Intravenous infection of zebrafish with the Mma mgtC mutant.

(A) Survival of 30 hpf embryos intravenously infected with 150–200 CFU of wild-type M. marinum, ΔmgtC mutant or complemented strain compared to non-injected controls (n = 24). Results are from a representative experiment (infection with 133 CFU for wild-type, 142 CFU for mgtC mutant and 205 CFU for complemented strain) out of three independent experiments. (B) Ratio of whole embryo bacterial counts between Mma M and mgtC mutant strain-infected embryos at 0, 3 and 5 dpi. A ratio of 1 indicates equal CFU values. A ratio >1 indicates that WT CFU are higher than mgtC mutant CFU. Results are expressed as mean CFU per embryo+SD from four independent experiments (0 and 5 dpi) or two independent experiments (3 dpi). The mild difference between mutant and wild-type strains is not statistically significant (Student Test). (C) Visualization of neutrophils in mpx:GFP infected larvae at late stages of infection (one day before embryo′s death). Neutrophils fluoresce in green while mcherry-expressing bacteria fluoresce in red. Neutropenia occurs in wild-type and complemented strains but not in the mgtC mutant.

Increased phagocytosis of mgtC mutant by neutrophils upon subcutaneous injection of zebrafish embryos.

(A) Survival of embryos subcutaneously injected at 3 dpf with approximately 50–100 CFU of wild-type M. marinum, mgtC mutant, complemented strain or PBS as control (n = 24). Results are from a representative experiment (infection with 119 CFU for wild-type, 74 CFU for mgtC mutant and 32 CFU for complemented strain) out of three independent experiments. A drawing of the injection site is shown. (B) Maximum intensity projection of neutrophil-phagocytosed bacteria at the site of injection, at 4 hpi by confocal microscopy. (C) Quantification of bacterial phagocytosis by neutrophils. The number of neutrophils containing phagocytosed bacteria at the site of injection was counted at 4 hpi using confocal microscope of a minimum of 10 embryos. Results are expressed+SD from a representative (104 CFU for wild-type, 82 CFU for mgtC mutant and 34 CFU for complemented strain) of three independent experiments. Asterisk indicates statistical significance (* P<0.05).