Measuring Intracellular Mtb cAMP Levels

Intracellular Mtb cAMP levels were measured using the Direct cAMP Enzyme Immunoassay Kit according to the acetylated version of the manufacturer's protocol (Sigma‐Aldrich). Sample culture aliquots were recovered and resuspended to ~1x10⁸ CFU/ml in TBST, pH 6.50, centrifuged at 4500 × g for 10 min at 4°C, resuspended in 0.10 M HCl, and boiled for 10 min at 100°C (Kahramanoglou et al., 2014 (link)). Whole‐cell lysates were transferred to 1.50 ml screw‐cap microcentrifuge tubes (USA Scientific) filled with 200 μl 0.10 mm diameter zirconia glass beads (BioSpec Products) and exposed to three rounds of bead beating (2400 oscillations in 30 s), using the Mini‐BeadBeater‐1 (BioSpec Products), with cooling on ice for at least 2 min in between each round. Bacterial cell debris was removed via centrifugation at 12,500 × g for 15 min at 4°C, and clarified lysates were stored at −20°C until further use. Intracellular cAMP levels were measured by reading the optical density at 405 nm (OD₄₀₅) of 100 μl of immunoassay whole cell lysates using an EnSpire Multimode microplate reader (PerkinElmer). Intracellular cAMP levels were estimated from standard curves generated from reading the OD₄₀₅ of 0–20 pmol/ml of cAMP in 0.10 M HCl, and cAMP per 10⁸ CFU was calculated by dividing pmol cAMP/ml by CFU/ml, similarly to prior reporting (VanderVen et al., 2015 (link)).