organisms.

<p>In IFNγ-stimulated mouse cells, GMS proteins localise mainly to endomembranes such as the ER and keep membrane-bound or cytosolic GKS proteins in a GDP-bound inactive state. Our current view is that the plasma membrane is protected by a hypothetical unknown factor that inhibits GKS protein-mediated damage. During host cell infection by <i>T. gondii</i> or <i>E. cuniculi</i>, invagination of the plasma membrane creates a parasitophorous vacuole that excludes the hypothetical factor and also does not carry GMS proteins. This “missing-self” allows GKS proteins to activate and accumulate on the PVM leading to the PV disruption, pathogen elimination and ultimately host cell death. However, bacteria entering via phagocytic mechanisms do not actively exclude the hypothetical factor and are therefore targeted for endolysosomal degradation.</p

<i>E. cuniculi</i> infection triggers IFNγ-dependent host cell death.

<p>(<b>A</b>) <i>Wt</i> MEFs were induced with IFNγ for 24 h and then infected with <i>E. cuniculi</i> spores for 24 h or left untreated. Without fixation, the cells were stained with Hoechst and Propidium iodide dye, photographed under live-cell conditions and automatically enumerated. (<b>B</b>) Quantification of A, graph represents mean values +/− SD from five independent experiments. (<b>C</b>) <i>Wt</i> MEFs were seeded in 96-wells and induced with IFNγ for 24 h (black bars) or left untreated (white bars). Cells were infected with <i>E. cuniculi</i> spores at different MOIs (MOI = 5–20) or with <i>T. gondii</i> Me49 tachyzoites (MOI = 5) as positive control. Cell viability was measured with a formazan-based colorimetric assay 24 h or 48 h post infection and expressed as percentages of uninduced uninfected control cells. Graph represents mean value +/− SD of triplicates of one representative experiment. Three independent experiments were performed; Significance was calculated with two-tailed T-Test: ** p>0.005, *** p>0.0005.</p

Scheme of different dynamics of IRG action.

<p>Upon <i>E. cuniculi</i> infection, a low but steady number of IRG-positive vacuoles can be detected over the first 24 h post infection accompanied by a continuous loss of viable meronts. Moreover, host cell death is triggered by the combination of <i>E. cuniculi</i> infection and IFNγ induction. Initiation of IRG loading might be a stochastic and asynchronous event that is followed by a rapid elimination of the pathogen. In contrast, IRG loading on PVs of avirulent <i>T. gondii</i> strains starts immediately after parasite invasion. IRG-positive vacuoles seem to accumulate reaching a maximum at about 2 h post infection. When fully loaded, the vacuoles disrupt followed by <i>T. gondii</i> death and host cell death in an invariant order.</p

Le Miroir des sports : publication hebdomadaire illustrée

17 août 19371937/08/17 (N964)-1937/08/17

Additional file 1: Figure S1. of Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile

Growth curves in BHIS of DSM 27638, DSM 27639 and DSM 27640. The isolates were grown in brain heart infusion medium containing 0.5% (w/v) yeast extract und 0.03% (w/v) L-cysteine. All isolates have the same growth rate under laboratory conditions as shown as the means of three replicates with standard deviation. (DOCX 55 kb