DOI: 10.5061/dryad.r4xgxd2j8
title: Time-lapse videos and snapshot images of mycobacterial cells alone or during infection under drug treatment

description: Original approaches are needed to cope with drug-recalcitrant infections
 that threaten global health, including tuberculosis. Here, we implemented
 a drug-discovery strategy based on a customized microfluidic dynamic
 single-cell screening for pheno-tuning compounds (µDeSCRiPTor). Our
 strategy assesses dynamic phenotypic changes occurring at the level of
 single mycobacterial cells upon exposure to pheno-tuning compounds, via
 time-lapse microscopy. The goal was to identify compounds that synergize
 with existing anti-tubercular drugs, ultimately making the bacterial
 population more homogeneously susceptible to therapy. Among the four
 identified hits, we focused on M06, which shows promise for improving
 tuberculosis therapy.
description: <strong>Dynamic microfluidic screening videos (Dataset
 1).</strong> A <em>Mycobacterium smegmatis</em>
 fluorescent reporter of DNA damage response (RecA-GFP), also
 constitutively expressing a red-fluorescent marker
 (mCherry<sub>cyt</sub>) was analyzed by multi-phasic
 time-resolved microscopy using a customized multi-condition microfluidic
 platform. Cells were dispensed into the 32 microfluidic growth chambers
 and imaged at regular time intervals using a motorized inverted
 epifluorescence microscope, equipped with a 100X oil objective.
 Fluorescence imaging conditions were: GFP/FITC [Excitation (Ex) at 475/28
 nm, Emission (Em) at 525/48 nm] 50% transmittance (T), 150 msec; mCherry
 (Ex 575/25, Em 625/45), 50% T, 150 msec. Coordinates of the location of
 individual bacteria or small groups of bacteria, referred to as points,
 were recorded and monitored throughout the three phases of the experiment:
 exponential growth in fresh medium; exposure to subinhibitory
 concentrations of 72 test compounds or control conditions; and washing of
 cells in fresh medium. Raw image stacks were extracted for each channel
 and smoothed. Binary masks of whole microcolonies were created from
 red-channel images, using K-means clustering to identify the regions of
 interest (ROI) containing only cells. All objects smaller than a single
 cell, separated from the main ROI, or placed on the edge of the image were
 omitted from the final mask. A shape complexity index was also calculated
 for each ROI, and images with an index lower than 14 were excluded. This
 process was reiterated for each stack of images (movie). Finally, binary
 masks were visually examined and only 54% of them were retained for
 further ROI analysis and statistics.
 <strong>Snapshot images of <em>M.
 tuberculosis</em> expressing different NAT variants under stress
 (Dataset 2). </strong>Secondary cultures of <em>M.
 tuberculosis </em>H37Rv control (WT), NAT (rv3566c) mutant (MUT,
 Leu81<sub>del</sub>), NAT-overexpressing (OE) were cultured in
 Middlebrook 7H9 broth until OD<sub>600</sub> 0.5, in
 duplicate. Secondary cultures of M. tuberculosis NAT-silencing (SI) and
 related control strain (CTsg) were diluted to OD<sub>600</sub>
 0.05 and induced with anhydrotetracycline (100 ng/mL) for 3 days prior to
 stress exposure, in duplicate. Mid-log phase secondary cultures were
 treated for 24 h with DMSO (CT),
 H<sub>2</sub>O<sub>2</sub> (30 mM), or with
 different drugs at 10X-MIC concentration: M06 (12.5 µg/mL); moxifloxacin
 (MOX, 500 ng/mL); isoniazid (INH, 250 ng/mL); rifampicin (RIF, 800 ng/mL).
 Next, bacilli were stained with either CellROX (5 µM) or FM464 (5 µg/mL)
 dyes for 30 min. Bacteria were imaged by phase-contrast and fluorescence
 using an inverted DeltaVision Elite Microscope (Leica) equipped with a
 UPLFLN100XO2/PH3/1.30 objective (Olympus). All samples were prepared by
 dispensing 0.6 μL of bacterial suspension between two #1.5 coverslips,
 sealed with glue. Exposure conditions: phase-contrast 50% T (channel 2),
 100 msec; Cy5 (Ex 632/22, Em 679/34 – channel 1) 50% T, 100 msec for
 CellROX detection; TRITC (Ex 542/27, Em 597/45 – channel 1) 50% T, 100
 msec for FM464 detection. Individual bacilli were automatically segmented
 in phase contrast, using Omnipose for cell segmentation in combination
 with a custom-trained model. The latter was obtained from training on a
 dataset consisting of the ground truth dataset provided as part of
 Omnipose and additional manually labeled images of <em>M.
 tuberculosis</em>. A Python notebook was written to extract
 single-cell fluorescence and cell area from snapshot images using the
 Omnipose-derived segmentation masks. Inverted masks were used to extract
 background fluorescence, which was subtracted from the fluorescence of
 each cell. <strong>Time-lapse videos
 of <em>M. tuberculosis</em> RecA reporter stressed with INH
 and M06 (Dataset 3). </strong>Log-phase <em>M.
