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GBS does not use transcytosis or phagocytes to cross the BBB. (A) Representative confocal images of red fluorescent brain vasculature in 20 hpi larvae infected with approximately 100 CFU GBS-GFP. White dashed box, GBS microcolony at a vessel bifurcation. Yellow dashed box, GBS microcolony in a straight vessel. Scale bars, 10 µm. (B) Proportion of GBS microcolonies at brain blood vessel bifurcations compared to expected hypothetical values; Fisher’s exact test. Representative of 2 independent experiments. (C) Sequential images from a time-lapse movie, showing a GBS-GFP microcolony clearing from a brain blood vessel. T, hours: minutes after start of time-lapse video recording. Scale bar, 10 µm. (D) Total GBS microcolonies in the brain over time (11–19 hpi) for a representative larva. (E) Representative confocal image (xy) with optical cross sections (yz and zx) of red fluorescent vessels in a 20 hpi larva infected with approximately 100 CFU GBS-GFP. White arrowhead, lack of co-localization of red and green fluorescence because GBS microcolony is in the blood vessel lumen and not the endothelial cell. Scale bar, 10 µm. (F) Proportion of vessels containing GBS in the lumen that are not inside of endothelial cells (EC) (black), or GBS inside of an endothelial cell (white) at 20 hpi. Representative of 3 independent experiments. (G) Representative confocal images of larvae brains with red fluorescent blood vessels infected with approximately 100 CFU GBS-GFP at 20 hpi, without dynasore treatment (top) and with 40 µM dynasore treatment (bottom). White arrowheads, GBS entering the brain. Scale bar, 10 µm. (H) Proportion of larvae with GBS in the brain, with or without dynasore treatment; ns: not significant, Fisher’s exact test. (I) Representative images of a 3 dpf larva with red fluorescent monocytes, intravenously injected at 2 dpf with PBS (top) or lipoclodronate (LC, bottom). Insets show monocytes in the caudal hematopoietic tissue. Scale bar, 100 µm. (J) Monocyte fluorescence per larva intravenously injected with PBS (left) or LC (right), quantified by FPC. Horizontal bars, means; Student t test. Representative of 2 independent experiments. (K) Representative images of a 20 hpi larva infected at 3 dpf with 100 CFU GBS-GFP and injected at 2 dpf with PBS (top) or LC (bottom). White arrowhead, GBS infection in brain. Scale bar, 100 µm. (L) GBS burden per larva at 20 hpi, intravenously injected with PBS or LC, quantified by FPC. Horizontal bars, means; ns: not significant, Student t test. (M) Representative confocal images of brains of 20 hpi larvae infected with approximately 100 CFU GBS-GFP. Larvae were injected with PBS (left) or LC (right). Scale bar, 10 µm. (N) Quantification of the volume of GBS in larvae brains after injection with PBS or LC. Horizontal bars, means; ns: not significant, Student t test. (O) Proportion of larvae with GBS in the brain, with or without LC treatment; ns: not significant, Fisher’s exact test. (P) Representative confocal images of red fluorescent brain vasculature and blue fluorescent polymorphonuclear (PMN) neutrophils in a 20 hpi larva infected with approximately 100 CFU GBS-GFP. White arrowhead, neutrophil in the brain, containing GBS. Yellow arrowhead, GBS entering brain without an associated neutrophil. Gray arrowheads, uninfected neutrophil in the brain. Scale bar, 10 µm. (Q) Proportion of larvae with neutrophils associated with GBS entering the brain. All underlying data in Fig 2 can be found in the supplemental Excel file entitled “S1 Data”.
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