Intravenous infection of zebrafish with the <i>Mma mgtC</i> mutant.

<p>(A) Survival of 30 hpf embryos intravenously infected with 150–200 CFU of wild-type <i>M. marinum</i>, Δ<i>mgtC</i> 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 <i>mgtC</i> mutant and 205 CFU for complemented strain) out of three independent experiments. (B) Ratio of whole embryo bacterial counts between <i>Mma</i> M and <i>mgtC</i> 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 <i>mgtC</i> 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 <i>mpx</i>:GFP infected larvae at late stages of infection (one day before embryo's death). Neutrophils fluoresce in green while <i>mcherry</i>-expressing bacteria fluoresce in red. Neutropenia occurs in wild-type and complemented strains but not in the <i>mgtC</i> mutant.</p

Increased phagocytosis of <i>mgtC</i> mutant by neutrophils upon subcutaneous injection of zebrafish embryos.

<p>(A) Survival of embryos subcutaneously injected at 3 dpf with approximately 50–100 CFU of wild-type <i>M. marinum</i>, <i>mgtC</i> 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 <i>mgtC</i> 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 <i>mgtC</i> mutant and 34 CFU for complemented strain) of three independent experiments. Asterisk indicates statistical significance (* <i>P</i><0.05).</p

Phagocytosis and replication of the <i>mgtC</i> mutant in the J774 macrophage cell line.

<p>(A) Phagocytosis of the <i>mgtC</i> mutant and complemented strains by J774 macrophages is normalized to 100% for the wild-type strain. Results are expressed as means+SD from four independent experiments. (B) Numeration of macrophages infected with <i>mcherry</i>-expressing bacteria, normalized to 100% for the wild-type strain. Results are expressed as means+SD from three independent experiments. (C) Replication of the <i>mgtC</i> mutant and complemented strains by J774 macrophages normalized to the wild-type strain. Results are expressed as means+SD from four independent experiments. (D) Kinetic of phagocytosis of the <i>mgtC</i> mutant and complemented strains by J774 macrophages normalized to 100% for the wild-type strain at 180 min. The <i>wbbl2</i> mutant strain is used as a positive control. Results are expressed as means ± SD (error bars) from three independent experiments. Asterisks indicate statistical significance (* <i>P</i><0.05; ** <i>P</i><0.01).</p

Expression of <i>Mma mgtC</i> and upstream genes in high Mg²⁺ and low Mg²⁺ conditions.

<p>(A) RT-PCR experiment on RNA isolated from <i>M. marinum</i> strains grown in high or low Mg²⁺ with primers specific for <i>mgtC</i>, <i>MMAR_2686</i>, <i>MMAR_2683</i> (PPE31) and <i>sigA</i>. Experiment was carried out with wild-type strain, <i>mgtC</i> mutant strain and complemented strain. Controls where reverse transcriptase was omitted (indicated by RT -) are done to verify the absence of genomic DNA contamination in the RNA sample. The <i>sigA</i> gene is used as control. (B) Quantification of <i>mgtC</i>, <i>MMAR_2686</i> and <i>MMAR_2683</i> RNA by Q-RT-PCR experiment using RNA isolated from <i>M. marinum</i> strains grown in high or low Mg²⁺. The sigma factor <i>sigA</i> was used as an internal standard. Results are expressed as means+standard deviations (SD) from a representative experiment performed in triplicate.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Growth of <i>Mma mgtC</i> mutant in Mg²⁺ deprived liquid medium.

<p>(A) Growth curves of <i>M. marinum</i> wild-type, Δ<i>mgtC</i> and Δ<i>mgtC</i>+<i>mgtC</i>::<i>attB</i> strains grown in Sauton's medium without magnesium. (B) or in regular Sauton's medium supplemented with magnesium. OD₆₀₀ is indicated over the growth period. The curves from two independent experiments are shown with SD.</p

Adherence of the <i>mgtC</i> mutant on J774 macrophages and internalization in HeLa cells.

<p>(A) Adherence of the <i>mgtC</i> mutant strain to J774 macrophages after a 3-hr period of infection at 4°C, as compared to the WT strain. Results are normalized to 100% for the wild-type strain and expressed as means+SD from three independent experiments. (B) J774 macrophage internalization of the WT, the <i>mgtC</i> mutant and the complemented strains after a 3-hr period of infection in the presence of cytochalasin D (10 µg/ml). Results are normalized to 100% for the wild-type strain and expressed as means+SD from three independent experiments. (C) Internalization of the <i>mgtC</i> mutant and complemented strains in HeLa cells. The <i>wbbl2</i> mutant strain is included as a positive control. Results are normalized to 100% for the wild-type strain and expressed as means+SD from four independent experiments. Asterisks indicate statistical significance (* <i>P</i><0.05).</p

Alignment of mycobacterial MgtC proteins and genetic environment of <i>mgtC</i> gene.

<p>(A) Alignment of <i>S.</i> Typhimurium MgtC (Accession Number AAL22622.1), <i>M. tuberculosis</i> MgtC (Accession Number NP_216327.1) and <i>M. marinum</i> MgtC (Accession Number ACC41130.1) using ClustalW. The upper line indicates the soluble C terminal part. Rectangles indicate four conserved residues that have been shown to be essential for <i>Salmonella</i> MgtC function. (B) Genetic environment of <i>mgtC</i> gene (striped arrows) in <i>M. tuberculosis</i> and <i>M. marinum</i> genomes. In both species, the <i>mgtC</i> gene is adjacent to <i>Rv1810</i> that is homologous to <i>MMAR_2686</i> (black arrows) and to PPE genes (grey arrows). The <i>MMAR_2688</i> gene is homologous to <i>Rv1812c</i>.</p

Expression of <i>P</i>. <i>aeruginosa mgtC</i> gene in liquid medium and intracellularly.

<p>The levels of <i>PA4635</i> (A) and <i>PA2558</i> (B) transcripts relative to those of the <i>rpoD</i> gene were measured by qRT-PCR. RNA was extracted from bacteria grown in liquid medium (<i>in vitro</i>) containing a high (1 mM) or low (10 μM) concentration of MgSO₄ and a pH of 7 or at 5.7. A pH of 5.7 is expected to mimic the pH faced by bacteria in the phagosome. Bacterial RNA was also extracted from infected J774 macrophages (<i>ex vivo</i>) that were treated (+ BAFI) or not (- BAFI) with bafilomycin A1. For all conditions, RNA were prepared two times independently. Results are expressed as means ± SD from at least three independent measurements (each performed in triplicate). The numbers indicate the induction fold relatively to the condition 1 mM MgSO₄ pH 7.</p

Behaviour of <i>mgtC</i> mutant towards J774 macrophages.

<p>(A) Strain cytotoxicity was evaluated using a LDH assay. A T3SS mutant (Δ<i>pscN</i>) is included as negative control. Error bars correspond to standard deviations from three independent experiments. (B) Survival of bacteria upon phagocytosis. Results are expressed as percentage of surviving bacteria at the indicated time comparatively to the number of bacteria internalized after 30 min of phagocytosis. Error bars correspond to standard errors (SE) from five independent experiments. (C) Effect of inhibition of vacuolar proton ATPase on bacterial survival. The survival of bacteria was measured from macrophages treated or not with bafilomycin A1. Error bars correspond to standard errors (SE) from three independent experiments. In all panels the asterisks indicate <i>P</i> values (Student’s t test, **<i>P</i> <0.01, ***<i>P</i> <0.001).</p