 tuberculosis</em> reporter of DNA damage response (RecA-mCherry),
 also constitutively expressing a green-fluorescent marker
 (GFP<sub>cyt</sub>) reporter (OD<sub>600nm</sub>
 0.5) was diluted ten times in fresh 7H9 medium in the absence of
 detergents and seeded into our microfluidic hexa-device. The microfluidic
 assembly was mounted on a DeltaVision Microscope at 37 °C. Growth medium
 in the absence or presence of M06 (1.25 µg/mL, 1X MIC) and/or INH (MIC:
 0.2 µ/mL, 8X MIC)<em> </em>was injected at 10-µl/min rate from
 a syringe pump, according to the different experimental phases. Images
 were acquired every 3 hours using a UPLFLN100XO2/PH3/1.30 objective
 (Olympus) and a high-speed sCMOS camera, 2,560 x 2,160 pixels, pixel size
 6.5 x 6.5 μm, 15-bit, spectral range of 370–1,100 nm. Exposure conditions
 were phase-contrast 100 % T (channel 2), 200 msec, FITC (Ex 492/28, Em
 523/23 – channel 1) 50% T, 50 msec; TRITC (Ex 556/25, Em 611/47 – channel
 3) 100% T, 250 msec. Individual cells were manually segmented at specific
 time points using ROI Manager Macro of ImageJ 2.0.0.-rc-59/1.51n or
 counted over time using the Cell Counter plugin. Total fluorescence was
 normalized to cell area and background fluorescence was also
 subtracted. <strong>Snapshot images of THP-1
 macrophages infected with <em>M. tuberculosis</em> RecA
 reporter and treated with M06 (Dataset 4). </strong>THP-1 monocytes
 (ATCC TIB-202) were cultured in RPMI 1640 (Gibco) supplemented with 10%
 FBS and 1X PS. Each stock of about 10<sup>6</sup> cells per mL
 in RPMI and 10% DMSO was defrosted at 37 °C, washed once in 10 mL, and
 inoculated in 30 mL of prewarmed complete RPMI using a T-175 flask. Cells
 were propagated at 37 °C in a humidified 5% CO<sub>2</sub>
 atmosphere until confluence, for up to four days. For final experiments,
 confluent cells were diluted to a concentration of
 10<sup>5</sup> cells/mL in complete RPMI without phenol red
 and without antibiotics and seeded into a 35-mm µ-Dish (ibidi) in the
 presence of PMA (30 ng/mL). After 36-hour incubation, cells were washed
 with fresh medium and re-incubated overnight before infection.
 Differentiated THP1 macrophages were infected with <em>M.
 tuberculosis</em> RecA-mCherry_GFP<sub>cyt</sub>
 reporter diluted in RPMI without phenol red to
 OD<sub>600</sub> 0.005 (MOI 1:5). After 3 hours of incubation
 at 37 °C in humidified 5% CO<sub>2</sub> atmosphere, cells
 were washed five times with complete RPMI without phenol red. Infected
 cells were either left untreated (CT) or were treated with M06 (M06, 1.25
 µg/mL, 1X MIC). Imaging was carried out at time zero (D0) and after 2 days
 (D2) of infection. Eight<em> </em>images were acquired for
 each field of view through a z-stack of 16 µm, starting the scan from the
 middle of the sample, using a 100X oil immersion objective, 1.4NA, WD
 0.12mm (Olympus). Exposure conditions on the Delta Vision microscope:
 bright field 10% T, 50 msec (channel 0); FITC (Ex 475/28, Em 525/48 –
 channel 1) 50% T, 25 msec; TRITC (Ex 542/27, Em 597/45 – channel 2) 100%
 T, 100 msec. Image stacks were projected by maximum intensity in SoftWorx,
 and processed on ImageJ, by segmenting intracellular bacterial foci based
 on their green fluorescence. Derived masks were used to measure the size
 and both fluorescence channels to assess RecA induction and control the
 fluorescence of intracellular bacilli. Inverted masks were used to
 subtract background fluorescence from each ROI. The customized ImageJ
 macro used for analysis is also provided
 (Macro_Analysis_THP1_MTUB.ijm).
description: Open-source image analysis software, such as ImageJ, can be used
 to open both .tif and .dv files.
description: # **Time-lapse videos and snapshot images of mycobacterial cells alone or
 during infection under drug treatment** This dataset includes four
 distinct datasets as described below. ## Dataset 1: Dynamic microfluidic
 screening videos The two directories contain 15 folders, one for each
 experiment defined by a unique alphanumeric code. Each folder contains 3
 associated .tif files per each experimental point: one image stack for the
 green channel (GFP); one image stack for the red channel (mCherry); and
 one image stack for the binary mask (Mask). Each file is named as follows:
