Fig 1

(A) Schematic diagram of the zebrafish Mm infection model for early stages of Tb. Fluorescently labelled Mm bacteria are microinjected into the blood island of embryos at 28 hpf. Red dots represent small clusters of Mm-infected cells visible from 1 dpi. At 3 dpi these Mm clusters have grown into early stage granulomas. (B) Representative confocal micrographs of GFP-Lc3 co-localization with mCherry-labeled Mm clusters in infected embryos/larvae at 1, 2 and 3 dpi. Scale bars, 10 μm. (C) Quantification of the percentage of Mm clusters positive for GFP-Lc3 at 1 and 2 dpi. The results were accumulated from two individual experiments (≥ 20 embryos/group). ns, non-significant, *p<0.05, **p<0.01, ***p<0.001. (D) Representative confocal micrographs of GFP-Lc3 co-localization with mCrimson-labeled Mm clusters in an mpeg1:mCherry-expressing macrophage at 3 dpi. Arrowheads indicate intra-macrophage GFP-Lc3-positive Mm. Scale bars, 10 μm. (E) Western blot determination of Lc3 protein levels in Mm-infected embryos/larvae and uninfected controls. Protein samples were extracted at 1, 2 and 3 dpi (>10 larvae/sample). The Western blot was probed with antibodies against Lc3 and Actin as a loading control and is representative for three independent experimental repeats. The Lc3-II/Lc3-I ratio is indicated below. (F) Representative confocal micrographs of Ubiquitin antibody co-localization with Mm clusters in infected embryos/larvae at 1, 2 and 3 dpi. Scale bars, 10 μm. (G) Quantification of the percentage of Mm clusters positive for ubiquitin staining at 1 and 2 dpi (≥ 15 embryo/group). The results are accumulated from two independent experiments. ns, non-significant, *p<0.05, **p<0.01, ***p<0.001. (H) Western blot analysis of ubiquitination levels in Mm-infected embryos/larvae and uninfected controls. Protein samples were extracted at 1, 2 and 3 dpi (>10 larvae/sample). The Western blot was probed with an antibody detecting both poly- and mono-ubiquitin and with anti-Actin antibody as a loading control and is representative for three independent experimental repeats. (I) Representative confocal micrographs of GFP-Lc3 and Ubiquitin co-localization with Mm clusters in infected larvae at 3 dpi. Scale bars, 10 μm.