DOI: 10.5061/dryad.cvdncjt90
title: Data from: Mechanical morphotype switching as an adaptive response in Mycobacteria

description: Invading microbes face a myriad of cidal mechanisms of phagocytes that
 inflict physical damage to microbial structures. How intracellular
 bacterial pathogens adapt to these stresses is not fully understood. Here,
 we report a new virulence mechanism by which changes to the mechanical
 stiffness of the mycobacterial cell surface confers refraction to killing
 during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to
 reveal a process of “mechanical morphotype switching” in mycobacteria
 exposed to host intracellular stress. A “soft” mechanical morphotype
 switch enhances tolerance to intracellular macrophage stress, including
 cathelicidin. Both pharmacologic treatment, with bedaquiline, and a
 genetic mutant lacking uvrA modified the basal mechanical state of
 mycobacteria into a “soft” mechanical morphotype, enhancing survival in
 macrophages. Our study proposes microbial cell mechanical adaptation as a
 critical axis for surviving host-mediated stressors.
description: AFM image datasets of Height,
 DMT modulus (stiffness), and Peak Force Error channels for M. smegmatis
 imaged in axenic conditions of growth and stress
 AFM
 imaging Coverslips were prepared as previously described
 (3). Polydimethylsiloxane (PDMS) (Sylgard 184, Dow
 Corning) at a ratio of 15:1 (elastomer:curing agent) and cut 1:10 with
 Hexane to reduce 10-fold the spin-coated layer, while equally increasing
 the hydrophobicity of the surface. Aliquots of mycobacteria isolated from
 axenic culture or from infection of macrophages were filtered through a
 0.5 µm pore size PVDF filter (Millipore) to remove cell clumps and enrich
 single cells. Aliquots were deposited on the hydrophobic surface of a
 PDMS-coated coverslip. 7H9 growth medium was supplied. Where indicated,
 antibiotic was added to the growth medium. The medium was maintained at
 37°C using a custom-made heating element within the sample space and a
 TC2-80-150 temperature controller (Bioscience tools). Bacteria were imaged
 by peak force tapping using a Nanoscope 5 controller (Veeco Metrology) at
 a scan rate of 0.25 – 0.5 Hz and a maximum Z-range of 5 µm. A ScanAsyst
 fluid cantilever (Bruker) was used. Continuous scanning provided snapshots
 at 2–30 min intervals. Height, peak force error, adhesion, dissipation,
 deformation modulus and log modulus were recorded for all scanned images.
 Peak force error yields a fine representation of the height on the order
 of 10 nm in the Z-axis; this is computed as the difference between the
 peak force setpoint and the actual value. Images were processed using
 Gwyddion (Department of Nanometrology, Czech Metrology Institute). ImageJ
 was used for extracting bacterial cell surface height and modulus values
 and generating dynamic and quantitative mean values of the mechanical
 properties of individual cells. Correlated fluorescence and
 AFM Correlated fluorescence and AFM images were
 acquired as described previously. Briefly, fluorescence images were acquired
 with an electron-multiplying-charge-coupled device (EMCCD) iXon Ultra 897
 camera (Andor) mounted on an IX71 inverted optical microscope (Olympus)
 equipped with a UAPON100XOTIRF x100 oil immersion objective (Olympus) with
 the x2 magnifier in place. Illumination was provided by a monolithic laser
 combiner (MLC) (Agilent) using the 488 or 561 nm laser output coupled to
 an optical fibre with appropriate filter sets: F36-526 for Calcein-AM and
 F71-866 for mCherry-Wag31 or cytosolic RFP. The AFM was mounted on top of
 the inverted microscope and images were acquired with a customised Icon
 scan head (Bruker) using ScanAsyst fluid cantilevers (Bruker) with a
 nominal spring constant of 0.7 N m<sup>-1</sup> in peak force
 tapping mode at a setpoint &lt;2 nN and typical scan rates of 0.5 Hz.
