Figure 3—figure supplement 2.

Figure 3—figure supplement 2. Macrophages are essential to control Salmonella infection in zebrafish.

(A) Schematic illustration showing the mode of action of the nitroreductase (NTR) enzyme (encoded by Escherichia coli nfsb) in macrophages that converts the metronidazole (MTZ) pro-drug into a cytotoxic drug. (B) Schedule of the macrophage depletion experiment. Tg(mpeg1:GAL4/UAS:nfsB-mCherry) larvae were treated with MTZ (NTR+MTZ+) at 48 hpf, 24 hr before infection. DMSO treatment on the same line (NTR+MTZ−) or MTZ treatment on WT siblings (NTR−MTZ+) were used as controls. After MTZ treatment, larvae were injected in the hindbrain ventricle (HBV) either with PBS or with a sublethal dose (500 CFU) of Sal-GFP in the HBV. Infected larvae were analyzed for their survival from 0 to 4 dpi. (C) Representative mCherry fluorescent images of DMSO or MTZ-treated transgenic larvae at 1 day post-treatment (dpT). Asterisk: auto-fluorescence of the yolk, dotted line outlines the larva. Scale bar: 200 μm. (D) Quantification of total NTR+ macrophages in DMSO (MTZ−) and MTZ+-treated larvae at 1 dpT. Data of one representative of two replicates (mean number of cells/larva ± standard error of the mean [SEM], n = 15 per condition, Mann–Whitney test, one-tailed, ****p < 0.0001). (E) Survival curves of DMSO or MTZ-treated NTR+ PBS-injected embryos. Data of one representative of two replicates (n = 24 larvae per condition). (F) Survival curves of DMSO or MTZ-treated NTR+ Sal-GFP-infected embryos. Data of one representative of two replicates (n = 24 larvae per condition), log rank test, ****p < 0.0001. (G) Bacterial load quantification by fluorescent pixel count (FPC) from 1 to 4 dpi in DMSO or MTZ treated in NTR+ Sal-GFP-infected embryos. Data of one representative of two replicates (mean values/larva ± SEM, at 1 dpi: n_NTR+MTZ+ = 17, n_NTR−MTZ+ = 16, Mann–Whitney test, two-tailed, ****p < 0.0001, ***p < 0.001). (H) Representative fluorescent images of DMSO and MTZ-treated Tg(mpeg1:GAL4/UAS:nfsB-mCherry) Sal-GFP-infected larvae at 1 dpi showing mCherry (macrophage) and GFP (Salmonella) fluorescence. Asterisk: auto-fluorescence of the yolk, white arrowhead: Sal-GFP in the HBV. Scale bar: 200 μm.