Extended Data Figure 5 |. Bacterial STING activation of TIR NADase activity.

a, HPLC analysis of chemical standards separated with an ammonium acetate:methanol gradient elution used to analyze bacterial TIR-STING activity (see Methods). The NAD⁺ and ADPr peaks have overlapping bases under these conditions. cADPr, cyclic adenosine diphosphate-ribose; ADPr, adenosine diphosphate-ribose; NAD⁺, β-nicotinamide adenine dinucleotide; NAM, nicotinamide.

b, HPLC analysis of SfSTING NAD⁺ cleavage activity with gradient elution. SfSTING at 500 nM protein with 2 μM c-di-GMP converts 500 μM NAD⁺ into ADPr and NAM in 30 min at ambient temperature. SfSTING does not generate any cyclized product and is therefore a standard glycosyl hydrolase. Inset: Schematic of NAD⁺ cleavage reaction.

c, HPLC analysis of chemical standards separated with an alternative isocratic elution strategy (see Methods) that results in clearer separation of NAD⁺ and ADPr peaks.

d, HPLC analysis of SfSTING NAD⁺ cleavage activity with isocratic elution. SfSTING NAD⁺ cleavage activity requires specific activation with c-di-GMP (30 min reactions).

e, HPLC analysis of SfSTING NAD⁺ cleavage activity and cyclic dinucleotide agonist specificity. Each reaction was tested with 500 nM SfSTING, 500 μM cyclic dinucleotide, and 500 μM NAD⁺ and sampled at 45, 90 or 180 min (gradient coloring in bars). SfSTING preferentially responds to c-di-GMP, but 3′,3′-cGAMP and c-di-AMP can function as weak agonists. Data are representative of 3 independent experiments.

f, HPLC analysis of SfSTING NAD⁺ cleavage activity in the presence of 3′,3′-c-UMP–AMP. 3′,3′-c-UMP–AMP is a >1000× weaker agonist than c-di-GMP. Data are representative of 3 independent experiments.

g, HPLC analysis of SfSTING NAD⁺ cleavage activity in the presence of a synthetic c-di-GMP analog with a noncanonical 2′–5′ linkage (2′,3′-c-di-GMP). 2′,3′-c-di-GMP is not capable of stimulating robust SfSTING activation even at very high concentrations (250 μM vs. 250 nM canonical c-di-GMP), confirming the specificity of bacterial STING for 3′–5′-linked cyclic dinucleotides. Data are representative of 3 independent experiments.

h, Plate reader analysis of SfSTING NAD⁺ cleavage activity using the fluorescent substrate ε-NAD. ε-NAD increases in fluorescence intensity after cleavage. SfSTING exhibits rapid catalysis with complete turnover at 500 nM protein with 500 nM c-di-GMP after 10 min at 25°C. No background activity is observed in the absence of ligand. Data are representative of 3 independent experiments.

i, Plate reader analysis of SfSTING NAD⁺ cleavage activity in the presence of 500 nM protein with increasing c-di-GMP concentration reveals that low nM c-di-GMP levels are sufficient to induce ε-NAD cleavage. Greater c-di-GMP concentrations are required for maximal activity consistent with binding data and the higher amount of c-di-GMP required for complete stabilization of the SfSTING–c-di-GMP complex (See Extended Data Figure 4e–h). Saturation occurs above 100 nM c-di-GMP with 40 min reactions. Data are ± s.d. of n = 3 technical replicates and are representative of 3 independent experiments.

j,k, TIR-domain NAD⁺ cleavage activity requires protein oligomerization. For other systems^21,22, TIR activation has been observed at very high in vitro protein concentrations or in the presence of affinity resins as an artificial oligomerization-inducing matrix^21,22. We used a GST-TIR construct to express the SfSTING TIR domain in absence of the STING cyclic dinucleotide binding domain and observed that even at >200× the concentrations for which the full-length protein shows c-di-GMP induced activity, or in the presence of multivalent affinity resin, no NAD⁺ cleavage activity occurs. These results demonstrate NAD⁺ cleavage activity specifically requires STING cyclic dinucleotide recognition for activation. Data are representative of 2 independent experiments.