 **Experiment ID_Point_nnn_Condition_File type**. Only those points that
 were used for analysis are included in the directory. **Dataset 1** is
 associated with **Figure 1** of the paper. Experiment N, Experiment ID,
 Image acquisition frequency (min), Exposure start-frame, Exposure
 end-frame 1 S1R1 30 14 24 2 S1R2 30 13 24 3 S2R1 30 10 21 4 S2R2 20 10 27
 5 S3R1 30 14 24 6 S3R2 30 13 23 7 S4R1 20 6 22 8 S4R2 30 13 24 9 SCR1 30
 13 24 10 SCR4 30 10 21 11 SCR5 30 10 21 12 SCR6 30 10 21 13 SCR7 30 9 20
 14 SCR9 30 13 25 15 SCR10 30 11 23 ## Dataset 2: Snapshot images of *M.
 tuberculosis* expressing different NAT variants under stress The directory
 contains individual .tif files with two channels (C): either CellROX or
 FM464 fluorescence (C1) and phase contrast (C2). The ID name of each file
 contains the experimental information necessary to identify the content
 type: **Dye_Strain_Condition_Point_Replica**. **Dataset 2** is associated
 with **Figure 4** of the paper. **Dye**: CellROX or FM464 fluorescent
 dyes. **Strain**: *M. tuberculosis* WT; MUT (NAT L81del mutant); OE (NAT
 overexpressing); SI (NAT silenced); CTsg (strain containing control
 plasmid for SI). **Condition**: CT (DMSO); H2O2 (30 mM); M06 12.5 µg/mL
 (10X MIC); MOX 0.5 µg/mL (10X MIC); INH 0.25 µg/mL (10X MIC); RIF 0.8
 µg/mL (10X MIC). **Point**: analyzed point (Pn). **Replica**: Two
 biologically independent cultures were processed separately, and pooled in
 equal amounts just before snapshot image acquisition. ## Dataset 3:
 Time-lapse videos of *M. tuberculosis* RecA reporter stressed with INH and
 M06 The directory contains individual .tif movie files, which were
 drift-corrected and manually analyzed on ImageJ. Each image stack is
 composed of three channels (C): GFPcyt (C1); phase contrast (C2);
 RecA-mCherry (C3). The ID name of each file contains the experimental
 information necessary to identify the content type:
 **Strain_Drug_Point_Replica**. **Dataset 3** is associated with **Figure
 5** of the paper. **Strain**: *M. tuberculosis* (Mtub) dual fluorescent
 reporter RecA-mCherry_GFPcyt (GMT37). **Drug**: either isoniazid alone
 (INH), or M06 alone followed by M06 in combination with INH (M06_INH).
 Bacilli were imaged every 3 hours under the following conditions. **For
 INH movies:** Start frame, End frame, Condition 1  17    Pre-growth in 7H9
 medium only 18 37    INH 0.2 µg/mL (8X MIC) 38 89 Washing in 7H9 medium
 only **For M06_INH movies:** Start frame, End frame, Condition 1 19
 Pre-growth in 7H9 medium only  20 35   M06 1.25 µg/mL (1X MIC) 36 55   M06
 1.25 µg/mL (1X MIC) + INH 0.2 µg/mL (8X MIC) 56 113 Washing in 7H9 medium
 only **Point**: analyzed movie point (Mn). **Replica**: Three biologically
 independent cultures were loaded on different areas of the microfluidic
 device, imaged over time, and analyzed. ## Dataset 4: Snapshot images of
 THP-1 macrophages infected with *M. tuberculosis* RecA reporter and
 treated with M06 The directory contains individual .dv files, which can be
 opened with open-source software like ImageJ, and the ImageJ macro
 generated for analysis (**Macro_Analysis_THP1_MTUB.ijm**). Each .dv file
 derives from maximum-intensity projection (PRJ) of eight z-stacks 2-µm
 spaced. PRJ .dv files are composed of three channels (C): bright field
 (C0); GFPcyt (C1); RecA-mCherry (C2). The ID name of each file contains
 the experimental information necessary to identify the content type:
 **Replica_Condition_Day_Cell_Strain_Point_Processing**. **Dataset 4** is
 associated with **Figure 5** of the paper. **Replica**: four biological
 replicates were analyzed. **Condition**: either untreated control (CT) or
 treated with M06 1.25 µg/mL (1X MIC). **Day**: days post infection, i.e.,
 D0 and D2. **Cell**: infected cell type THP-1 macrophages. **Strain**: *M.
 tuberculosis* (Mtub) dual fluorescent reporter RecA-mCherry_GFPcyt
 (GMT37). **Point**: analyzed point (Pn). **Processing**: projected stacks
 (PRJ). ## Sharing/access Information The dataset is associated with a
 preprint, which has just been accepted for publication: Mistretta M,
 Cimino M, Campagne P, Volant S, Kornobis E, Hebert O, Rochais C,
 Dallemagne P, Lecoutey C, Tisnerat C, Lepailleur A, Ayotte Y, LaPlante SR,
 Gangneux N, Záhorszká M, Korduláková J, Vichier-Guerre S, Bonhomme F,
 Pokorny L, Albert M, Tinevez J-Y, Manina G.  Dynamic microfluidic
 single-cell screening identifies pheno-tuning compounds to potentiate
 tuberculosis therapy. bioRxiv 2023.03.31.535085.