Recombinant human HBP (rHBP, Cat.No.2200-SE-050/CF), polyclonal goat anti-human HBP antibody (Cat.No.AF2200), and monoclonal mouse anti-human HBP antibody (Cat.No.MAB2200) were purchased from R&D (Minneapolis, MN, USA). CLI-095 [a specific inhibitor of Toll-like receptor 4 (TLR4), Cat.No.tlrl-cli95] and OxPAPC (a TLR4 non-specific inhibitor, Cat.No.tlrl-oxp1) were obtained from Invivogen (Hong Kong, China). Escherichia coli O26:B6 lipopolysaccharide (LPS, Cat.No.L2654), EGTA (a calcium chelator, Cat.No.E3889), U73122 (a phospholipase C inhibitor, Cat.No.U6756), PD98059 (a mitogen-activated protein kinase pathway inhibitor, Cat.No.P215), SB202474 (a negative control for SB203580), SB203580 (a p38 MAPK inhibitor), Wortmannin (a PI3K inhibitor, Cat.No.F9128) and Genistein (a receptor tyrosine kinase inhibitor, Cat.No.G6649) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pertussis toxin (PTX; a receptor inhibitor coupled to G protein) was kindly provided by the National Vaccine and Serum Institute (Beijing, China). The PMN Elastase Human ELISA Kit (Cat.No.ab119553) and the Lactoferrin Human ELISA kit (Cat.No.ab108882) were from Cayman Chemical (Ann Arbor, MI, USA).

All the blood specimens were kindly provided by the Chinese Center for Disease Control and Prevention and kept anonymous. The Chinese Center for Disease Control Human Research Protection Office approved the retrospective testing using anonymous samples. Blood specimens from 13 healthy individuals and 14 patients, including eight with meningitis and six with STSS, were used in this study.

Human PMNs were isolated from freshly heparinized blood that was collected by gradient centrifugation. Red blood cells were separated by 6% dextran (Sigma–Aldrich), and then the PMNs were isolated using density gradient media containing 70% Percoll (Pharmacia, New York, NY, USA) and Ficoll (GE Healthcare, Little Chalfont, UK). The purified PMNs were suspended in Hanks’ balanced salt solution (HBSS, Invitrogen, Carlsbad, CA, USA) at a concentration of 2 × 10⁶ cells/mL. The cellular purity was greater than 97% as indicated by Wright–Giemsa staining. The survival rate was greater than 97% as assessed by Trypan blue staining. The purified PMNs were used in the following experiments.

One hundred microliters of human whole blood or 1.0 × 10⁶ isolated PMNs were diluted in HBSS to a final volume of 1.0 mL and incubated with various putative stimulants at 37°C for 30 min. The HBP levels in the blood samples or the release of HBP from PMNs were subsequently determined. Cells were centrifuged at 300 × g for 15 min and HBP levels in the supernatant were analyzed by a sandwich ELISA or a Western blot. The amount of HBP in the cell lysate of the whole blood samples or the PMNs obtained by treatment with 1% Triton X-100 was determined and considered as the total HBP. The percentage of the HBP level in the supernatant relative to the total HBP level was calculated.

The HBP levels in the supernatant of the whole blood sample or the PMN suspension were determined by a sandwich-based ELISA as previously described (Tapper et al., 2002). Briefly, microtiter plates (Costar, Corning Inc., Corning, NY, USA) were coated with a polyclonal goat anti-human HBP antibody (100 μL of 0.5 μg/mL antibody solution). After washing and blocking, 100 μL of sample was added to duplicate wells and incubated at 37°C for 1 h or 4°C overnight. The standard for quantifying the HBP levels was rHBP. After a thorough washing, the plates were incubated with a monoclonal mouse anti-human HBP antibody (100 μL of 1 μg/mL antibody solution) at 37°C for 1 h or 4°C overnight. A horseradish peroxidase (HRP)-conjugated antibody against mouse IgG (1:8,000, ZSGB-Bio, Beijing, China) was added to detect the primary antibody. The plate was read in a microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.

