Effects of Suilysin on Streptococcus suis-Induced Platelet Aggregation

Abstract

Blood platelets play important roles during pathological thrombocytopenia in streptococcal toxic shock syndrome (STSS). Streptococcus suis (S. suis) an emerging human pathogen, can cause STSS similarly to S. pyogenes. However, S. suis interactions with platelets are poorly understood. Here, we found that suilysin (SLY), different from other bacterial cholesterol-dependent cytolysins (CDCs), was the sole stimulus that induced platelet aggregation. Furthermore, the inside-out activation of GPIIb/IIIa of platelets mediated SLY-induced platelet aggregation. This process was triggered by Ca²⁺ influx that depend on the pore forming on platelets by SLY. Additionally, although SLY induced α-granule release occurred via the MLCK-dependent pathway, PLC-β-IP3/DAG-MLCK and Rho-ROCK-MLCK signaling were not involved in SLY-induced platelet aggregation. Interestingly, the pore dependent Ca²⁺ influx was also found to participate in the induction of platelet aggregation with pneumolysin (PLY) and streptolysin O (SLO), two other CDCs. It is possible that the CDC-mediated platelet aggregation we observed in S. suis is a similar response mechanism to that used by a wide range of bacteria. These findings might lead to the discovery of potential therapeutic targets for S. suis-associated STSS.

Keywords: Ca2+ influx; Streptococcus suis (S. suis); platelet aggregation; streptococcal toxic shock syndrome (STSS); suilysin (SLY).

Figure 1

SLY is the sole stimulus of S. suis-induced platelet aggregation. (A) The culture supernatant of S. suis and recombinant proteins or (B) the washed bacteria cells were added to PRP in a stirred cuvette. Platelet aggregation was expressed as a final percentage of light transmission detected by Platelet Aggregometer se-2000. ADP (20 μM) and GAS bacterial cells were used as the positive controls. Unpaired two-tailed Student's t-tests were used and the threshold for significance was P < 0.05; ns, not significant. Data are expressed as the mean ± SD of three independent experiments, with each experiment using blood from a different donor. (C) Platelet aggregations were observed when detecting the light transmission of PRP stimulated by bacterial supernatant (at OD₆₀₀ = 1.0) or rSLY. (D) Wright's staining of cytospin preparations of S. suis culture supernatant or rSLY protein-treated whole blood samples. 05ZYH33, wild type strain; Δsly, isogenic mutant of sly; Δmrp, isogenic mutant of mrp (which was used as an irrelevant control for Δsly); 1330, SLY-negative Canadian strain; H-05ZYH33, heat-inactive 05ZYH33; Sup, supernatant; rSLY, recombinant SLY; rSLY^P353V, recombinant non-hemolytic mutant of SLY^P353V; rFhb, recombinant Factor H-binding protein, which was used as an irrelevant protein and was purified by the same procedure as that used for rSLY.

Figure 2

Platelet activation by SLY, and GPIIb/IIIa (CD41a) mediated the SLY-induced platelet aggregation. (A) SLY induces dense granule release from platelets. PRP was incubated with S. suis culture supernatant or SLY protein (1 μg/ml). The Mann–Whitney U-test was used for statistical analysis. (B,C) S. suis culture supernatant or rSLY protein (1 μg/ml)-induced surface GPIIb/IIIa (CD41a) increase and fibrinogen binding to platelets in human blood was assessed by flow cytometry (Methods Section). Representative histograms for the MFI of CD41a/fibrinogen binding are shown in (B, left panel). Unpaired two-tailed Student's t-test was used for (B) statistical analysis. Mann–Whitney U-test was used for (C) statistical analysis. (D) PRP was preincubated with eptifibatide (10 μM) for 15 min prior to addition of S. suis supernatant or SLY protein. Platelet aggregation was expressed as a final percentage of light transmission. Unpaired two-tailed Student's t-test was used for statistical analysis. ADP (20 μM) was used as the positive control for platelet activation. THB and PBS were the negative controls for the culture supernatant and proteins, respectively. Data in panels (A–D) are expressed as the mean ± SD of three independent experiments, with each experiment using blood from a different donor. P < 0.05 is considered to be the threshold for statistical significance; ns, not significant; 05ZYH33, wild type strain; Δsly, isogenic mutant of sly; Sup, supernatant; rSLY, recombinant SLY.

Figure 3

SLY-induced platelet activation and aggregation dependent on pore formation on platelets. (A) The hemolytic activity of 05ZYH33 supernatant and rSLY. Culture supernatants of S. suis 05ZYH33 and rSLY were tested the hemolytic activity as described by Materials and Methods. One hemolytic unit is defined as the reciprocal of the suilysin titer, which was calculated as the highest dilution of the supernatant/rSLY which caused at least 50% hemolysis. (B) Dose response of rSLY-induced platelet aggregation and the cholesterol inhibiting effect. (C,D) The cholesterol inhibiting effect of rSLY-induced surface GPIIb/IIIa (CD41a) increase and fibrinogen binding to platelets in human blood were assessed by flow cytometry (Methods Section). A total of 1 μg/ml of rSLY and 100 μg/ml of cholesterol were used. (E) The cholesterol (100 μg/ml) effect on S. suis supernatant-induced platelet aggregation was detected using a platelet aggregometer. Unpaired two-tailed Student's t-test was used for (C) statistical analysis. Unpaired t-test with Welch's correction was used for (B,D,E) statistical analysis. THB and PBS were the negative controls for culture supernatant and proteins, respectively. Cholesterol was dissolved in ethanol. rSLY, recombinant SLY; Cho, cholesterol; 1+Cho, 1 μg/ml of rSLY added to cholesterol. Data in panels (A–E) are expressed as the mean ± SD of three independent experiments, with each experiment using blood from a different donor. P < 0.05 is considered to be the threshold for statistical significance; ns, not significant; 05ZYH33, wild type strain; Δsly, isogenic mutant of sly; Sup, supernatant.

