Fig 8

Fig 8 Streptococcus pneumoniae perforates blood vessels to invade the brain in zebrafish larvae.

(A) Representative confocal image of red fluorescent brain vasculature in a 24 hpi larva infected with approximately 1,000 CFU green fluorescent S. pneumoniae D39 (SPN-GFP). White arrowhead, SPN microcolony in vessel. (B) Representative confocal image (top) and 3D rendering (bottom) of an infected red fluorescent brain blood vessel, from a 24 hpi larva infected with approximately 1,000 CFU SPN-GFP. White dashed line, diameter of vessel. Yellow dashed circle, border of a vessel perforation at the microcolony site. (C) Quantification of vessel diameter (µm) at the SPN microcolony for infected vessels compared to contralateral uninfected vessels from the same animal at 24 hpi. Horizontal bars, means; paired t test. (D) Proportion of SPN-infected vessels with perforations. (E) Representative confocal image of 3D rendered red fluorescent brain blood vessels from 24 hpi larvae infected with approximately 1,000 CFU SPN-GFP and injected intravenously with far red fluorescent 0.02 µm beads (pseudocolored magenta). White arrowhead, escaped beads. (F) Proportion of vessels with retained beads or escaped beads in the brain, in uninfected and infected vessels from larvae in (E); Fisher’s exact test. (G) Representative confocal images of an uninfected (top) or infected red fluorescent brain blood vessel (bottom) from a 24 hpi larva infected with approximately 1,000 CFU SPN-GFP and injected intravenously with annexin V (pseudocolored magenta). White arrowhead, positive annexin V staining in vessel endothelial cell. (H) Proportion of uninfected and SPN-infected vessels with positive annexin V staining at 24 hpi; Fisher’s exact test. Scale bar, 10 µm throughout. All underlying data in Fig 8 can be found in the supplemental Excel file entitled “S1 Data”.