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. 2023 Mar 15;42(9):e113490. doi: 10.15252/embj.2023113490

Figure 6. ESX‐1‐secreted proteins and PDIM are required for uptake‐independent killing of macrophages by Mtb aggregates.

Figure 6

  • A–D
    BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time‐lapse microscopy at 1‐h intervals for 60 h. The plots represent the percentage of macrophages that die within the first 12 h after stable contact with an Mtb aggregate. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P‐values were calculated using a one‐way ANOVA test (A, C, D) or t‐test (B) comparing each strain to the wild‐type reference strain (A–D); ns, P‐values > 0.05. (A) Macrophages in contact with aggregates of Mtb H37Rv wild‐type (WT), ∆RD1 mutant, and complemented ∆RD1 mutant (∆RD1::2F9). (B) Macrophages in contact with aggregates of Mtb Erdman wild‐type (WT) or PDIM‐deficient (fadD26) strains. (C) Macrophages in contact with aggregates of Mtb Erdman wild‐type (WT) or mutant strains (espI eccD1, esxA, espA) and the complemented strains (esxA::esxBA, espA::espA). (D) Macrophages in contact with aggregates of Mtb Erdman wild‐type (WT) or mutant strains (espB, espA espB) and the espB‐complemented strains (espB::espB, espA espB::espB).
  • E
    Schematic representation of EsxA, EsxB, EspA, and EspB secretion pattern in the mutant strains used in this study (based on Western Blot and quantitative proteomics data shown in Fig EV4 and in Appendix Figs S13 and S14).
  • F
    BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains, incubated with Annexin V, and imaged by time‐lapse microscopy at 1‐h intervals for 48 h. Percentage of macrophages that show Annexin V—positive membrane domains within the first 12 h after entering in contact with Mtb aggregates. Each symbol represents a single biological replicate (n ≥ 3 replicates with ≥ 90 cells per replicate). Bars represent means and standard deviations. P‐values were calculated using a one‐way ANOVA test.
  • G
    Cytochalasin D‐treated BMDMs were infected with aggregates of Mtb Erdman WT, stained with the membrane‐permeable dye Oregon Green 488 Bapta‐1 AM to visualize cytosolic Ca2+ and imaged by time‐lapse microscopy at 20‐min intervals for 24 h. Oregon Green 488 Bapta‐1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells. Values in the plot correspond to the time of death after first contact or 16 h post‐contact for cells that survive. Each symbol represents a single macrophage. (n = 123, 64, 48, 71, 78, and 55, respectively). Black bars represent the median and interquartile range. P‐values were calculated using a one‐sample Wilcoxon test; ns, P‐values > 0.05.
  • H
    BMDMs were treated and imaged as in (A–D). Percentage of macrophages that die within the first 12 h after interaction with an Mtb aggregate. Infected cells are incubated with different concentrations of BTP15 (0, 2, 10, and 50 μM) during the course of the experiment. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P‐values were calculated using a one‐way ANOVA test comparing the treated vs. untreated samples.

Source data are available online for this figure.