Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors

doi: 10.3389/fcimb.2022.981827

Figure Lengend Snippet: Strains and plasmids utilized in this study.

Article Snippet: pST5552 , Bacterial Expression vector, Kan R GFP under control of the theophylline inducible riboswitch , Addgene, USA (Plasmid #36255); .

Techniques: Construct, Control, Expressing, Plasmid Preparation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors

doi: 10.3389/fcimb.2022.981827

Figure Lengend Snippet: Flow cytometry gating strategy. (A) A primary gate was applied to the bacterial population according to forward scatter (FSC) and side scatter (SSC) properties. For cell enumeration, a secondary gate was applied to the non-fluorescent bead population. (B) M. tuberculosis Δ leuD Δ panCD ::pTiGc was cultured in the presence of 4 mM theophylline to allow induction of the riboswitch-based promoter for expression of TurboFP635. Constitutive expression of GFP is observed, whilst fluorescence of TurboFP635 is observed following induction with theophylline. (C) Selecting on the bacterial population, a rectangle gate was created to select for live cells, according to their GFP positivity. Single colour controls ensured optimal voltage settings for positive fluorescence of GFP (pST5552) and TurboFP635 (pSTCHARGE), above that of the autofluorescence of unstained cells. (D) The fluorescence dilution technique allows monitoring of bacterial replication for 5 generations. To improve detection of the fluorescent signal to allow measurement over 5 days, theophylline, was retained in culture for 24 hours. Bacterial replication could thus effectively be monitored from day 2 onwards since the fluorescent signal remained stable in vitro and intracellularly between day 0 and day 1. (E) Following harvesting of intracellular bacteria, cells were stained with CV-AM for analyses of metabolic esterase activity. CV-AM is a non-polar, cell permeable fluorogenic probe that is rapidly hydrolysed to a polar, fluorescent compound by intracellular esterases of live cells. Dead cells no longer possess esterase activity, and will thus not convert to the fluorescent calcein, whilst calcein is stably retained in live cells. (F) Selecting on the live cells population, dilution of the TurboFP635 fluorescent signal provided an indication of bacterial replication following removal of the inducer, theophylline (Theo). The high red gate was created based on maximum TurboFP635 fluorescence observed at D0 using the range tool and used to detect mycobacteria that retain their TurboFP635 fluorescence from later time points (D3 and D5), representing slow or non-growing bacteria. The low TurboFP635 gate was created to distinguish replicating intracellular bacteria, as visually assessed when overlayed with in vitro day 3 or day 5 bacteria. (G) Selecting on the high and low TurboFP635 subpopulations, the esterase activity of intracellular bacteria was assessed by overlaying on a histogram plot. (H) Fluorescence dilution of the TurboFP635 signal over time. The geometric median fluorescent intensity (MFI) of TurboFP635 enabled determination of the number of bacterial generations during infection. CV-AM, calcein violet AM.

Article Snippet: pST5552 , Bacterial Expression vector, Kan R GFP under control of the theophylline inducible riboswitch , Addgene, USA (Plasmid #36255); .

Techniques: Flow Cytometry, Cell Culture, Expressing, Fluorescence, In Vitro, Bacteria, Staining, Activity Assay, Stable Transfection, Infection