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GBS microcolonies distort vessels and form obstructions. (A) Representative confocal image of a bifurcated blood vessel in a 20 hpi larva infected with approximately 100 CFU GBS-GFP, showing the uninfected contralateral vessel (left) and infected vessel (right). White dashed lines, diameter of the vessel. (B) Quantification of vessel dilation (µm) at the microcolony for infected vessels compared to uninfected vessels from the same animal at 20 hpi. Horizontal bars, means; paired t test. Representative of 2 independent experiments. (C) Quantification of vessel constriction (µm) adjacent to the microcolony for infected vessels compared to uninfected vessels from the same animal at 20 hpi. Horizontal bars, means; paired t test. Representative of 2 independent experiments. (D) Quantification of vessel diameter (µm) over time (4–11 hpi) for GBS infected and uninfected vessels; Student t test. (E) Representative confocal image of a brain blood vessel infected with approximately 100 CFU GBS-GFP from a 20 hpi gata1:dsRed larva, with red fluorescent red blood cells collecting at the microcolony. Larvae were injected intravenously with far red fluorescent dextran to visualize vessel perfusion (pseudocolored magenta) just prior to imaging. White arrowhead, absence of dextran perfusion in vessel. (F) Representative confocal image of a brain blood vessel infected with approximately 100 CFU red fluorescent GBS-mCherry (pseudocolored green) from a 20 hpi larva, with a green fluorescent platelet (pseudocolored red) attached to the microcolony. Larva was injected intravenously with far red fluorescent dextran to visualize perfusion (pseudocolored magenta). White arrowhead, lack of dextran in vessel. (G) Representative confocal images of larvae brains with red fluorescent red blood cells infected with approximately 100 CFU GBS-GFP at 20 hpi, without warfarin (top) and with 31.25 µM warfarin (bottom). White arrowhead, a red blood cell trapped in a brain blood vessel GBS microcolony. (H) Quantification of the number of obstructions associated with a GBS microcolony in larvae with or without warfarin at 18 hpi. Red blood cells (RBCs). Horizontal bars, means; Student t test. Representative of 2 independent experiments. (I) GBS burden per larva at 20 hpi, treated at 3 dpf with or without warfarin, quantified by FPC. Horizontal bars, means; Student t test. (J) Representative confocal image of a larva brain with fluorescent red blood vessels and GBS-GFP exiting a vessel to enter the brain. Inset shows area of the brain where GBS is entering the brain though a blood vessel. White arrowheads, GBS that has crossed the blood–brain barrier to enter the brain. (K) Proportion of larvae with or without GBS in the brain, with or without warfarin treatment, at 18 hpi; Fisher’s exact test. Representative of 2 independent experiments. (L) 30 hpi survival curve of larvae with and without GBS infection, and with and without warfarin treatment; Kaplan–Meier test, compared to infected, untreated group. Representative of 3 independent experiments. Scale bar, 10 µm throughout. All underlying data in Fig 7 can be found in the supplemental Excel file entitled “S1 Data”.
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