Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells
- KwaZulu-Natal Research Institute for TB-HIV, South Africa
- University of KwaZulu-Natal, South Africa
- Max Planck Institute for Infection Biology, Germany
- Massachusetts General Hospital, United States
- Nelson R Mandela School of Medicine, University of KwaZulu-Natal, South Africa
- Inkosi Albert Luthuli Central Hospital, South Africa
Figures
Figure 1 with 5 supplements
Number of Mtb internalized determines probability of macrophage death.
The death frequency for 720 infected MDM was graphed over time as a function of the sum of Mtb internalized (raw data provided in Figure 1—source data 1). To obtain death frequencies, MDM were …
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Figure 1—source data 1
Intracellular Mtb fluorescence through time, Movie frames of Mtb phagocytosis, and MDM frame of death, if it occurs, for IFNγ untreated MDM.
- https://doi.org/10.7554/eLife.22028.005
Figure 1—figure supplement 1
Measures of cell death.
Time of cell death induced by Mtb and determined by macrophage dynamics was correlated to macrophage death as determined by the uptake of the dead cell stain DRAQ7. Time of DRAQ7 uptake was …
Figure 1—figure supplement 2
Fluorescence as a measure of Mtb numbers.
we tracked the increase in Mtb by fluorescence (blue squares) versus by colony forming units (CFU, red circles) over three days of growth in culture. Means and standard deviations of triplicates. …
Figure 1—figure supplement 3
MDMs were ranked according to the sum of Mtb internalized (black), number of Mtb in the last aggregate internalized (red), number of Mtb in the first aggregate internalized (green), or randomly (blue).
Ranks (x-axis) were then plotted against the cumulative number of dead cells present up to each rank (y-axis). Ranking according to the sum of Mtb internalized gave the best separation between the …
Figure 1—figure supplement 4
Outcomes of macrophage-Mtb interactions.
(A) A subset of cells is shown as an example. Macrophages were numbered on the y-axis based on the sum of Mtb internalized, with cells internalizing the largest number at the top of the chart. The …
Figure 1—figure supplement 5
Analysis of differences between macrophages grouped by the sum of Mtb internalized.
(A) Fraction of cells which died over time in each of the 10 infected cell groups (n = 72, group mean and standard deviation of Mtb per macrophage shown in the legend on the right), and the …
Figure 2
Internalization of single large aggregates is more cytotoxic than several smaller aggregates.
(A) Fraction of dead MDM after phagocytosing one large aggregate of Mtb (black line) or multiple small aggregates (red line) with a similar cumulative sum of Mtb to the single internalizations, but …
Figure 3
Mtb aggregate size determines timing of macrophage death.
(A) MDMs which internalized exactly one clump before the halfway point of the movie were ranked based on the amount of Mtb internalized (n = 72 cells). The blue circles are internalizations of …
Figure 4
Mtb induced macrophage death does not resemble apoptosis.
(A) Cell death was caused by internalization of Mtb clumps (top row, Mtb in red), the pyroptosis inducers Nigericin+LPS (middle row), or the apoptosis inducer cisplatin (bottom row). Time of cell …
Figure 5 with 1 supplement
Mtb grows robustly inside dead macrophages.
(A) In silico synchronized images of representative macrophages infected with Mtb and imaged before and after death. Each horizontal set of images represents the same macrophage over time, with the …
Figure 5—figure supplement 1
Differences in growth measured by CFU between Mtb in dead cells and extracellular Mtb.
(A) A homogeneous population of Mtb in dead cells was produced by infecting at a high multiplicity per cell (MOI = 30 according to CFU). (B) the same input dose of concentrated Mtb culture was …
Figure 6
Mtb growth in macrophages before and after macrophage death.
Median (circle with dot) and interquartile range (blue rectangle) of Mtb signal in live (A) and dead cells (B). Insets show exponential fits of Mtb dynamics in the individual live (n = 101) or dead …
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Figure 6—source data 1
pHrodo signal over time.