Thirty microliters of the supernatant of a whole blood sample or a PMN suspension were separated by 12% (w/v) polyacrylamide gel electrophoresis in the presence of 1% (w/v) sodium dodecyl sulfate (SDS-PAGE) and transferred to a polyscreen polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA). The membrane was first blocked in 5% (w/v) non-fat milk in phosphate-buffered saline (PBS) supplemented with 0.05% (v/v) Tween 20 at room temperature for 1 h, and incubated with the polyclonal goat anti-human HBP antibody (1 μg/mL) at 4°C overnight. After intensive washing, the membrane was incubated with HRP-labeled mouse anti-goat IgG (1:8,000, ZSGB-Bio) for 1 h. The immunoblots were analyzed using a Gel Imaging System and the Quantity One Software version 4.0 (Bio-Rad, Hercules, CA, USA).

The highly virulent S. suis serotype type 2 strain 05ZYH33 (GenBank accession number NC_009442), which was originally isolated from a patient who died from STSS during the S. suis outbreak in 2005 in Sichuan, China, was used in this study. A suilysin gene (sly) deletion mutant of 05ZYH33 (Δsly) was constructed via the in-frame replacement of the SSU05_1403 gene with a chloramphenicol (Cm) resistance cassette. The complement strain of the Δsly mutant (CΔsly) was established by transforming the mutant with a pAT18 vector that expressed the sly gene. These strains were previously constructed in our laboratory (He et al., 2014). All the bacterial strains were grown on Colombia agar plates (BD Biosciences, San Jose, CA, USA) containing 5% sheep’s blood at 37°C under 5% CO₂. The bacterial suspensions were grown in Todd-Hewitt broth (THB, BD) for 8 h without agitation, and the supernatants were harvested for future experiments. Five microgram per mL of Cm or 8 μg/mL Em (Sigma–Aldrich) were added to the media for selection.

Native SLY was purified as previously described (Jacobs et al., 1994; Lv et al., 2014). Briefly, the supernatant from a large-scale 05ZYH33 culture was collected by continuous-flow centrifugation and filtered through a ceramic filer with a 0.8 μm pore size at 4°C, and concentrated to 150 ml using 10,000-nominal-molecular-weight-limit filters (PTCG; Minitan; Merck Millipore). The concentrated supernatants were sterilized by filtration through a 0.2 μm pore size filer (Falcon, Thermo Fisher Scientific), loaded onto a Superose-12 gel filtration column (FPLC, Pharmacia), and eluted into 40 mM PBS (pH 7.2) supplemented with 0.5 M NaCl. The elution fractions were collected and analyzed by SDS-PAGE. The recombinant SLY and the non-hemolytic sly mutant SLY (P353V) used in this study were previously constructed and purified in our laboratory (Ren et al., 2012). The endotoxin level in the native suilysin (SLY) was less than 0.03 EU/mL. The endotoxins that remained in the purified recombinant SLY were removed with Triton X-114 (Liu et al., 1997) and were less than 0.5 EU/mL before the purified recombinant SLY was used in experiments.

Superoxide anion production was determined as previously described (Nilsson et al., 2006). PMNs were stimulated with 0.50, 0.75, or 1.0 μg/mL native SLY or with the supernatants from 05ZYH33 cultures at 37°C for 20 min. The PMNs were kept on ice for 10 min to terminate the stimulation, collected by centrifugation at 200 × g for 15 min and re-suspended in 0.25 ml Krebs-Ringer phosphate (KRG, 120 mM NaCl, 4.9 mM KCl, 1.7 mM KH₂PO₄, 1.2 mM MgSO₄⋅7H₂O, 8.3 mM Na₂HPO₄⋅2H₂O, 10 mM glucose, pH 7.3) buffer containing 0.1 mM cytochrome C. Superoxide production from the PMNs was induced by incubation with 200 ng/mL phorbol myristate acetate (PMA) at 37°C for 20 min. The PMNs were pelleted, and the absorbance of the supernatants was determined at 550 nm. To determine baseline levels of superoxide anion production, the PMNs were incubated with KRG buffer without cytochrome C. KRG buffer without PMNs was used as the blank reference. The HBP in the supernatant was also determined by Western blotting.

The integrity of the PMNs was assessed by electron microscopy. After incubation with 1.0 μg/mL native SLY, the PMNs were spread onto poly-L-lysine coated coverslips, incubated at 37°C for 1 h, and fixed in 2.5% (v/v) glutaraldehyde at room temperature for 30 min. The PMNs were dehydrated in an ascending ethanol series from 50% (v/v) to absolute ethanol (10 min per step). The specimens were subjected to critical point drying in CO₂ with absolute ethanol as an intermediate solvent, mounted on copper holders, sputtered with 30 nm palladium and gold, and examined in an S-3400N scanning electron microscope.

