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.

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^-1 in peak force tapping mode at a setpoint <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