Fig. 3. NADase acts intracellularly to inhibit ifnβ transcription via an ATP-depletion dependent mechanism.

a RTqPCR analysis of ifnβ expression in transiently permeabilized (10 µg/ml digitonin) C57Bl/6 macrophages treated with 0.5 µM purified enzymatically active (NADase) or inactive (G330D) recombinant NADase, or untreated (UT), in combination with c-di-AMP (7.5 µM), c-di-GMP (7.5 µM), dsDNA (2.5 ng/ml), pI:C (2.5 µg/ml) or UT, as indicated. Analysis was performed 2,5 h post treatment. b LPS (2.5 µg/ml)-induced ifnβ expression in permeabilized (+digitonin) or non-permeabilized (- digitonin) macrophages treated with enzymatically active NADase (0.5 µM), as indicated. ifnβ expression is presented as % of LPS alone. c Expression of ifnβ and tnfα in permeabilized macrophages treated with enzymatically active NADase (0.5 µM) in combination with c-di-AMP (7.5 µM), and with the addition of titrated amounts of ATP (0.1 mM and 1 mM), as indicated. d Kinetic analysis of cellular ATP concentration in transiently permeabilized (+digitonin) macrophages treated with NADase, G330D, or untreated (UT), as indicated. e Kinetic analysis of LDH-release (as a measure of cytotoxicity) in cells treated as in d above. All results (mean and SD; n = 3) are representative of three independent experiments. a–c One-way ANOVA with Dunnett’s test, d, e two-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Schematic representation of our results. SLO pores allow diffusion of S. pyogenes-derived c-di-AMP into the macrophage cytosol where it activates STING to drive ifnβ expression. However, simultaneous CMT of the enzymatically active NADase expressed by epidemic strains suppresses type I IFN production at the transcriptional level via a mechanism that is dependent on ATP depletion but unrelated to cell death.