 The samples were maintained at 37°C in 7H9 growth medium heated by a
 custom-made coverslip heating holder controlled by a TC2-80-150
 temperature controller (Bioscience tools).
   Analysis of TnSeq experimentation to
 identify conditionally essential Tn Insertion Mutants isolated from Low
 and High Buoyancy Fractions 
 <strong>Transposon library construction, genomic DNA
 Extraction, and sequencing.</strong> Transposon
 library after buoyancy centrifugation was collected and resuspended in
 400ul 10mM Tris (pH=9). After beads-beating, genomic DNA was extracted by
 Phenol-Chloroform method. DNA concentration was measured and quantified by
 Nanodrop and Qubit. For building transposon sequencing library,
 approximately 5ug genomic DNA was resuspended in 150ul TE buffer and
 transferred to a Covaris tube, Genomic DNA was disrupted to 200-500bp size
 range by sonication with the following parameters: duty cycle (10%),
 intensity (4), cycles/burst (200), time (80s). The fragmented genomic DNA
 was size-selected and purified by AMPure XP beads. The fragmented genomic
 DNA was further subjected to end repair and dA tailing. Annealed adapter
 was ligated to the dA tailed fragmented genomic DNA and the linked ligated
 DNA fragment was used as template of 1<sup>st</sup> round
 nested PCR to amplify fragments containing adaptor and transposon
 junction. Indexed barcoded sequencing and illumina sequencing adaptor was
 added by 2<sup>nd</sup> nested PCR. All sequence libraries
 were examined by Agilent 2100 Bioanalyzer and subjected to next generation
 sequencing.   
 <strong>Transposon mutagenesis.</strong>
 <em>Mycobacterium smegmatis</em>
 mc<sup>2</sup>155 strain was grown to stationary phase
 (OD&gt;6) in 50ml of 7H9 growth medium. Bacterial cultures were washed
 and resuspended in 5ml MP buffer (50mM Tris, 150mM NaCl, 10mM MgSO4, 2mM
 CaCl2). To transduce bacteria with MycoMarT7 phage, approximately
 10<sup>11</sup> plaque forming units of phage (PFU) was added
 to the bacterial suspension in MP buffer and incubated at 37°C for 4h.
 Immediately after transduction, ~300-400ul of the transduction mixture was
 plated on 15-cm LB agar plates, containing 20ug/ml kanamycin and 0.1%
 Tween80. After 3 days, library size was determined, and bacteria was
 scrapped and stored in 7h9 medium plus 15% glycerol as library stock. The
 transposon library was made in triplicate. The transposon library was
 cultured to an OD<sub>600nm</sub> of 0.8 and 1ml of sample was
 loaded onto 10ml of stock isotonic percoll medium, with buoyant density
 beads as fiducial markers. Buoyancy centrifugation was conducted at 18°C,
 and spinning at 20k rpm (~50,000 g), for 1h20m. Three buoyancy fractions
 were isolated: “high” (&gt;1.02 g cm<sup>-3</sup>,
 &lt;1.064 g cm<sup>-3</sup>), “middle” (&gt;1.064 g
 cm<sup>-3</sup>, &lt;1.102 g cm<sup>-3</sup>),
 and “low” (&gt;1.102 g cm<sup>-3</sup>). Three biological
 replicates of the buoyancy centrifugation were conducted for the
 transposon library made in triplicate each of the three transposon
 libraries: 3 (libraries) x 3 (buoyancy centrifugation experiments) x 3
 (buoyancy fractions) = 27 individual samples.
   <strong>Transposon mapping and
 analysis.</strong> Reads processing and TA loci
 mapping were performed through software TRANSIT (<em>31</em>).
