<i>Mtb</i>-mediated phagosome disruption in different phagocyte types, relationship with infection dose and cell death.

<p>(A) Comparative infectivity of WT and ΔESX-1 <i>Mtb</i>. BM-DC were infected with DsRed-WT or-ΔESX-1 strain and the red fluorescence was assessed by cytometry at 1 dpi. Percentages of cells having phagocytized DsRed-mycobacteria are indicated. (B) Detection of phagosomal rupture subsequent to infection with WT or ΔESX-1 strains, as determined by green <i>vs</i>. blue CCF-4 signals in BM-DC or BM-MΦ, infected with untagged <i>Mtb</i>, WT or ΔESX-1 (MOI = 1) at different time points, as detected after exclusion of cell debris and free bacteria by FSc/SSc gating and inclusion of CD11b⁺ cells. (C) MFI_{520 nm}, MFI_{447 nm} and MFI_{447 nm}/MFI_{520 nm} ratios in infected BM-DC or BM-MΦ at different time points. (D) Phagosomal rupture, monitored at 4 dpi by CCF-4 staining, in BM-DC infected with different MOI of WT or ΔESX-1 <i>Mtb</i>. (E) Percentages of dead cells, as determined by the use of Pacific Blue Dead/Live reagents, compared to those of cells displaying a CCF-4 blue shift. Due to the emission overlap of CCF-4-Coumarin and Pacific Blue fluorochromes, the two different assays were performed in separate tubes in parallel, in cells from the same BM-DC cultures. The results are representative of 2 independent experiments. Of note, the decrease of the dead cell percentage for WT <i>Mtb</i> at very high MOI is likely due to generation of cell debris, not anymore measurable by cytometry.