A group A Streptococcus ADP-ribosyltransferase toxin stimulates a protective interleukin 1β-dependent macrophage immune response

Abstract

The M1T1 clone of group A Streptococcus (GAS) is associated with severe invasive infections, including necrotizing fasciitis and septicemia. During invasive M1T1 GAS disease, mutations in the covRS regulatory system led to upregulation of an ADP-ribosyltransferase, SpyA. Surprisingly, a GAS ΔspyA mutant was resistant to killing by macrophages and caused higher mortality with impaired bacterial clearance in a mouse intravenous challenge model. GAS expression of SpyA triggered macrophage cell death in association with caspase-1-dependent interleukin 1β (IL-1β) production, and differences between wild-type (WT) and ΔspyA GAS macrophage survival levels were lost in cells lacking caspase-1, NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), or pro-IL-1β. Similar in vitro findings were identified in macrophage studies performed with pseudomonal exotoxin A, another ADP-ribosylating toxin. Thus, SpyA triggers caspase-1-dependent inflammatory cell death in macrophages, revealing a toxin-triggered IL-1β-dependent innate immune response pathway critical in defense against invasive bacterial infection.

Importance: Group A Streptococcus (GAS) is a leading human pathogen capable of producing invasive infections even in healthy individuals. GAS bacteria produce a toxin called SpyA that modifies host proteins through a process called ADP ribosylation. We describe how macrophages, frontline defenders of the host innate immune system, respond to SpyA by undergoing a specialized form of cell death in which they are activated to release the proinflammatory cytokine molecule interleukin 1β (IL-1β). Release of IL-1β activates host immune cell clearance of GAS, as we demonstrated in tissue culture models of macrophage bacterial killing and in vivo mouse infectious-challenge experiments. Similar macrophage responses to a related toxin of Pseudomonas bacteria were also shown. Thus, macrophages recognize certain bacterial toxins to activate a protective immune response in the host.

FIG 1

SpyA-deficient GAS bacteria evade macrophage killing. To measure total and intracellular killing, BMDMs isolated from C57BL/6 mice were activated through overnight incubation in DMEM supplemented with 2% FBS. (A) Total killing was measured by recovering total CFU of cells infected with GAS (AP) at an MOI of ~10 after 2 and 4 h. (B) Intracellular killing was assessed by recovering CFU from cells infected with GAS for 30 min followed by 100 µg/ml of gentamicin treatment (1 h); levels of internalized bacteria were monitored over 2 and 4 h in serum-free media. (C) BMDMs were infected with S. aureus RN4220 at an MOI of ~5, and intracellular killing was monitored at 4 and 6 h. (D) Transfected J774 cells were infected with ΔspyA GAS for 2 h in 2% FBS media for a total killing assay. Data shown are representative of the results of multiple repeats. Error bar; SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant; n = 3 (Student’s two-tailed unpaired t test or one-way ANOVA and Tukey’s multiple-comparison test).

FIG 2

SpyA promotes cell death and caspase-3 activation in a manner independent of MAPK and NF-κB signaling. (A) LIVE/DEAD staining illustrating BMDM viability after 4 h of GAS (AP) infection. Cells were infected at an MOI of ~25 or remained uninfected (UI). Scale bar, 100 µM. (B) Numbers of dead cells were counted from multiple field of views (n = 12). (C) Relative levels of LDH release (percentages compared to uninfected [UI] cells). ΔspyA-infected BMDMs exhibit reduced 2-h cell cytotoxicity at an MOI of 10 or 25. Infection with the SpyA-complemented strain restored LDH release to the WT level (n = 4). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s two-tailed unpaired t test). (D to H) Western blot analysis of whole BMDM lysates after 2 h of GAS infection. β-Actin was used as a loading control. (D) Phospho-ERK1/2 (42 or 44 kDa). (E) Phospho-p-p65 (65 kDa). (F) Apoptosis-inducing factor (AIF; 58 or 68 kDa). (G) Cytochrome c (15 kDa). (H) Caspase-3 (FL, full length [35 kDa]; CLVD, cleaved [17 or 19 kDa]). (I) Capsase-3 and caspace-7 (Caspace-3/7) activity assay. Error bars, SEM. *, P < 0.05; n = 4 (Student’s two-tailed unpaired t test).

FIG 3

SpyA stimulates a caspase-1-dependent inflammatory response and IL-1β production in macrophages. (A) BMDMs were infected with GAS (AP) at an MOI of ~10. At the end of the assay, cells were washed and stained for 1 h with FAM YVAD-FMK to visualize activated caspase-1 (green) and for 5 min with Hoechst 33342 to visualize DNA (blue) under an epifluorescent microscope. Scale bar, 100 µm. (B) Quantification of FMK YVAD-FMK-positive cells (per 100 cells) from multiple random fields of view (n = 10) per sample. (C) Western blot illustrating the abundance of pro-caspase-1 and its p10 subunit in GAS-infected BMDM cell lysates and caspase-1 p10 subunit released in the supernatants (Sup). Data represent densitometry quantifications illustrating average levels of active caspase-1 p10 expression in lysates and supernatants relative to pro-caspase 1 expression. Error bars, SEM. *, P < 0.05 (n = 3). Experiments were performed in duplicate using BMDMs isolated from four different C57BL/6 mice. (D and E) Real-time qPCR (D) and ELISA (E) quantification of IL-1β and TNF-α transcripts and proteins produced by BMDMs infected with GAS 2 h after total killing (n > 3). (F) IL-1β released by BMDMs after 4 h infection with S. aureus RN4220 expressing pSpyA or control vector (n = 4). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s two-tailed unpaired t test). UI, uninfected.