Figure 4

Pore dependent Ca²⁺ influx by SLY triggers platelet activation and aggregation. (A) rSLY induces Ca²⁺ influx in human platelets. The purified platelets marked with fluo-8 were resuspended in HBSS (with 2 mM Ca²⁺), and rSLY (1 μg/ml), rSLY (0.1 μg/ml), rSLY^P353V (1 μg/ml), cholesterol (10 μg/ml), or other control reagents were added. Ca²⁺ influx to platelets was detected using a Varioskan Flash Multiplate Reader. (B,C) The EGTA effect on rSLY-induced surface GPIIb/IIIa (CD41a) increase and fibrinogen binding to platelets in human blood was assessed by flow cytometry (Methods Section). Human blood was preincubated with EGTA (3 mM) for 10 min prior to addition of rSLY (1 μg/ml). Unpaired two-tailed Student's t-test was used for (B) statistical analysis. (D) The EGTA (3 mM) effect on S. suis supernatant-induced platelet aggregation was detected using a platelet aggregometer (Methods Section). Unpaired t-test with Welch's correction was used for (C,D) statistical analysis. THB and PBS were the negative controls for culture supernatant and proteins, respectively. EGTA was dissolved in H₂O. Data in panels (A–D) are expressed as the mean ± SD of three independent experiments, with each experiment using blood from a different donor. P < 0.05 is considered to be the threshold for statistical significance; ns, not significant; Cho, cholesterol; rSLY, recombinant SLY; rSLY^P353V, recombinant non-hemolytic mutant of SLY^P353V; 05ZYH33, wild type strain; Δsly, isogenic mutant of sly; Sup, supernatant.

Figure 5

Platelet signaling in response to SLY. (A,B) rSLY (1 μg/mL)-induced platelet aggregation in PRP pretreated with or without MLCK inhibitor ML-7 (100 μM), ROCK inhibitor Y27632 (100 μM), and PLC-β inhibitor U73122 (20 μM). Unpaired t-test with Welch's correction was used for (A,B) statistical analyses. (C) The effects of ML-7 (100 μM) and eptifibatide (10 μM) on rSLY-induced surface GPIIb/IIIa (CD41a) increase. Unpaired two-tailed Student's t-test was used for statistical analysis. PBS is the negative control for rSLY protein. ML-7, Y27632, and eptifibatide were dissolved in H₂O. U73122 was dissolved in ethanol. Data in panels (A–C) are given as the mean ± SD of 3–8 independent experiments from different blood donors. P < 0.05 is considered to be the threshold for statistical significance; ns, not significant.

Figure 6

SLY contributes to the intravascular thrombosis and its associated liver injury caused by S. suis. Female BALB/c mice (6–8 weeks old) were challenged with (A,B) 05ZYH33; (C) mutant Δsly (~1 × 10⁸ CFU); (D) PBS control through the caudal vein. Pathological changes were observed by hematoxylin and eosin pathological staining of tissue sections at 48 h post-inoculation. Panels numbered II are the enlarged images from the white boxes in panels numbered I. a, b, c, and d in the panels represent blood vessels, intravascular thrombosis, coagulative necrosis, and leukocyte infiltration, respectively.

Figure 7

rPLY and rSLO induce platelet aggregation via pore dependent calcium influx. The cytotoxicity of rPLY (A) and rSLO (B) against platelets and the cholesterol inhibiting effect were assessed by an LDH assay (Methods Section). (C) The cholesterol (100 μg/mL) effect on rPLY (0.8 μg/mL)- and rSLO (1.5 μg/mL)-induced platelet aggregation. (D) The EGTA (3 mM) effect on rPLY (0.8 μg/mL)- and rSLO (1.5 μg/mL)-induced PNA formation. PBS acted as the negative control for the recombinant proteins. Cholesterol and EGTA were dissolved in ethanol and H₂O, respectively. Unpaired t-test with Welch's correction was used for (A,B,D) statistical analyses. Unpaired two-tailed Student's t-test was used for (C) statistical analysis. Data in panels (A–D) are given as the mean ± SD of three independent experiments, with each experiment using blood from a different donor. P < 0.05 is considered to be the threshold for statistical significance; ns, not significant; rPLY, recombinant pneumolysin; rSLO, recombinant streptolysin O; Cho, cholesterol; 0.8+Cho, 0.8 μg/mL of rPLY added to cholesterol; 1.5+Cho, 1.5 μg/mL of rSLO added to cholesterol.

Figure 8

Schematic representation of platelet activation and aggregation induced by S. suis. Ca²⁺ influx across transmembrane pores created by SLY, the CDC of S. suis, can trigger inside-out signaling leading to integrin GPIIb/IIIa activation and α-granule (GPIIb/IIIa) or dense granule (ATP) secretion. Subsequently, GPIIb/IIIa activation leads to platelet aggregation. Additionally, Ca²⁺-MLCK signaling is involved in α-granule release induced by SLY.