Colocalization of pneumococci with VWF in vascular system of Danio rerio. (A) Colocalization of red fluorescent pneumococci with zebrafish-derived VWF was microscopically visualized after fixation, embedding, and staining of wild type larvae with VWF-specific rabbit FITC-conjugated antibodies and pneumococcus specific rabbit antibodies followed by goat Alexa 568-conjugated antibodies. Visualization of three representative larvae (no. 1, 2, and 3) is shown after digital zoom in magnification of the region marked by white and subsequently by black squares. All larvae are displayed from ventral side. The white square encircles intersegmental vessels of the trunk muscles in larva 1, the heart vasculature of larva no. 2 and the vessels of the gill arches of larva no. 3. White arrows of R1–R5 point to VWF colocalization with single diplococci or short pneumococcus chains appearing as yellow overlay. Colocalizations within selected regions of interest (zoom in ROI) are confirmed by corresponding overlay histograms. Scale bars represent 10 μm. (B) Visualization of RFP-expressing serotype 35A pneumococci in zebrafish larvae without and after pre-incubation of pneumococci with human VWF prior to injection of 600 cfu into the heart chamber. The red square in the zebrafish illustration depicts the region of the zebrafish tail, which is focused for visualization after immunostaining. Using a transgenic zebrafish larva, which expresses red-fluorescing endothelium (FLK1:mCherryCAAX), the position of the main blood vessel and its branching circular microvasculature within the tail region are visualized in b. After 5 h of infection with non-VWF-pre-incubated RFP-expressing pneumococci (without VWF), only single diplococci are detected within the intersegmental blood vessels of the tail region of cross linked larvae after immune staining of VWF (green) (without VWF, merge, white arrows). The yellow appearance in the merged illustration points to the recruitment of zebrafish-derived VWF to the surface of the bacteria. After 5 h of infection with VWF pre-incubated pneumococci, several bacterial aggregates were detected after immunostaining of VWF in green and bacteria in red as yellow clumps of up to 10 μm in size in the vasculature of the zebrafish tail (+VWF, merge and BF + merge, yellow arrows). (C) A further representative example of VWF-mediated pneumococcal aggregate formation with the vasculature of an infected zebrafish larva. A zoom in into the zebrafish region in close proximity to the larval heart (white square) visualizes a cluster of pneumococci (+VWF, merge, red arrows). A histogram of fluorescence signal overlaps in the aggregates is added in Figure S3B. Scale bars represent 10 μm.
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