Journal: bioRxiv

Article Title: A genetically encoded biosensor reveals heterogenous cAMP dynamics coordinating growth and resuscitation in Mycobacterium tuberculosis

doi: 10.64898/2026.06.02.729633

Figure Lengend Snippet: ( A ) Ratiometric rmgCarvi fluorescence of Mtb-rmgCarvi epi (Erdman) cultured in 7H9+OADC or resuspended in DMEM cell culture-based media or immediately upon infection of RAW264.7 macrophages, were measured using a plate reader. cAMP content increases slightly in cell culture media but is significantly increased upon infection (N=4; biological replicates color-coded). Symbols represent means ± SD. One-way ANOVA with Holm-Sidak’s correction ** - p < 0.01, *** - p < 0.001. ( B ) rmgCarvi fluorescence dynamics in Mtb-rmgCarvi epi during the first 24 hours of infection of RAW264.7 macrophages, with or without IFN-ψ activation, or in macrophages where the phagosomal pH was deacidified using 10 mM ammonium chloride (NH 4 Cl), measured by flow cytometry. Intra-mycobacterial cAMP levels drop within 3 hours and is maintained stably over the next 21 hours. cAMP induction upon infection was transient, as indicated by cpGFP and mCherry fluorescence measurements using flow cytometry (N=2-8, 10000 cells per replicate per time point). ( C, D ) Ratiometric rmgCarvi fluorescence values in intracellular Mtb-rmgCarvi epi bacteria upon infection of murine RAW264.7 ( C ) or human U937 ( D ) macrophages, captured by fluorescence microscopy and quantified using FIJI (N=3, >300 events per replicate per time point). Kruskal-Wallis test with Dunn’s correction **** - p < 0.0001. ( E ) Mtb-rmgCarvi epi , cultured in 96-well plates, were exposed to a library of ∼240 compounds over 2 days. Correlation plot showing the rmgCarvi fluorescence ratios after 1 h vs 24 h exposure. The dotted black horizontal and vertical lines represent the medians at the corresponding time points. The colored lines indicate + 3 median absolute deviation values. ( F ) Heat-map showing the validation of the activity of some of the chosen compounds from the screen. Compounds 3 and 6 were identified as putative cAMP agonists, similar to GSK286 and Rifampicin (RIF). Compound 9 was included as a negative control and GlutaMAX represents the media control. The time points are indicated on the x-axis , including the fluorescence prior to exposure to compounds (Pre). All data shown are mean of at least three biological replicates. Biological replicates in panels ( C, D ) are color coded.

Article Snippet: Propagation and infection of RAW264.7 (ATCC: TIB-71) and U937 cells (ATCC: CRL-1593.2) was done as previously described, with some modifications ( ).

Techniques: Fluorescence, Cell Culture, Infection, Activation Assay, Flow Cytometry, Stable Transfection, Bacteria, Microscopy, Biomarker Discovery, Activity Assay, Negative Control, Control