Fig 3

A) Scheme of 72hpf with body (light blue) and yolk region (pink) highlighted; the yolk sustaining L. pneumophila growing has been indicated with green dots. The scheme of the zebrafish larvae has been adapted from [35] and has been previously modified from [87]. B) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth in the yolk of 72 hpi mfap4: mCherry larva (red macrophages) injected in the bloodstream with HD of WT-GFP at 72hpf, shown laterally. Inset shows the maximum intensity projection of the L. pneumophila foci of the same larva mounted ventrally. Scale bar = 20mm. Related to S3 Movie. C) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth in the yolk of lyz:DsRed (red neutrophils) infected larva at 72 hpi, showed laterally. Scale bar = 20mm. Related to S4 Movie. D) Quantification of bacterial burden in the yolk over time. Larvae injected with LD, HD WT, or ΔdotA HD were imaged over time, and then scored for the GFP + bacteria absolute presence in the yolk for each larva over time. Larvae were scored as “infected” when they showed at least one small detectable GFP+ dot. Data obtained were plotted as % of larvae with bacteria in the in the yolk upon LD (blue curve), HD (red curve) WT or ΔdotA HD (green curve) injection over time. 11 independent experiments have been plotted (representing a total of n = 69 WT LD; n = 58 WT HD; n = 54 ΔdotA HD infected larvae) E) Time frame extracted from a 4D acquisition of L. pneumophila growth in the yolk between 48 and 72 hpi of mfap4: mCherry larva (red macrophages) injected in the bloodstream with HD of WT-GFP shown laterally. Imaris 4D reconstruction and volume rendering of the bacteria aggregate and interaction with macrophages. The yolk has been manually highlighted (in blue) with Imaris. Scale bar: 10. Related to S5 Movie. F) Quantification of bacterial burden in the whole body, in the body or in the yolk region versus macrophage or neutrophil quantification in the body or in the yolk region in HD WT-GFP infected larvae followed over time. Two independent experiments plotted for each phagocyte type (total of 11 larvae for macrophage and 11 larvae for neutrophil quantification). Quantification of the fluorescence images (GFP bacteria and RFP leukocytes) was done using CellProfiler software. P < 0.05 was considered statistically significant (symbols: **** P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05). No symbol on graphs means that not statistically differences were observed.