- https://doi.org/10.7554/eLife.22028.021
Figure 7
Mtb grows in IFNγ treated MDM and alveolar macrophages after cell death.
(A) MDM was exposed to IFNγ for 18 hr, then infected with Mtb and imaged (raw data provided in Figure 7—source data 1). Shown is the median fold change (circle with dot) and interquartile range …
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Figure 7—source data 1
Intracellular Mtb fluorescence through time in dead IFNγ treated MDM.
- https://doi.org/10.7554/eLife.22028.024
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Figure 7—source data 2
Intracellular Mtb fluorescence through time in dead alveolar macrophages.
- https://doi.org/10.7554/eLife.22028.025
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Figure 7—source data 3
Extracellular Mtb fluorescence through time.
- https://doi.org/10.7554/eLife.22028.026
Figure 8 with 1 supplement
Internalization of dead infected cells leads to rapid cell death.
Cells are ranked according to the time of last pickup, with cells ranked highest internalizing earliest. The frequency of cell death was 89%. The blue circles are internalizations of aggregates of …
Figure 8—figure supplement 1
Correlation of time of last pickup and time to death in the cells internalizing a dead infected cell.
R2 = 0.93 (p=8×10−23), with a median time to cell death of 3.2 hr (interquartile range 1.5 to 6 hr).
Figure 9 with 1 supplement
Positive feedback in Mtb infection.
(A) Schematic of the positive feedback loop. Mtb are in red, live cells are in grey and dead cells are outlined by dashed borders. Time is from left to right. Probabilities of death at time points …
Figure 9—figure supplement 1
Sensitivity of Mtb expansion to doubling time in dead cells.
The stochastic simulation described in Figure 9 was repeated for doubling times ranging from 1 to 3 days, with 10,000 iterations per doubling time value. The fraction of iterations where Mtb …
Author response image 1
Videos
Video 1
Macrophage internalization of Mtb aggregates.
Mtb infections of human macrophages were imaged by time-lapse microscopy at a resolution of 10 min between image acquisitions. Macrophage borders and locations were tracked using a custom-written, …
Video 2
Death of an Mtb-infected macrophage.
MDMs were infected with Mtb H37Rv-RFP (red) and imaged in the presence of the viability dye DRAQ7 (green). The entry of DRAQ7 corresponds to the time of cell death. Scale bar is 20 µm.
Video 3
Pyroptotic death of macrophages.
Macrophages were sensitized with 1 µg/ml LPS for 3 hr, then treated with 20 µM nigericin to induce pyroptosis. Cells were imaged in the presence of DRAQ7 (green).
Video 4
Cisplatin-Induced cell death.
Macrophages were treated with 50 µM cisplatin and imaged in the presence of DRAQ7 (green). Scale bar is 20 µm.
Video 5
Mtb grows inside a dead macrophage treated with IFNγ prior to infection.
The left panel of the montage is a movie of macrophage infection at a resolution of 10 min between image acquisitions. The border of the analyzed macrophage is shown as a green outline, and the …
Video 6
Loss of phagosome acidification upon Mtb induced host cell death.
Mtb were stained with the pH detection dye pHrodo at 100 µM and used to infect MDM in the presence of the cell death indicator dye DRAQ7. pHrodo fluorescence is shown in green, mCherry-expressing …
Video 7
Cell death cascade following phagocytosis of a dead infected macrophage.
The borders of four macrophages are shown as green, yellow, magenta, or blue outlines. RFP-expressing Mtb are shown in red. Time is hours:minutes. Scale bar is 20 µm.
Additional files
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Source Code 1
Source code 1- Image analysis script.
- https://doi.org/10.7554/eLife.22028.032
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Source Code 2
Bootstrap script used Figure 1—figure supplement 5B.
- https://doi.org/10.7554/eLife.22028.033
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Source Code 3
Figure 9B simulation.
- https://doi.org/10.7554/eLife.22028.034