A total of 2 × 10⁶ PMNs were incubated with 1.0 μg/mL native SLY at 37°C for 30 min with rotation and subsequently kept on ice. The expression of markers for PMN degranulation was assessed with PE conjugated mouse anti-human CD63 (1:100), APC conjugated mouse anti-human CD11b (1:100), and FITC conjugated mouse anti-human CD66b (1:100, BD Biosciences). Isotype-matched antibodies were used as negative controls. The fluorescence intensity of each sample was determined on an Accuri C6 flow cytometer (BD Biosciences). The data were analyzed using the FlowJo software (FlowJo LLC, Ashland, OR, USA).

Polymorphonuclear neutrophils were loaded with 5 μM Fura-3 AM (Invitrogen) in HBSS at 37°C for 1 h and plated into a confocal dish (Thermo Fisher Scientific). Putative PMN stimulants were added to the confocal dish. The PMNs were monitored with a FV1000 confocal laser-scanning microscope (Olympus, Tokyo, Japan) for 200 s. The mean fluorescence intensity of at least eight cells was recorded.

The Miles assay was performed on 8-week-old female C57BL/6J mice to evaluate the effects of SLY on vascular permeability. C57BL/6J mice were purchased from the Animal Care Center of the Academy of Military Medical Science (AMMS; Beijing, China) and housed in a clean room with unlimited access to food and water. TLR4 knockout mice C57BL/10ScNJNju and control mice were obtained from the Model Animal Research Center of Nanjing University. The mice were randomly divided into four groups and intradermally injected in the abdomen with 100 μL THB, 05ZYH33-supernatant, Δsly-supernatant, or CΔsly-supernatant. Four hours after the injection, 100 μL Evans blue dye solution (2.5%, Ourchem, Sinopharm) was injected via the tail vein. The mice were sacrificed by cervical dislocation 30 min after the Evans blue injection, and equal areas (20 mm × 20 mm) of skin surrounding the intradermal injection site were removed from each mouse and completely dried in an oven. The Evans blue dye was eluted from the oven-dried skin into 1 mL formamide (Sinopharm) at 55°C for 2 days, and quantified by spectrophotometry at 630 nm.

Statistical analyses were performed using the Prism 5 software (GraphPad Software, La Jolla, CA, USA). For normally distributed data, a comparison between two groups was made using Student’s t-test, and comparisons among multiple groups were done with a one-way analysis of variance. The P-value was for a 2-sided test, and P < 0.05 was considered as statistically significant. All data were expressed as the mean ± standard deviation. A rank-sum test was performed to compare the HBP levels in the patients versus the controls.

All the protocols for handling the patient blood specimens or experiments using blood from healthy donors were approved by the Institutional Medical Ethics Committee of AMMS. A signed informed consent form was obtained from each patient or a guardian and healthy volunteer. The entire study was conducted in accordance with the Declaration of Helsinki.

All experimental procedures involving mice were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and State Key Laboratory of Pathogens and Biosecurity of the Beijing Institute of Microbiology and Epidemiology and approved by the Institutional Animal Care and Use Committee of the AMMS under Permit No. IACUC of AMMS-2014-031. Animal welfare was considered and suffering was minimized.

Heparin-binding protein has been reported as a potential biomarker of severe sepsis and septic shock (Linder et al., 2009, 2012; Chew et al., 2012) and as an indicator of vascular leakage (Johansson et al., 2009). A comparison of serum HBP levels between patients with S. suis-associated STSS versus patients without STSS or healthy individuals revealed that the serum HBP levels in patients with STSS were significantly higher than those in patients with meningitis or healthy controls (46.61 ± 9.49 ng/mL vs. 11.52 ± 5.20 ng/mL or 1.84 ± 0.96 ng/mL, respectively; P = 0.0007, Figure 1), which suggested that HBP is released during the progression of STSS in patients infected with S. suis. Both the ELISA and Western blotting analyses demonstrated that the bacterial culture of the highly virulent S. suis strain 05ZYH33 significantly increased the release of HBP in the whole blood of healthy individuals (Figure 2A) and in purified PMN suspension (Figures 2B,C), whereas THB media without 05ZYH33 failed to induce the release of HBP in a whole blood sample or a PMN suspension. The positive control for the Western blot was rHBP. The molecular weight of native HBP is approximately 31 kDa, while rHBP appears to be approximately 39 kDa on a Western blot owning to its carboxyl-terminal polyhistidine tag (Figure 2C). These results suggest that strain 05ZYH33 stimulates the release of HBP from PMNs.

