Suilysin Stimulates the Release of Heparin Binding Protein from Neutrophils and Increases Vascular Permeability in Mice

Abstract

Most of the deaths that occurred during two large outbreaks of Streptococcus suis infections in 1998 and 2005 in China were caused by streptococcal toxic shock syndrome (STSS), which is characterized by increased vascular permeability. Heparin-binding protein (HBP) is thought to mediate the vascular leakage. The purpose of this study was to investigate the detailed mechanism underlying the release of HBP and the vascular leakage induced by S. suis. Significantly higher serum levels of HBP were detected in Chinese patients with STSS than in patients with meningitis or healthy controls. Suilysin (SLY) is an exotoxin secreted by the highly virulent strain 05ZYH33, and it stimulated the release of HBP from the polymorphonuclear neutrophils and mediated vascular leakage in mice. The release of HBP induced by SLY was caused by a calcium influx-dependent degranulation. Analyses using a pharmacological approach revealed that the release of HBP induced by SLY was related to Toll-like receptor 4, p38 mitogen-activated protein kinase, and the 1-phosphatidylinositol 3-kinase pathway. It was also dependent on a G protein-coupled seven-membrane spanning receptor. The results of this study provide new insights into the vascular leakage in STSS associated with non-Group A streptococci, which could lead to the discovery of potential therapeutic targets for STSS associated with S. suis.

Keywords: Streptococcus suis-associated streptococcal toxic shock syndrome; heparin binding protein; suilysin; vascular permeability.

Figures

FIGURE 1

The serum HBP levels were significantly increased in patients with STSS compared with patients without STSS or healthy individuals. The serum HBP levels of healthy individuals (n = 13), patients infected with S. suis presenting meningitis (n = 8), and patients infected with S. suis presenting STSS (n = 6) were determined by ELISA. Data were analyzed by a rank-sum test. ∗∗∗ indicates a significant difference between patients with STSS vs. controls, P = 0.0007.

FIGURE 2

05ZYH33 cultures induced the release of HBP in whole blood samples and purified PMN suspension. (A,B) 05ZYH33 cultures significantly induced the release of HBP in human whole blood samples (A) and purified PMN suspensions (B). Whole blood samples or purified PMN suspensions were incubated with 05ZYH33 cultures or THB (control) for 30 min at 37°C. The HBP level in the supernatants was measured by ELISA. The total cell lysate following Triton X-100 treatment was used as the positive control. Data are expressed as the mean ± SD. An unpaired Student’s t-test was used. ∗∗P < 0.01, ∗P < 0.05. (C) Western blotting results of release of HBP from human purified PMN suspensions. rHBP and Triton X-100 were used as positive controls.

FIGURE 3

Suilysin secreted from 05ZYH33 induced the release of HBP from PMNs. (A) The release of HBP from whole blood samples was induced by supernatants of 05ZYH33 but not by the bacterial cells. Human whole blood samples were incubated with PBS, THB, 05ZYH33 bacterial cells, or 05ZYH33 supernatants for 30 min at 37°C. The HBP level in the supernatants was measured by ELISA. PBS was considered as the background and THB was used as a negative control. An unpaired Student’s t-test was used for the statistical analysis. (B) Heat treatment of the supernatants significantly reduced the release of HBP from whole blood samples. The supernatants were treated at 100°C for 5 min before being added to whole blood samples. (C) A SDS-PAGE analysis of the elution fraction from anion-exchange and hydrophobic interaction chromatography, which induced the maximal release of HBP. (D) LDH failed to induce the release of HBP from PMNs. The PMNs were incubated with His-LDH or Trix-LDH at 0.1, 1.0, or 10 μg/mL for 30 min at 37°C. (E) Suilysin in the 05ZYH33-supernatant stimulated the PMNs to secret HBP. Human whole blood samples were incubated with PBS, THB, the 05ZYH33-supernatant, the Δsly-supernatant or the CΔsly-supernatant for 30 min at 37°C. (F) Western-blot analysis of the HBP that was released from PMNs incubated with cultures of wild-type 05ZYH33, the Δsly mutant, or purified nSLY. A representative image is presented. ∗∗∗P < 0.001, ∗∗P < 0.01, n.s., not significant. ELISA data are expressed as the mean ± SD of at least four independent experiments.

