Fig. 1

M. marinum WT and esx‐1 mutant traverse across an intact blood–brain barrier within macrophages. (a) Lateral view of a casper zebrafish larva at 3 dpi (corresponds to 7 dpf), infected with M. marinum E11:mEos3.1 (green). Red arrow marks the caudal vein injection spot. Boxed area represents the brain region of which representative images are shown in this figure. (b) Tg(kdrl:mCherry)^is5 zebrafish larva, uninfected, 1 hr after 3‐kDa Alexa 680 tracer injection at 2 dpf, showing massive leakage of tracer to the ventricles, confirming the immaturity of the BBB. (c) Injection of tracer at 7 dpf, showing localisation in brain blood vessels, indicating that the BBB is functionally intact at this moment. Blood vessels in close proximity to the eyes and gills do not possess a BBB and do not restrict diffusion of the dye, leading to extravasation of the dye at these locations. Scale bars bc = 250 μm. (d) Tg(kdrl:mCherry)^is5 zebrafish larvae infected at 2 dpf (before formation of a functional BBB), 1 dpi single nonphagocytosed bacteria (green) can be found in blood vessel (red; open arrow) and in brain tissue (arrow), showing that M. marinum can enter brain tissue at this time point. (e) Also at 2 dpi, phagocytosed bacteria (colocalisation of green bacteria and cyan phagocytes labelled with anti‐L‐plastin) in blood vessel (open arrow) and single nonphagocytosed bacteria in brain tissue (arrow) are found. (f) Systemic infection at 4 dpf (larvae with a functional BBB), M. marinum (green) is solely found in brain blood vessels (red) at 1 dpi and (g) 2 dpi (arrows). (h–j) From 3 dpi onwards, phagocytosed mycobacteria (colocalisation of green bacteria and cyan phagocytes) are able to leave the bloodstream (arrows). (k) Representative image of section of brain in Tg(Fli1:GFP)^y1 zebrafish larvae, systemically infected with the esx‐1 mutant (red) at 4 dpf, showing that mutant bacteria also cross the BBB within phagocytic cells (anti‐L‐plastin, cyan). (l,m) Examples of spot with high expression of mCherry tagged VEGFr2 in Tg(kdrl:mCherry)^is5 larva, colocalising with phagocytosed (cyan) M. marinum E11 (green) in blood vessel (red). (l) Merged image, (m) corresponding single red channels. Scale bars d–m = 25 μm