S. pyogenes establishes subpopulation heterogeneity in their expression of speB..

(A) speB regulatory circuit schematic. RopB induces speB expression in the presence of SIP peptide and Vfr inhibits speB expression by blocking RopB-SIP complex. CovS kinase-mediated CovR phosphorylation (CovR∼P) derepresses speB, while CovS phosphatase-mediated CovR dephosphorylation represses speB. MgCl2 and LL-37 mediate CovS kinase and phosphatase, respectively. Induction of speB expression was evaluated with GFP fluorescence and hasABC expression was evaluated with RFP fluorescence. (B, C, D) Wild-type Spy was treated with LL-37 (300 nM) or MgCl2 (15 mM). (B) Live-cell fluorescent microscopy with brightfield, GFP, and RFP channels on Spy culture grown at stationary phase. Scale bars, 100 µm. (C) Measurement of fluorescence over cell density after 10 h of growth. (D) Flow cytometry demonstrating speB expression (GFP; horizontal axis) and hasABC expression (RFP; vertical axis) of Spy growing at stationary phase. Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001, ***P<0.001, ns: not significant.

S. pyogenes regulators are required for heterogenous speB expression.

Spy genetic control strains of the speB regulatory circuit were evaluated. (A) Measurement of fluorescence over cell density after 10 h of growth. (B) Flow cytometry demonstrating speB expression (GFP; horizontal axis) and hasABC expression (RFP; vertical axis) of Spy growing at stationary phase. Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001, **P<0.01, ns: not significant.

Vfr is a dominant repressor for speB.

(A, B) Wild-type Spy was grown to early exponential (EE), mid-late exponential (MLE), and stationary phase (SP). Spent media from each stage of growth were collected and filter sterilized through 0.2µm filter and inoculated with Spy to detect GFP (speB) and RFP (hasABC) fluorescence. (C) Spy Δvfr was grown in the presence or absence of recombinant Vfr (rVfr). (D) Wild-type Spy was grown with rVfr (0 - 10 ug/mL) and either LL-37 (0 - 300 nM) or MgCl2 (0 - 15 mM) for 10 h. (E) AlphaFold structure of Vfr with potential SpeB cleavage sites (blue). (F) SDS-PAGE of Vfr (0.3 mg/mL) incubated with incremental concentrations of SpeB for 2.5 h. Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001, *P<0.05, ns: not significant.

speB expression is induced in the presence of immune effectors.

(A) Evaluating the effects of neutrophil lysate on speB and hasABC expression. Spy grew in the presence of neutrophil lysates (106 cells/mL) compared to Spy growing in RPMI 5% THY. Treatment with LL-37 (300 nM) and MgCl2 (15 mM) served as controls. (B) Flow cytometry demonstrating speB (GFP fluorescence; horizontal axis) and hasABC induction (RFP fluorescence; vertical axis) of 108 CFU of Spy during mouse intradermal and human blood infections after 24 h and 4 h, respectively.

Immune effectors induce speB through Vfr.

(A, B) Neutrophil lysates were fractioned based on net surface charge through anionic exchange. (A) Fractions were used to supplement Spy growth and speB expression was evaluated with GFP fluorescence at late log phase. Protein content within each elution (mL) was detected by UV (mAU) (Upper left). (B) Fractions were also used to evaluate protease activity. (C) AlphaFold structure of Vfr with potential NET protease cleavage sites (blue). (D) SDS-PAGE of Vfr (0.3 mg/mL) incubated with lysate from neutrophils (106 cells/mL) treated with inhibitors AEBSF, Chymostatin, or PMSF. PBS served as a vehicle control. (E) Spy grew in the presence of neutrophil lysates (106 cells/mL) and AEBSF (0.6 mM) compared to Spy growing in RPMI 5% THY. Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001, ***P<0.001, **P<0.01.

SpeB and neutrophils are sufficient to induce speB expression.

(A) Diagram of mouse intradermal infection model in three different conditions: neutrophil-depleted mouse (anti-Ly6G treatment); NET-deficient mouse (PAD4-/-); and neutrophil competent (wild-type) control. (B, C) Flow cytometry on mouse skin lesions from 24 h intradermal infection of 108 CFU of Spy or Spy ΔspeB. Population of speB expressors was determined based on fluorescent intensity during flow cytometry. No expressors range was based on the negative empty vector control. (B) C57Bl/6 mice were treated with neutrophil-depleting antibody, anti-Ly6G, (blue) or control (black) for 24 h then infected. (C) NETosis-deficient mice (PAD4-/-, yellow) or control (C57Bl/6 mice, black) were infected. Statistical significance of high speB expressors was determined using a two-way ANOVA with Dunnett’s multiple comparisons test. ***P<0.001, *P<0.05, ns: not significant.

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(A) Wild-type Spy growth kinetics determined through optical density at 600 nm (O.D. 600) in RPMI, 5% THY with LL-37 (300 nM) or MgCl2 (15 mM). (B) SpeB activity was measured using the fluorescent peptide sub103. (C) Flow cytometry gating strategy for measuring Spy GFP and RFP fluorescence. Samples were selected based on particle size (FSC-Area, SSC-Area), then selected for into single cells (FSC-Area, Height; SSC-Area, Height). Sample containing Group A Carbohydrate (APC-A positive population; right peak) were selected. Lastly, live cell population was selected (APC-Cy7-A negative population; left peak). (D) Flow cytometry demonstrating speB expression (GFP; horizontal axis) and hasABC expression (RFP; vertical axis) of Spy growing at stationary phase separated in panels based on treatment.

(A) Spy growth kinetics determined through optical density at 600 nm (O.D. 600) in RPMI, 5% THY. (B) Lionheart live-cell fluorescent microscopy with brightfield, GFP, and RFP channels on of Spy culture grown at stationary phase. Scale bars, 100 µm. (C) Measurement of fluorescence over cell density after 10 h of growth. Wild-type and Δvfr Spy were treated with LL-37 (300 nM) or MgCl2 (15 mM).

(A) Regulation of speB and hasABC during mouse intradermal and human blood infections. Flow cytometry demonstrating speB (GFP fluorescence; horizontal axis) and hasABC induction (RFP fluorescence; vertical axis) of 108 CFU of Spy strains. (B) Colony Forming Units (CFU) of Spy within 4 h human blood infection was measured by plating. (C) SDS-PAGE of Vfr (0.3 mg/mL) incubated with recombinant Neutrophil Elastase (rNE) or neutrophil lysate (106 cells/mL) with inhibitor BAY-678. (D) Measurement of RFP fluorescence (hasABC) over cell density after 10 h of growth. (E) Spy growth kinetics determined through optical density at 600 nm (O.D. 600).

(A) Flow cytometry gating strategy for neutrophil depletion model with anti-Ly6G. Singlets were selected through side (SSC-H, SSC-W) and forward (FSC-H, FSC-W) scatter. Population of live cells were selected based on BUV480-A fluorescence (left). Granulocytes were selected based on the presence of CD45, and neutrophils were identified based on presence of Ly6G and CD11. (B, C) Flow cytometry demonstrating speB (GFP fluorescence; horizontal axis) and hasABC induction (RFP fluorescence; vertical axis) of 108 CFU of Spy. SpyΔvfr strain in anti-Ly6G neutrophil depletion model (B). Control strains, Spy empty vector and Δvfr, during mouse intradermal infection with PAD4-/-(C).