The molecule responsible for inducing the release of HBP was identified. Supernatants of the 05ZYH33 culture, but not the 05ZYH33 bacterial cells, significantly induced the release of HBP in whole blood samples (P < 0.001, Figure 3A). Heat treatment of the supernatants significantly reduced the release of HBP in the whole blood samples (P < 0.001, Figure 3B), indicating that the specific factor(s) could be proteins. Therefore, the proteins in the supernatants were isolated by anion-exchange and hydrophobic interaction chromatography. The proteins in the elution fractions that markedly stimulated the release of HBP were separated by SDS-PAGE, and two major protein bands at approximately 58 and 35 kDa appeared on the gel (Figure 3C). Mass spectrometry revealed that the 58 kDa protein was SLY, and the 35 KDa protein was L-lactate dehydrogenase (LDH; Supplementary Table S1). Recombinant LDH failed to stimulate the release of HBP from PMNs (Figure 3D), which suggests that LDH was not the molecule that induced the release of HBP. Therefore, SLY is a likely candidate for the substance that releases HBP. Supernatants of the sly gene deletion mutant strain of 05ZYH33 (Δsly) did not stimulate the release of HBP, while the supernatants of the complement strain for Δsly (CΔsly) restored the stimulation of the release of HBP (P < 0.01, Figure 3E). Furthermore, Western blotting showed that purified native SLY (nSLY) stimulated the release of HBP from the PMNs, but a bacterial culture of Δsly strain failed to do so (Figure 3F). Taken together, these findings suggest that SLY secreted by 05ZYH33 induces the release of HBP from the PMNs.

Heparin-binding protein is usually stored in azurophilic granules and secretory vesicles in the PMNs and released by cell lysis or via degranulation after the activation of a series of signal transduction pathways (Borregaard and Cowland, 1997; Tapper et al., 2002). We can infer that a high concentration of SLY could lyse the PMNs and release the stored HBP, while a sub-cytolytic concentration would interact with the cell membrane receptor and could possibly trigger PMN degranulation. Different concentrations of nSLY (0.50–1.0 μg/mL) did not affect the production of superoxide anions from the PMNs (Figure 4A), while the release of HBP from the PMNs was SLY dose-dependent (Figure 4B), suggesting that the PMNs remained viable when treated with nSLY. These findings indicate that cell lysis is not related to the release of HBP mediated by SLY. Scanning electron microscopy demonstrated that the PMNs exhibited membrane blebs when treated with 1.0 μg/mL nSLY (Figure 4D) and the control did not exhibit such blebs (Figure 4C), suggesting that the degranulation might be induced by nSLY. A flow cytometry analysis revealed the significant up-regulation of markers for azurophilic granules (CD63), secondary/tertiary granules and secretory vesicles (CD11b), and specific granules (CD66b, also named CD67) at the plasma membrane after the PMNs were incubated with recombinant SLY (rSLY), but the recombinant factor H-binding protein (rFhb – a S. suis cell wall protein that was purified using the same methods) was not up-regulated. This implies that the granular vesicles in the PMNs were mobilized for degranulation (Figures 4E–G). Other proteins stored in the neutrophil granules, including elastase from the azurophilic granules and lactoferrin from the secondary granules, were released when the neutrophils were incubated with nSLY or the 05ZYH33-supernatant (Figures 4H,I). All of these results suggest that SLY induces the release of HBP via PMN degranulation.