FIGURE 4

Suilysin induced PMN degranulation. (A) Superoxide anion production was not affected by treatment with nSLY or 05ZYH33 supernatants. Purified PMNs were incubated with 0.50, 0.75, or 1.0 μg/mL nSLY, the 05ZYH33-supernatant, or HBSS for 30 min at 37°C. Following treatment, the cells were stimulated with 200 ng/ml PMA in the presence of 0.1 mM cytochrome C. The superoxide anion production was determined by measuring the absorbance at 550 nm. Data are expressed as the mean ± SD of three independent experiments. (B) Western blot analysis of release of HBP from PMNs that were treated as described for the measurement of superoxide anion production. Thirty microliters of each sample was loaded in each lane. A representative image is presented. (C,D) Scanning electron microscopy images of purified PMNs that were pre-incubated with HBSS (C) or 1 μg/mL nSLY (D). Red arrows indicate excessive membrane ruffles. Representative images are presented. The scale bar represents 5 μm. (E–G). Levels of membrane-associated marker proteins for PMN degranulation, CD63 (E), CD11b (F), and CD66b (G) were increased by 1 μg/mL rSLY. PMNs were stimulated with HBSS as a negative control (—), SLY-isotype (—), rSLY (—), or rFhb (—) and analyzed by flow cytometry. (H,I) Levels of granule marker proteins for PMN degranulation, elastase (H) and lactoferrin (I) were increased by 1 μg/mL nSLY or the 05ZYH33 supernatant. ∗∗∗P < 0.001, ∗∗P < 0.01. ELISA data are presented as the mean ± SD of at least three independent experiments.

FIGURE 5

SLY induced a Ca2+ influx and the release of HBP was dependent on the Ca2+ influx. (A) The SLY-induced release of HBP from human whole blood samples was abolished by EGTA. PMNs were pre-incubated with EGTA (10 mM) for 1 h at 37°C. ∗∗∗P < 0.001. (B) SLY induced a Ca2+ influx. Positive control fMLP, negative control LPS, nSLY, nSLY with pre-incubation of EGTA or with Ca2+-free HBSS, rSLY, non-hemolytic SLY mutant P353V was added to a confocal small dish containing purified PMNs loaded with the fluorescent Ca2+ indicator Fluo-3/AM at the indicated time (60 s, arrow), and Ca2+ mobilization was monitored by real-time fluorescence microscopy for 250 s. (C) SLY in the 05ZYH33-supernatant stimulated a Ca2+ influx. The positive control was fMLP, the negative control was LPS, and the 05ZYH33-supernatant, THB, the Δsly-supernatant, the 05ZYH33-supernatant with pre-incubation of EGTA were used as stimulants. The fluorescence intensity of at least eight cells in one vision was measured and the mean increase intensity is presented.

FIGURE 6

Receptor and signal molecule were involved in the Suilysin induced release of HBP. (A,B) The release of HBP was blocked by the TLR4-specific inhibitor CLI-095 (3 μM; A) and the TLR4-non-specific inhibitor OxPAPC (30 μg/mL; B). (C,D) The release of HBP was not affected by the PLC and PLA2 inhibitor U73122 (10 μM; C) and the ERK1/2 inhibitor PD98095 (20 μM; D). (E–H) The release of HBP was blocked by the p38 MAPK inhibitor SB203580 (10 μM; E), the PI3K inhibitor Wortmannin (1.0 μM; F), the GPRP inhibitor PTX (0.15 μg/mL; G) but not by the RTK inhibitor Genistein (100 μM; H). The PMNs were pre-treated with or without the inhibitors at 37°C for 1 h (PTX for 3h and CLI-095 for 6 h) before a 30 min incubation with the stimulants at 37°C. The HBP level was measured by ELISA. The HBP level relative to the total amount of HBP in the cell lysate from treatment with Triton X-100 was calculated. The data are expressed as the mean ± SD of at least four independent experiments. ∗∗∗P < 0.001. n.s., not significant.

FIGURE 7

Suilysin increased the vascular leakage in C57BL mice. (A,B) SLY increased the vascular leakage in mice. C57BL/6J mice were intradermally injected with THB, the 05ZYH33-supernatant, the Δsly-supernatant, or the CΔsly-supernatant. Four hours later, Evans blue dye (2.5%, 100 μL) was injected via the tail vein. After 30 min, photographs of the skin area containing the extravasated protein-bound dye were taken, and the dye was extracted from the skin using formamide. Dye concentrations were measured at 630 nm using a spectrophotometer. (C,D) The 05ZYH33 supernatant did not induce vascular leakage in Tlr4 knockout mice. TLR4+/+ and TLR4-/- were treated in the similar ways as in (A,B). ∗∗∗P < 0.001, ∗∗P < 0.01, n.s., not significant.