Osman et al., 2020 - Mycobacterium marinum phthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage. PLoS One   15:e0233252 Full text @ PLoS One

Fig 1 PDIM transport to the outer mycomembrane is required for optimal phagosomal permeabilization by <italic>M</italic>. <italic>marinum</italic>.

Representative dot plots of (A) wildtype, (B) ΔESX-1, and (C) ΔmmpL7 infected THP-1 macrophages 24 hours post infection with overlays of uninfected (blue) and infected (red) cells within each sample. Gating outlined in black, % permeabilization is as labeled. Plots are representative of two independent experiments. (D) Quantification of permeabilization events. Each point represents an independent experiment, symbols indicate matched experiments.

Fig 2 PDIM transport deficiency reduces <italic>M</italic>. <italic>marinum</italic>-induced phagosomal membrane damage.

(A) Maximum intensity projections of confocal micrographs showing galectin-8 labeling of PMA-differentiated THP-1 cells infected with tdTomato-expressing wildtype or mutant M. marinum strains 22 hours post infection. Arrows indicate co-localization of Galectin-8 (green) and M. marinum (magenta) fluorescence. Bottom panels show close-up views of the corresponding boxed regions in the top panels. Scale bars, 50μm. (B) Percent of infected cells that have mycobacterial foci labeled with galectin-8. Symbols represent individual imaging fields. Horizontal lines depict mean values. The respective means and 95% confidence intervals for wildtype, ΔESX-1 and ΔmmpL7 infections are 28.9 (22.0–35.8), 0.7 (0.2–1.2), and 4.7 (3.1–6.3). Statistical significance was determined using a Kruskal-Wallis nonparametric test with Dunn’s post-hoc test to control for multiple comparisons, **** p < 0.0001, * p < 0.05. Data are representative of two experiments.

Fig 3 PDIM-deficient <italic>M</italic>. <italic>marinum</italic> retain hemolytic activity.

(A) 2-hour hemolysis of sheep red blood cells following incubation with wildtype, ΔESX-1 and ΔmmpL7 M. marinum relative to a Triton X-100 control. Statistical significance was determined by one-way ANOVA with Tukey's post-test. *p < 0.05, ** p < 0.01, otherwise, p > 0.05. Symbols represent matching experimental replicates. Pink circles, green squares, dark purple triangles and light purple diamonds are the first, second, third, and fourth replicates, respectively. (B) ΔESX-1 and ΔmmpL7 M. marinum hemolysis from (A) normalized to wildtype for each of four experiments. p-values, one-sample t-test against a hypothetical mean of 100% Error bars, mean +/- S.E.M.

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