Quantification of c-di-AMP in cell extracts by ELISA.

The quantity of c-di-AMP in cell extracts was measured by competitive enzyme-linked immunosorbent assay (ELISA) as described previously (57, 58). S. pyogenes strains were grown to the exponential phase (OD₆₀₀ = ∼0.4) in 10 ml THY medium, washed three times with PBS, resuspended in 1 ml PBS, and lysed by PlyC treatment. The clear supernatant of the culture was collected in a fresh tube after centrifugation at 7,000 relative centrifugal force (rcf) at 4°C for 10 min. The cell lysates were then boiled for 10 min. Clear supernatants were collected after centrifugation and stored at −80°C until used to measure intracellular c-di-AMP concentration. Since E. coli does not produce c-di-AMP, E. coli TOP10 cells (Invitrogen) were used as a negative control for the assay. TOP10 cells were cultured to the exponential phase (OD₆₀₀ = ∼0.4) in 10 ml LB broth with continuous shaking at 37°C, washed three times with PBS, resuspended in 1 ml 50 mM Tris buffer (pH 8), lysed with beads using Fast Prep 24 5G (MP Biomedical) at 6.0 m/s for 40 s, and stored at −80°C until used to measure intracellular c-di-AMP concentration.

CabP protein was purified as previously described (57). Briefly, E. coli BL21(DE3) with the recombinant plasmid pET28b-His-CabPspT from the Grundling lab was grown to an OD₆₀₀ of ∼0.6 in 600 ml LB medium containing 30 μg/ml kanamycin at 37°C in a shaker. The culture was then incubated at room temperature for an additional 4 hours with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to induce CabP production. Next, cells were harvested, washed, and resuspended in 20 ml lysis buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 10 mM imidazole, and 10% glycerol). The cells were then lysed by bead beating using the Fast Prep 24 5G (6.0 m/s for 40 s twice). The beads and cell debris were removed by centrifugation at 13,000 rcf at 4°C for 20 min, and then 8-ml cell lysates containing His-tagged CabP proteins were loaded onto the column with 1 ml HisPur cobalt resin (Thermo Scientific) and incubated for 1 h. The resin was washed with 10 ml of lysis buffer containing 10 mM imidazole, 20 mM imidazole, and finally 30 mM imidazole, successively. Subsequently, the proteins were eluted in 4 ml lysis buffer containing 250 mM imidazole. The eluted proteins were then dialyzed twice against 1 liter of PBS at 4°C for 1 h and then overnight against 1 liter of PBS containing 10% glycerol. The proteins were concentrated using a Centriprep centrifugal filter unit (Sigma-Aldrich) at 2,000 × g at 4°C until they reached ∼0.2 mg/ml. The protein concentration was measured with the Bradford assay kit (Sigma-Aldrich). Finally, the purity of the protein was determined by SDS-PAGE, and the protein samples were stored in small aliquots at −80°C until used.

The purified CabP was diluted to 50 μg/ml in coating buffer (50 mM Na₂CO_3, 50 mM NaHCO₃, pH 9.6), and 100 μl of the solution was added to each well to coat the wells of a 96-well flat-bottom plate (Dot Scientific Inc.). The coated plates were sealed with plastic wrap and incubated overnight (14 to 18 h) at 4°C. The coated wells were then washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 5% bovine serum albumin (BSA). The cell extract samples were diluted (5 times) with 50 mM Tris buffer (pH 8). For preparing a standard curve, c-di-AMP (Biolog) solutions containing 0.5 nmol to 250 nmol were prepared in 50 mM Tris buffer (pH 8). Biotinylated c-di-AMP (Biolog) was added to all the controls and samples to give the final amount of 25 nmol. 100 μl of all the controls and samples were added to the coated wells (in triplicate). The plates were incubated for 2 h at room temperature. Each well of the plates was washed three times with 200 μl PBST. Next, 100 μl of 0.1-μg/ml high-performance streptavidin (Thermo Fisher Scientific) in PBS was added and incubated for 1 h. Wells were washed three times with PBST before adding substrate. One hundred microliters of the substrate (0.5 mg of o-phenylenediamine dihydrochloride [Sigma-Aldrich] in citrate buffer [pH 5] containing 20 μl H₂O₂) was added to each well and incubated for 30 min at room temperature. Finally, the reactions were stopped with 100 μl of 2 M H₂SO₄. The OD₄₉₂ was measured for each well using a plate reader (EL-808 Ultra microplate reader; Bio-Tek Instruments, Inc.). A standard curve was generated to measure the level of c-di-AMP. The concentrations of the samples were plotted along the x axis against OD₄₉₂ on the y axis, and a polynomial standard curve was generated to find the concentration of c-di-AMP in the samples.