FIG 4

GAS SpyA expression is associated with increased bacterial clearance and reduced mortality in mice. CD1 mice were infected with 2 × 10⁵ to 4 × 10⁵ CFU of WT, ΔspyA, or ΔspyA plus pSpyA GAS (AP) via tail-vein injection. (A) Survival kinetics of infected mice monitored over 20 days (WT strain n = 20, ΔspyA strain n = 21). *, P = 0.02. Statistical significance was evaluated using the log rank test with a 95% confidence interval. (B) Bacteria recovered from organs or blood of GAS-infected mice 48 h postinfection for CFU enumeration to assess bacterial burden (n > 12). (C) CFU levels recovered from heart and lung of mice infected with ΔspyA plus pSpyA GAS (48 h) are significantly lower than those from ΔspyA mutant-infected mice (n = 6). (D) ELISA of proinflammatory cytokines from mouse blood serum isolate 6 h and 48 h postinfection (n = 5 to 9). UI = uninfected control, WT = wild-type GAS. Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s two-tailed unpaired t test).

FIG 5

GAS SpyA activation of a caspase-1-dependent inflammasome response is required for bacterial clearance by cultured BMDMs and in vivo. (A) BMDMs were coincubated with GAS (AP) at an MOI of 20 plus 10 µM pan-caspase inhibitor zVAD-FMK (ZVAD) or 25 µM caspase-1 inhibitor Ac-YVAD-CHO (YVAD) for 2 h. Data shown are representative of the results of multiple experiments. (B) Relative levels of LDH release (percentages compared to levels in uninfected [UI] cells; left panel) and bacterial killing (right panel) by WT and Casp-1^−/− BMDMs 2 h postinfection. (C) Relative levels of LDH release (left panel) and bacterial killing (right panel) 2 h postinfection (MOI = 5) by BMDMs isolated from wild-type (C57bl6), Nlrp3^−/−, Asc^−/−, and IL-1β^−/− mice (n = 3). Data shown are representative of the results from two or more animals. (D) ELISA shows IL-1β secreted by BMDMs isolated from wild-type (C57bl6), Nlrp3^−/−, Asc^−/−, and IL-1β^−/− mice after 2 h coincubation with GAS at an MOI of 20. Data shown are representative of the results from two animals. (E) CD1 mice were infected with 7 × 10⁵ CFU of WT or ΔspyA GAS (AP) via tail-vein injection (n = 7 per group). Immediately after infection, anakinra (IL-1 receptor antagonist) was subcutaneously injected at 100 mg/kg over a 12-h interval until the end of survival curve. WT plus saline solution versus ΔspyA plus saline solution, *, P = 0.04; WT plus saline solution versus WT plus anakinra. **, P = 0.0036; WT plus anakinra versus ΔspyA plus anakinra, P = 0.06; ΔspyA plus saline solution versus ΔspyA plus anakinra, P = 0.07 (log rank test with 95% confidence interval); N.S., not significant (P > 0.05). (F) GAS-infected mice (n = 8 to 10) were sacrificed 2 days postinfection; heart, spleen, and liver were harvested for CFU enumeration; no statistically significant differences were observed between WT and ΔspyA-infected animals that received anakinra treatment or for ΔspyA-infected animals with saline solution treatment. Closed symbols = WT; open symbols = ΔspyA. Black = control mice, red = anakinra-treated mice. Error bars, SEM. Statistical analysis: *, P < 0.05; N.S., not significant (P > 0.05) (Student’s two-tailed unpaired t test). (G) BMDMs in RPMI plus 2% FBS were treated with 1 or 10 ng/ml of recombinant mouse IL-1β (PeproTech, Rocky Hill, NJ) at the time of infection. Cells were washed and CFU recovered 2 h postinfection to assess bacterial killing. Error bars, SEM. *, P < 0.05; **, P = 0.01; ***, P < 0.001; NS, not significant; n = 3 (Student’s unpaired two-tailed t test).

FIG 6

Pseudomonas aeruginosa ADP-ribosyltransferase exotoxin A triggers an inflammasome-dependent macrophage response to enhance bacterial killing. BMDMs were seeded in 24 wells in 2% FBS media a day before infection. (A) LDH released by BMDMs treated with 100 ng or 500 ng of P. aeruginosa exotoxin A (PEA) for 18 h. (B to E) RNA and supernatants from BMDMs were isolated for real-time qPCR and ELISA to assess transcript and protein levels of TNF-α and IL-1β in BMDMs treated with PEA. (F) LDH assay of supernatants harvested from BMDMs 2 h after P. aeruginosa infection (n = 4). (G and H) Total and intracellular killing of P. aeruginosa infected for 30 min at an MOI of ~20, followed by a 1-h streptomycin treatment (150 µg/ml). Cells were washed and incubated in serum-free media for 2 h to establish intracellular killing. (I) ELISA of IL-1β produced by P. aeruginosa-infected BMDMs 2 h postinfection. (J) BMDMs from Nlrp3^−/−, Asc^−/−, and IL-1β^−/− mice were isolated to assess intracellular killing of P. aeruginosa (2 h post-gentamicin treatment). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3 (Student’s unpaired t test or one-way ANOVA followed by Dunnett’s test).