Fig. 7

E. faecalis long cell chains are less virulent in the zebrafish model of infection and more prone to phagocytosis than diplococci.

A. Survival of zebrafish larvae (n>20) following infection with E. faecalis OG1RF (WT) and atlA isogenic deletion mutant before (?atlA) and after (?atlA^S) sonication to disperse long chains. Statistical significance was determined by Log-rank test; **P = 0.0011; *** P = 0.0002; NS, P>0.05. B. Quantification of E. faecalis uptake by zebrafish phagocytes. Embryos were infected with 1,200 E. faecalis cells expressing GFP and fixed in 4% paraformaldehyde 1.5h post infection. Phagocytes were immunolabelled using rabbit anti L-plastin antibodies and detected with goat anti-rabbit antibodies conjugated to Alexafluor 647. Fluorescent bacteria and phagocytes were imaged by scanning confocal microscopy. The area of GFP fluorescence signal outside and inside phagocytes was measured using a dedicated Fiji plugin. The ratio of GFP fluorescence area outside to inside phagocytes was used to quantify bacterial uptake. Phagocytosis was significantly higher for long chains (?atlA) when compared to their sonicated counterparts (?atlA^S) (**P = 0.0098) or the wild-type cells (*P = 0.0438). No difference in uptake was found between short chains corresponding to the wild-type or sonicated ?atlA mutant (NS, P>0.05). Representative images of phagocytes (magenta) following infection with ?atlA, sonicated ?atlA^S and wild-type OG1RF cells shown. Phagocytes labeled with L-plastin appear in magenta, GFP-producing bacteria in green. Scale bar is 20?m. C. Survival of phagocyte-depleted zebrafish larvae (n>20) following injection with E. faecalis OG1RF (WT) or ?atlA. D. Pairwise comparisons of phagocytosis indexes corresponding to E. faecalis OG1RF and ?atlA uptake by human monocyte-derived macrophages (MDM). Statistical significance was determined by paired t-test; ?atlA cells were more efficiently phagocytosed by MDM than WT cells (**P = 0.0024; n = 7).