The P. aeruginosa ΔoprF mutant is attenuated in zebrafish embryos in a macrophage-dependent manner. (A) Diagram of 50 h post-fertilization (hpf) zebrafish embryo showing the caudal vein injection site (green arrow). (B) Survival curves of embryos infected with PAO1 wild-type stain or PAO1 ΔoprF mutant. Non infected embryos (NI) were used as control. Embryos were either treated with LipoPBS (left panel) or with LipoCld (right panel) to deplete macrophages. Approximately 3500 CFU P. aeruginosa were microinjected into the caudal vein (n = 50–70 for infected embryos and n = 30 for NI embryos, pool of three independent experiments). Results are expressed as the percentage of surviving embryos at different times post-infection. (C) Representative fluorescence images of embryos infected by either wild-type or ΔoprF P. aeruginosa (at 20 hpi) in the context of LipoPBS or LipoCld treatment. The efficiency of macrophages depletion is shown in the upper panels with the visualization of red macrophages. Scale bar 400 µm. (D) Quantification of bacterial loads (fluorescence pixel counts) from two independent experiments. Each symbol represents individual embryo (n = 18–20) and horizontal lines indicate the median values. Statistical significance was determined by log-rank test (B) or one-tailed Mann–Whitney’s test (D).

Real-time visualization and quantification of phagocytosis of P. aeruginosa after local injection into muscle of wild-type and ΔoprF strains. (A) Illustration of zebrafish larva with the muscle (in red) injection site (green arrow). (B) Confocal time-lapse images of tg(mfap4:mCherry-F) larva (red macrophages) infected with ΔoprF P. aeruginosa (green) by injection in the muscle. The same area was imaged every hour from 1.5 hpi. White arrows depict GFP-expressing P. aeruginosa that will be taken up by macrophages, which are recruited at the bacterial location. Maximum intensity projection of 96 sections every 1 µm, scale bar 50 µm. (C) Confocal time-lapse images of tg(mfap4:mCherry-F) larvae (red macrophages) infected with wild-type or ΔoprF P. aeruginosa (green) by injection in the muscle. The left panel is at 1.5 hpi and the right panel a 4.5 hpi. White arrows depict GFP-expressing P. aeruginosa phagocytosed by macrophages. Maximum intensity projection of 96 sections every 1 µm, scale bar 50 µm. (D) Quantification of recruited macrophages at 1.5 and 4.5 hpi, from three independent experiments. Each symbol represents individual embryo (16 to 18 embryos for each strain) and horizontal lines indicate the mean values ± SEM. Statistical significance was determined by two-tailed t test, ns—not significant, *p < 0.05. (E) Quantification of infected macrophages, from three independent experiments. Mean ± SEM, two-tailed Mann–Whitney’s test, ns—not significant, *p < 0.05. (F) Quantification of bacterial loads (fluorescence pixel counts) from three independent experiments, presented as total value (left) or percentage of the FPC value at first time point (1.5 hpi), counted for each embryo. Mean ± SEM, two-tailed Mann–Whitney’s test, ns—not significant, *p < 0.05.

Visualization and quantification of intracellular P. aeruginosa within macrophages. GFP-expressing PAO1 WT and ∆oprF strains were used for infecting J774 macrophages. Cells were fixed after phagocytosis and 20 min (T₀) or 2.5 h (T_3h) treatment with gentamycin and imaged with fluorescent microscope. DAPI was used to stain the nucleus. (A) Visualization of intracellular bacteria. The merged image shows Differential Interference Contrast (DIC), nucleus staining (blue) and bacteria expressing GFP (green). The scale bars depict 10 µm. (B) Count of the number of bacteria in infected macrophages from images obtained. The numbers of bacteria per cell were classified in three groups (1–2, 3–5 and > 6 bacteria per cell) and percentage of each class is shown. Count is done from 100 cells per condition and results are expressed as means from three independent experiments.

Transmission electron micrographs (TEM) of P. aeruginosa within macrophages. J774 macrophages were infected with P. aeruginosa wild-type strain or oprF mutant for 50 min after phagocytosis and subjected to TEM to visualize intracellular bacteria. For the wild-type strain, most of bacteria were found inside membrane bound vacuoles (A–C) whereas oprF mutant was mostly found in phagolysosomes (D–F). (B) and (C) are showing details of the cell shown in (A) (white rectangles) at higher magnification, (E) and (F) show details of the cell shown in (D) (white rectangles).

Colocalization of P. aeruginosa with a probe that labels acidic compartments. J774 macrophages were infected with PAO1 and ∆oprF strains expressing GFP. After 2.5 h of gentamicin treatment, infected J774 cells were incubated with Lysotracker for 10 min, a red fluorescent weak base that accumulates in acidic compartments. Cells were then fixed and imaged with fluorescence microscope. (A) The image shows individual panels for Differential Interference Contrast (DIC), lysosomal compartment (red), bacteria expressing GFP (green), the nucleus (blue) and merged image of all channels. White arrows show colocalization of bacteria with lysotracker while yellow arrows show non-colocalization. Scale bar is equivalent to 5 µm. (B) Values indicate the percentage of bacteria that colocalized with Lysotracker red (white arrows). Data are the mean (± SEM) of 3 independent experiments, with a minimum of 100 bacteria counted per experiment for each sample. *, p ≤ 0.05 (Two-tailed unpaired t test).

Effect of bafilomycin on the virulence of wild-type and ΔoprF mutant in zebrafish embryos. Embryos were infected with either wild-type or ΔoprF P. aeruginosa (2000–2500 CFU) in the hindbrain ventricle at 2 dpf or not infected (NI) (n = 60–70 for infected embryos and n = 45 for NI embryos, pool of three independent experiments). Survival curves of embryos treated with 50 nM bafilomycin (Bafil) (B) or treated with DMSO as control (A). Statistical significance was determined by log-rank test. Imaging (C) and quantification (D) of bacterial loads (fluorescence pixel counts) representative of two independent experiments at 20 hpi (n = 11–14). Each symbol represents individual embryo and horizontal lines indicate the median values. Statistical significance was determined by one-tailed Mann–Whitney’s test.