Fig. 1. Bacteria-derived c-di-AMP access the macrophage cytosol via SLO pores and activates STING directly.

a Schematic overview of the S. pyogenes enzymes involved in the synthesis (DacA) and degradation (GdpP and Pde2) of c-di-AMP. b Analysis of c-di-AMP concentration in culture supernatants (i.e. secreted c-di-AMP) from S. pyogenes M14 wild type (HSC5 WT) and deletion mutants, as indicated. c, d Gene expression analysis of ifnβ (c) and tnfα (d) in C57Bl/6 macrophages infected as indicated, at 4 h post infection (hpi). Uninfected (UI) macrophages were analyzed as control. e, f ELISA-based analysis of secreted IFNβ (e) and CXCL1 (f) from macrophages infected, as indicated, at 20 hpi. g ifnβ expression in transiently permeabilized macrophages (±10 µg/ml digitonin) treated with pure c-di-AMP (7.5 µM) or sterilized culture supernatant (culture filtrate; CF) from ΔgdpPΔpde2 bacteria, with or without phosphodiesterase I (±PD; 20 U/ml) pre-incubation. Untreated (UT) macrophages were analyzed as control. h Analysis of c-di-AMP secreted from S. pyogenes M3 WT and its isogenic SLO deletion mutant (Δslo). i Gene expression analysis of ifnβ in macrophages infected, or UI, as indicated, at 4 hpi. j, k ELISA-based analysis of secreted IFNβ (j) and TNFα (k) at 20 hpi, as indicated. l Western blot analysis, at the indicated hpi, of activated (i.e., phosphorylated; pSTAT1) and total amount of STAT1 in macrophages infected with M3 WT or Δslo. Shown is one experiment representative of two. Molecular size (kDa), as indicated. m ifnβ expression at 4 hpi in M3 WT or Δslo infected macrophages ± digitonin treatment. Results for gene expression analysis (mean and SD; n = 3), secreted c-di-AMP (mean and SD; n = 3), and ELISA analysis (mean and SD; n = 3 in f and n = 4 in e, j, k) representative of at least three independent experiments. One-way ANOVA with Dunnett’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.