Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress (2019)
Figure 6

Figure 6 - RecA pulses and growth rate influence M. tuberculosis fate during ciprofloxacin exposure.

A Representative time-lapse image series of the dual reporter of RecA and rRNA expression in exponential phase. Pre-CIP growing (PG-S) and arrested (PA-S) survivors and resistant (R) cells are indicated by arrows. CIP exposure (1 µg/ml, 4X-MIC) and SX (0.5 µM) death assay (blue line) are indicated. Phase-contrast (gray), Prrn-GFPdes (green), and RecA-mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Cells were imaged at 3-hour intervals and numbers indicate days. Scale bar, 5 µm. See also Movie EV6.

B Representative time traces of cell lineages with different fates. Vertical bars represent fresh 7H9 medium (white), CIP (yellow), and SX (blue).

C-G Comparison of growth rate (C), cell size averaged over the lifetime of the cell (D), microcolony size (E) before CIP exposure, fluorescence range over the lifetime of the cell before CIP exposure (F), and mean fluorescence during CIP exposure (G) of cells that died (n = 174) and cells that survived (n = 164). Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.

H Single-cell fate as a function of VMR of RecA-mCherrywt fluorescence prior to CIP exposure. Significance by Chi-square test of independence: cells that died (n = 174), cells that survived (n = 155), and resistant cells (n = 9). The data shown are from 2 independent experiments. More survivors arise from the subpopulation that does not experience DNA damage before drug exposure.

I The intact architecture of the recA locus is required for pulsatile expression, which decreases in the absence of positive regulation and is abrogated in the absence of negative regulation. The presence of DNA damage is the main factor triggering RecA pulses. Schematic of M. tuberculosis reporters of DNA damage (magenta) and rRNA expression (green) during CIP treatment. In this study we classified three subpopulations of bacilli under optimal growth conditions, with no, moderate, or high DNA damage response (magenta gradient scale). The rRNA reporter helped us to visualize the metabolic state of the bacilli and was crucial for early detection of survivors. Following the first drug exposure, 62% of DNA-healthy cells survived, whereas 64% of cells with moderate and 82% of cells with high DNA damage died, suggesting that pre-existing DNA damage in cells treated with ciprofloxacin is detrimental. During the recovery phase, all survivors resumed metabolic activity first, and then underwent multiple divisions until they formed a new microcolony. At the time of second drug exposure most survivors died, indicating that survival of the first drug exposure was not due to drug resistance. However, 0.3% of cells that survived both the first and second drug exposure exhibited drug resistance. In sum, the random state of DNA damage experienced by M. tuberculosis before exposure to a drug targeting DNA replication has consequences for the onset of drug tolerance and the ultimate efficacy of treatment.

Expanded View Movie legends

Expanded View movie 1 - Time-lapse microscopy of M. smegmatis P2P1recA-GFPwt and P2P1recA-GFPdes reporters from exponential phase cultures.

Representative movies of exponential-phase bacteria grown inside the hexa-device and continuously perfused with 7H9 medium for 24 hours. Images were acquired every 15 minutes. Phase contrast and green fluorescence are merged. Time elapsed is indicated in hours. Scale bar, 10 μm.

Expanded View movie 2 - Time-lapse microscopy of M. smegmatis P2P1recA-GFPdes, P2P1recA-GFPdes_∆ClgR, P2wt-mCherrydes-recA, and P2mut-mCherrydes-recA reporters exposed to MMC.

Representative movies of exponential-phase bacteria grown inside the hexa-device under constant perfusion of 7H9 medium for 18 hours, stressed with 0.5 µg/ml MMC for 2 hours, then returned to normal (MMC-free) conditions. Images were acquired every 15 minutes. Phase-contrast images and green or red fluorescence images are merged. Fluorescence images of each channel are scaled to the brightest frame. Time elapsed is indicated in hours. Scale bar, 10 μm.

Expanded View movie 3 - Time-lapse microscopy of M. smegmatis P2P1recA-GFPdes_attB::P2P1recA-linker-mCherrywt, P2P1recA-GFPdes_PruvC-mCherrydes, P2P1recA-GFPdes_PradA-mCherrydes, P2P1recA-GFPdes_attB::PCt-mCherrydes, and attB::P2P1recA-linker-mCherrywt_attT::PssbA-linker-mCitrinewt reporters exposed to MMC.

Representative movies of exponential-phase bacteria grown inside the hexa-device under constant perfusion of 7H9 medium for 18 hours, stressed with 0.5 µg/ml MMC for 3 hours, then returned to normal (MMC-free) conditions. Images were acquired every 15 minutes. Phase contrast, green or yellow fluorescence, and red fluorescence images are merged. Fluorescence images of each channel are scaled to the brightest frame. Time elapsed is indicated in hours. Scale bar, 10 μm.

Expanded View movie 4 - Time-lapse microscopy of M. smegmatis P2P1recA-GFPdes_attB::P2P1recA-linker-mCherrywt reporter cells cultured with fresh or spent medium and treated with ciprofloxacin.

Representative movie of bacteria, from exponential-phase batch cultures, cultured inside the hexa-device under a constant flow of fresh 7H9 medium for 12 hours (A). CIP (1 µg/ml, 4-fold MIC) was added to the fresh flow medium for 6 hours then washed out with fresh flow medium (no CIP) for 24 hours. Cells were stained with SX 0.5 µM for 30 minutes then exposed to fresh flow medium containing 1 µg/ml CIP for 15 hours.

Representative movie of bacteria, from stationary-phase batch cultures, cultured inside the hexa-device under a constant flow of spent 7H9 medium for 20 hours (B). CIP (1 µg/ml, 4-fold MIC) was added to the spent flow medium for 6 hours then washed out with fresh flow medium (no CIP) for 12 hours. Cells were stained with SX 0.5 µM for 30 minutes, then exposed to fresh flow medium containing 1 µg/ml CIP for 18 hours.

Images were acquired every 30 minutes. Phase contrast, green fluorescence, and red fluorescence images are merged. Fluorescence images of each channel are scaled to the brightest frame. Time elapsed is indicated in hours. Scale bar, 10 μm.

Expanded View movie 5 - Time-lapse microscopy of M. tuberculosis Prrn-GFPdes_attB::P2P1recA-linker-mCherrywt reporter from exponential phase. Representative movies of exponential-phase bacteria grown inside the hexa-device and continuously perfused with 7H9. Images were acquired every 3 hours. Phase contrast, green fluorescence, and red fluorescence images are merged. Time elapsed is indicated in hours. Scale bar, 5 μm.

Expanded View movie 6 - Time-lapse microscopy of exponential-phase M. tuberculosis Prrn-GFPdes_attB::P2P1recA-linker-mCherrywt reporter treated with ciprofloxacin.

Representative movies of exponential-phase bacteria grown inside the hexa-device under a constant flow of 7H9 medium for 4 days. CIP (1 µg/ml, 4-fold MIC) was added to the flow medium for 2 days then washed out for 7 days. Cells were stained with SX 0.5 µM for 1 hour then exposed again to 1 µg/ml CIP for 5 days. Images were acquired every 3 hours. Phase contrast, green fluorescence, and red fluorescence images are merged. Fluorescence images of each channel are scaled to the brightest frame. Time elapsed is indicated in hours. Scale bar, 5 μm.