A Streptococcus Quorum Sensing System Enables Suppression of Innate Immunity

Abstract

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-β) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses.IMPORTANCEStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

Keywords: NF-κB; TLR; biosynthetic gene cluster; cytokines; immunosuppression; innate immunity; macrophage; macrophages; pheromone.

FIG 1

Macrophage responses to GAS are attenuated when Rgg2/3 QS is active. (A) NF-κB responses after infecting RAW246.7 cells containing a chromosomally integrated NF-κB-inducible secreted embryonic alkaline phosphatase reporter (RAW-Blue cells) with wild-type (WT), Δrgg2 (QS-OFF), or Δrgg3 (QS-ON) GAS. All cells were infected at an multiplicity of infection (MOI) of 10 unless otherwise indicated. Reporter activity (absorbance at 625 nm) is shown. (B) TNF-α and IL-6 production by RAW264.7 cells 8 h after infection. (C) LDH release as a measurement of macrophage cell death 8 h after infection. The percentage of dead cells was quantified using a 100% lysed cell control. (D) NF-κB activity after infecting RAW-Blue cells for 20 h with different MOIs of Δrgg2 (black bars) or Δrgg3 (blue bars) GAS. (E) TNF-α production by RAW264.7 cells 8 h after infection with different serotypes of GAS grown in the presence of revSHP (black bars) or SHP (blue bars). (F) TNF-α production by BMDM, BMDC, or THP-1 cells 8 (BMDM, BMDC) or 12 (THP-1) h after infection with GAS (strain NZ131) grown in the presence of revSHP (black bars) or SHP (blue bars). Means plus standard deviations (SD) are shown from a representative of three independent experiments conducted in triplicate. Statistical significance is indicated as follows: ***, P = 0.001; ****, P < 0.0001 by two-tailed unpaired t test (A and B) or ordinary one-way ANOVA with Tukey’s multiple-comparison test (C to E); ns, not significantly different.

FIG 2

QS-ON GAS actively suppresses inflammatory responses. (A) NF-κB activity after infecting RAW-Blue cells with Δrgg2 or Δrgg3 GAS or both. (B) TNF-α, IL-6, and IFN-β production after RAW264.7 cells were infected with the described MOIs of Δrgg2 and Δrgg3 GAS. (C) TNF-α production by BMDM, BMDC, and THP-1 cells 8 (BMDM, BMDC) or 12 (THP-1) h after infection at an MOI of 10 of Δrgg2 GAS or coinfected with an MOI of 10 of each Δrgg2 and Δrgg3 GAS or Δrgg2 and WT GAS grown in the presence of SHP. (D) TNF-α production after RAW264.7 cells were coincubated with TLR agonists and Δrgg3 or Δrgg2 GAS. LPS, lipopolysaccharide (TLR4); HKLM, heat-killed Listeria monocytogenes (TLR2); ODN1826, CpG oligodeoxynucleotide (TLR9). Means plus SD are shown from a representative of two (D) or three (A to C) independent experiments conducted in triplicate. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple-comparison test (A to C) or two-way ANOVA with Sidak’s multiple-comparison test (D).

FIG 3

Suppression requires contact with live QS-ON GAS. (A) NF-κB activity after stimulating RAW-Blue cells with an MOI of 10 of Δrgg2 GAS and treating with 200 nM SHP or revSHP or an MOI of 10 of Δrgg3 GAS. (B) NF-κB activity after infecting RAW-Blue cells directly with the described strains or with Δrgg3 GAS separated by a transwell with 0.4-μm pores (TW). (C) NF-κB activity after infecting RAW-Blue cells with live Δrgg2 GAS and/or GAS that was inactivated with heat treatment (HK), short wave UV light treatment (UV), or 4% paraformaldehyde (PFA), or OD-normalized sterile filtered supernatants (Supe). Means plus SD are shown from a representative of three independent experiments conducted in triplicate. ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Sidak’s multiple-comparison test (A and B) by ordinary one-way ANOVA with Tukey’s multiple-comparison test (C).

FIG 4

A QS-regulated biosynthetic gene cluster (BGC) is required for cytokine suppression. (A) NF-κB activity of RAW-Blue cells infected with GAS isogenic mutants lacking various virulence factor genes and grown in the presence of SHP. Following 30 min of infection, macrophages were stimulated with LPS. (B) Diagram depicting the QS-regulated genetic programs in GAS (strain NZ131), putative functions of the genes, and the isogenic mutants used in subsequent assays. (C) NF-κB activity after RAW-Blue cells were infected with the described GAS genetic mutant strains grown in the presence of either inactive reverse SHP (revSHP) or active SHP. (D) NF-κB activity after RAW-Blue cells were coinfected with Δrgg2 GAS at an MOI of 10 of the described BGC mutants grown in the presence of SHP. (E and F) NF-κB activity after RAW-Blue cells were stimulated with Δrgg2 GAS and infected with BGC mutants and complementation strains. All GAS strains were grown in the presence of SHP to induce Rgg2/3 QS activity. Means plus SD are shown from a representative of three independent experiments conducted in triplicate. *, P < 0.05; ****, P < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple-comparison test (A and D to F) or two-way ANOVA with Sidak’s multiple-comparison test (panel C). ns, not significantly different.

FIG 5

Rgg2/3 QS can be targeted pharmacologically to restore immunity. TNF-α production after RAW264.7 cells were infected with GAS that was treated with the QS inhibitor valspodar (10 μM) or not treated with valspodar. Means plus SD are shown from a representative of three independent experiments conducted in triplicate. ****, P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test.