 Loci that were differentially disrupted by transposon were analysed using
 resampling test in TRANSIT with default parameter. Different buoyancy
 fractions were compared with input libraries and genes that are
 over-represented (log2FC &lt; -1, adjusted p-value &lt;
 0.05) and under-represented (Log2FC &gt;1, p-value
 &lt; 0.05) were plotted.
description: # AFM Datasets Long-Term
 Time-Lapse Atomic Force Microscopy (LTTL-AFM) imaging datasets include
 images of *M. smegmatis* in axenic conditions of growth and stress. ##
 Description of the AFM data and file structure AFM images obtained using
 Bruker AFM systems are identified by a ".001" and can be opened,
 edited, and analysed using the open source software, Gwyddion (Department
 of Nanometrology, Czech Metrology Institute). Image files provided are in
 PNG format in folders labeled "AFM Images". Height images are
 described as "_height" in the filename while stiffness (DMT
 modulus) images are represented with "_dmt" or
 "_stiffness" and peak force error images with "_pfe".
 Cell dimensions of individuals segmented from AFM images are found in
 folders labeled "Cell Dimensions..." and in tabular format saved
 as ".csv" and ".xls" files. Individual cells are
 identified by the image number followed by a 1 (old pole daughter) or 2
 (new pole daughter). Dual-AFM and Optical Fluorescence datasets of *M.
 smegmatis* mutant ∆*3187* have AFM images in folders labeled as such
 ("AFM") while optical fluorescence images are in folders labeled
 "OFM". Optical fluorescence image files are ".tif".
 Text files ".docx" or ".rtf" exhibit information about
 the experimental conditions for which the imaging was conducted. Text
 files labeled ".txt" and entitled "Image_Time_"
 contain infromation about AFM image files and the time that they represent
 in a time-lapse experiment. AFM analysis was conducted using
 custom-written matlab scripts, which are labeled as ".m" files.
 **Directories** AFM data are partitioned into 2 folders &lt;2Gb in size
 and labeled as: "Mechanical Morphotype Switching LTTL-AFM
 Datasets" and "Mechanical Morphotype Switching LTTL-AFM Datasets
 2". The first directory contains exclusively AFM data while the
 second directory contains dual AFM and OFM data or AFM data, alone. **AFM
 dataset Folders and Pathes** AFM dataset folders are organized by the *M.
 smegmatis* (Ms) strains imaged. "WT mc2 155" refers to wildtype
 strain mc^2 155 originating from William Jacobs' lab at Albert
 Einstein School of Medicine. "delta lamA" refers to Ms∆*lamA*
 originating from Hesper Rego at Yale University School of Medicine.
 "delta 3187" refers to Ms∆3187 originating from Mary
 Jackson's lab at the University of Colorado at Fort Collins. Raw
 datasets are labeled in subfolders with the date when the data was taken.
 ex. "03-09-2014" (day-month-year) refers to September 3rd, 2014.
 Any subfolders are labeled for the data of the images represented in the
 folder (i.e. "Height" represents AFM height images and
 "Stiffness" represents the DMT modulus). ### Sharing/Access
 information # TnSeq
 Analysis Interpretation of Tnseq raw datasets are supplied in spreadsheet
 form that depicts comparisons of conditionally essential Tn insertion
 mutants enriched in discrete buoyancy fractions. ## Description of the
 TnSeq data and file structure A spreadsheet format depicts comparisons of
 conditionally essential Tn insertion mutants enriched in discrete buoyancy
 fractions. The data for two comparisons each are tabulated in two
 individual sheets. A high buoyancy fractionated sampe is compared to the
 middle fraction for conditional essentiality: "High vs Middle
 tri". A low buoyancy fractionated sample is equally compared to the
 middle fraction for conditional essentiality: "Low vs Middle
 tri". Column labels: 1. "#Orf" = open reading frame 2.
 "Name" = gene name (if any) 3. "Desc" = gene ontology
 4. "Sites" = Number of transposon insertion locations 5.
 "Mean Ctrl" = mean control 6. "Mean Exp" = mean
 exponential 7. "Log2FC" = Log 2 fold change 8. "Sum
 Ctrl" = sum control 9. "Sum Exp" = sum exponential 10.
 "Delta Mean" = change in the mean 11. "p-value" = P
 value 12. "Adj. p-value" = adjusted P value ### Sharing/Access
 information