An increased cytosolic Ca²⁺ concentration has been shown to promote PMN degranulation via the mobilization of intracellular granules and cytoskeleton rearrangements (O’Flaherty et al., 1991; Lacy, 2006; Lacy and Eitzen, 2008). Thus, the SLY-induced release of HBP might also be Ca²⁺-dependent. Indeed, the release of HBP induced by the bacterial culture supernatants was completely abolished in the presence of EGTA (P < 0.0001, Figure 5A), suggesting that the release of HBP is dependent on extracellular Ca²⁺. The measurement of intracellular Ca²⁺ revealed that nSLY (1 μg/mL) induced a rapid increase in the intracellular Ca²⁺ concentration in standard HBSS, and that the Ca²⁺ increase was completely abolished by EGTA or in Ca²⁺-free HBSS (Figure 5B). N-formylmethionyl-leucylphenylalanine (fMLP) served as a positive control and LPS was used as a negative control (Figures 5B,C). These results indicate that SLY induced the Ca²⁺ influx. A constructed non-hemolytic sly mutant SLY (P353V; Xu et al., 2010; Du et al., 2013) could neither induce calcium mobilization (Figure 5B), nor evoke the secretion of HBP (Supplementary Figure S1), suggesting that the SLY-induced release of HBP was dependent on the Ca²⁺ influx. Supernatants of 05ZYH33 consistently induced a Ca²⁺ influx, whereas THB media and supernatants of the Δsly mutant did not induce a Ca²⁺ influx (Figure 5C). The Ca²⁺ influx induced by the 05ZYH33 supernatant was also completely abolished by EGTA (Figure 5C). All of these results indicated that the Ca²⁺ influx plays a critical role in the release of HBP induced by SLY.

To investigate possible mechanisms and the key molecules involved in release of HBP, inhibitors were commonly used in this study because of the lack of suitable cell lines for investigating the release of HBP induced by SLY, and direct siRNA transfection into PMNs could cause activation of the PMNs and the release of HBP (data not shown).

Some cholesterol-dependent cytolysin (CDC) family members, such as anthrolysin O, perfringolysin O, listeriolysin O, streptolysin O, and pneumolysin, have been reported to interact with TLR4 (Malley et al., 2003; Park et al., 2004; Srivastava et al., 2005). SLY also belongs to the CDC family, and results from our previous study have shown that SLY interacts with TLR4 to induce an inflammatory response (Bi et al., 2015). In this study, our results demonstrated that both the TLR4 specific inhibitor CLI-095 (Figure 6A), which blocks the intracellular domain of TLR4, and the TLR4 non-specific inhibitor OxPAPC (Figure 6B), which competes with other ligands including CD14, LBP, and MD2 for receptor binding, significantly reduced the release of HBP induced by the 05ZYH33 supernatant. The results for both inhibitors were significant at P < 0.0001. This suggests that the release of HBP induced by SLY could be dependent on TLR4. We then used a pharmacological approach to further investigate the signaling pathway underlying the SLY-induced release of HBP. The phospholipase C inhibitor U73122 (Figure 6C) and the MAPK pathway inhibitor PD98059 (Figure 6D) did not affect the release of HBP, whereas the p38 MAPK inhibitor SB203580 (Figure 6E) and the PI3K inhibitor Wortmannin (Figure 6F) markedly reduced HBP secretion (both P < 0.0001). We confirmed that a time-dependent increase in the level of phosphorylated p38 MAPK occurred in the presence of nSLY (1.0 μg/ml; Supplementary Figure S2). Other receptor inhibitors such as the GPCR inhibitor PTX significantly inhibited the release of HBP (P < 0.0001, Figure 6G), but Genistein, an inhibitor of the receptor tyrosine kinases (RTK), had no effect (Figure 6H). Therefore, a GPCR might also be involved in the release of HBP induced by SLY in addition to TLR4. The mechanism could be complex, and further studies are necessary to explain why two receptors are involved in the release of HBP induced by SLY.

The effects of SLY on vascular leakage were tested in 8-week-old female C57BL mice using the Miles assay. Supernatants of the wild-type 05ZYH33 strain and the CΔsly mutant significantly increased the leakage of Evans blue dye in mice compared with a THB control or the supernatants from the Δsly mutant (05ZYH33-sup: 18.96 ± 1.35 μg/mL vs. Δsly-sup: 12.38 ± 0.57 μg/mL, P = 0.0008, Figures 7A,B), implying that SLY contributes to vascular leakage. In addition, the 05ZYH33 supernatant failed to induce vascular leakage in Tlr4 knockout mice (TLR4^+/+: 26.80 ± 1.96 μg/mL vs. TLR4^-/-: 11.35 ± 0.98 μg/mL, P < 0.0001, Figures 7C,D). These in vivo data support the essential role of TLR4 in vascular leakage mediated by SLY.