Human Serum Albumin Binds Streptolysin O (SLO) Toxin Produced by Group A Streptococcus and Inhibits Its Cytotoxic and Hemolytic Effects

Abstract

The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo. Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity "orchestra." We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.

Keywords: Streptococcus pyogenes; human serum albumin; red blood cells; streptolysin O; toxin.

Figure 1

HSA binds SLO toxin produced by Streptococcus pyogenes. (A) Molecular docking of SLO binding to HSA. Left: overall view of the putative complex between HSA (PDB ID: 1AO6) (deep pink) and SLO (PDB ID: 4HSC) (light blue) obtained by docking simulations. Right: residues involved in HSA-SLO interactions are highlighted. Images were drawn with the UCSF Chimera-package (35). (B) SLO binding to wt-HSA (filled circles) and to L305A/F374A-HSA (open circles) evaluated by spectrofluorimetric analysis. The continuous lines were calculated according to Eqn. 1 with the following parameters: wt-HSA - ΔF _tot = 20.1 and K _d = (2.5 ± 0.2)×10⁻⁹ M; and L305A/F374A-HSA - ΔF _tot = 21.1 and K _d = (7.3 ± 0.5)×10⁻⁹ M. Where not shown, the error bar is smaller than the symbol. (C) SLO binding to HSA evaluated by ELISA. Either 1.0×10⁻⁸ M or 2.0×10⁻⁸ M of activated SLO were added to HSA-coated wells, and the detection of HSA:SLO interaction was performed after incubation with anti-SLO antibody. Readings were performed using the TMB colorimetric substrate at 370 nm until reaction saturation. Microtiter wells coated with 100 μL of 1.6×10⁻⁷ M commercial HSA, without the subsequent addition of SLO, were used as a negative control. Results are representative of triplicates and are expressed as mean ± SD (One-way ANOVA, **P < 0.01 and ***P < 0.001). (D) SLO binding to recombinant wt-HSA evaluated by magnetic beads assay. Activated SLO (2.9×10⁻⁷ M) was incubated with 4 μg of wt-HSA-conjugated (+) or unconjugated (−) beads. Thirty microliters of magnetic beads eluates were resolved on a 12.5% SDS-PAGE and SLO bound to HSA was detected by immunoblot using anti-SLO antibody. Different quantities of SLO (i.e., 45, 90, 135, and 270 ng corresponding to 3%, 6%, 9%, and 18% of 1.5 μg SLO) were loaded as inputs. Results show the presence of the bands corresponding to SLO in the eluates.

Figure 2

HSA protects human HEp-2 cells from intoxication with SLO toxin. (A) Dose- and time-course experiments to evaluate SLO cytotoxic effects in HEp-2 and A549 cells. Cells were treated for 0.5, 3, 6, and 24 h with 75, 150, 300, 600, and 1000 U/mL of activated SLO. The percentage of viable cells was determined by the MTT metabolic test, considering that untreated cells were taken as 100%. Data represent the mean value ± SD derived from three replicates. (B) IC50 values were calculated by plotting cell survival (%) versus SLO concentration (U/mL) in HEp-2 and A549. (C) HEp-2 cells and A549 cells were treated for 0.5, 6, and 24 h with 600 U/mL of activated SLO, in the absence or presence of 1.0×10⁻⁵ M and 1.0×10⁻⁴ M HSA. The percentage of viable cells was determined by MTT test, considering that untreated cells (Control) were taken as 100%. Data represent the mean value ± SD derived from three replicates (Two-way ANOVA and Tukey’s post hoc test [****P < 0.0001 compared with control; °P < 0.05, °°P < 0.01, °°°P < 0.001, and °°°°P < 0.0001 compared with cells treated with 600 U/mL SLO in the absence of HSA]). (D) IC50 values were calculated assuming a linear fit in HSA-treated Hep-2 and A549 cells. (E) Exemplificative images show the morphologies of HEp-2 cells untreated (i.e., 0 U/mL SLO and 0 M HSA) and treated for 24 h with 600 U/mL of activated SLO in the absence or presence of 1.0×10⁻⁵ M and 1.0×10⁻⁴ M HSA. Images were acquired using a Leica microscope (Leica Microsystems, Heidelberg, Germany); magnification 20×.

Figure 3

HSA inhibits SLO-induced cells membrane permeabilization. HEp-2 cells were stained with the PKH67 fluorescent cell linker dye solution (green signal), in order to visualize cells membrane. After 24 h, stained cells were treated with 100 U/mL of activated SLO, in the absence or presence of 1.0×10⁻⁴ M HSA. Propidium iodide (PI) staining, which is not permeant to live cells, allowed to detect dead cells. Cells were immediately analyzed and acquired using the LCS Leica confocal microscope (Leica Microsystems, Heidelberg, Germany).

Figure 4

Effect of HSA and BSA on the SLO toxin-mediated red blood cells (RBCs) hemolysis. RBCs were isolated from the human whole blood of four healthy donors and treated with (A) 0, 1, 2.5 and 5 U of activated SLO for 1 h at 37°C. (B) Considering that 1 U of activated SLO causes a 50% lysis of 50 μL of 2% human RBCs, we used 5 U of activated SLO to cause the 50% lysis of 190 mL of 2% human RBCs. RBCs were treated with the toxin for 1 h at 37°C, in the absence or presence of 1.0×10⁻⁶ M to 1.0×10⁻⁴ M HSA. The protective effect of 1.0×10⁻⁴ M BSA was tested. As negative control, RBCs were incubated with PBS, in the absence of activated SLO. As positive control, RBCs were disrupted with 2% Triton X-100. The concentration of released hemoglobin was quantified by measuring the absorbance at 541 nm. Results were represented as the percentage of lysed RBCs (assuming as 100% the positive control) derived from three independent experiments ± SD (One-way ANOVA, ***P < 0.001 and ****P < 0.0001 compared with cells treated with 600 U of activated SLO in the absence of HSA).