The bacterial strains and plasmids used in this study are described in Table 1. S. aureus strains were cultured in brain-heart infusion (BHI) medium, which was supplemented with chloramphenicol (10 μg/ml) when required. Escherichia coli strains were cultured in Luria–Bertani (LB) medium, which was supplemented with ampicillin (100 μg/ml) when required.

The full-length coding sequence of mature vWbp was amplified by PCR from genome of S. aureus Newman using the primers 5′-GAACTCGAGGCATTATGTGTATCACAAATTTGGG-3′ (for ward) and 5′-GAAGGATCCGCAGCCATGCATTAATTATTTG CC-3′ (reverse). The PCR product was inserted into the XholI and the BamHI restriction sites of the pET15b vector, yielding pET15b-vWbp. For the overexpression of the vWbp, pET15b-vWbp was transformed into E. coli BL21 (DE3). Production of recombinant vWbp protein was induced with 0.5 mM IPTG after 12 h. Following induction, the cells were centrifuged at 4000 r/min for 30 min, suspended in 1 × column buffer (0.1 M Tris–HCl (pH 7.5), 0.5 M NaCl) and lysed with an ultrasonic disrupter. Lysates were centrifuged at 12000 r/min for 1 h, and the supernatant was subjected to Ni-NTA affinity chromatography. The column was washed with column buffer containing 40 mM imidazole, and the recombinant His-tagged vWbp protein was eluted with 500 mM imidazole and stored at –80°C.

For the tube coagulation assay, follow the protocol, sterile NaCl (0.9%) was added to Silin bottle containing freeze-dried rabbit plasma powder (purchased from Qingdao Hope Bio-Technology Co., Ltd.) and shake slightly to dissolve completely. 10 μL of recombinant vWbp (50 μM) and 490 μL above solution was added to borosilicate glass tubes and mixed. The tubes were incubated at 37°C, and coagulation was monitored by laying the tube down on its side every 10 min.

The plate coagulation assay was performed as described by Hwang et al. (1989) with minor modification. Agarose solution (0.9%) containing 0.4% PEG 8000, 3 mg/ml bovine fibrinogen and 1% plasma was added into 60-mm plates. Twenty microlitres of recombinant protein at concentrations ranging from 10 to 0.625 mg/ml was added to small wells punched out in the plates. Coagulation areas were measured after the plates were incubated at 37°C for 12 h.

The MIC of ABBA against S. aureus was measured by broth microdilution (Macia et al., 2014). To plot the growth curves of S. aureus, 1 ml of overnight-cultured S. aureus was added to 100 ml of sterile BHI broth with or without appropriate concentrations of ABBA. The absorbance value at 600 nm (OD₆₀₀) was measured every 30 min for 24 h using an Infinite^® F200 PRO instrument.

Western blotting was performed as previously described (Dong et al., 2013). S. aureus was cultured with different concentrations of ABBA to an OD₆₀₀ of 0.6–0.8. Supernatant samples were collected by centrifugation, separated by SDS-PAGE, and transferred to polyvinylidene membranes. The membranes were blocked with 5% BSA for 2 h at room temperature. A specific anti-vWbp primary antibody was added at a 1:5000 dilution, and the membranes were then incubated overnight at 4°C. Next day, after washes thrice with PBS containing 0.05% Tween-20 (PBS-T), HRP-conjugated secondary goat anti-rabbit antiserum was added at a 1:2000 dilution, and the immunoreactive bands were visualized by an ECL Western blot detection system (GE Healthcare, United Kingdom) followed by 2 h of incubation.

SYPRO orange (5000×) was diluted into the buffer solution (1:100) and mixed with an equal volume of vWbp. Four microliters of the above mixture and 2 μL of ABBA were mixed with 14 μL of buffer solution in PCR tubes. Thermal scanning (25–95°C at 1°C/min) was performed using a Bio-Rad iQ5 real-time PCR instrument, and fluorescence values were measured every 10 s. The melting curve was plotted, and the temperature of the derivative curve peak was the Tm value of the protein. A Tm shift greater than 2°C compared to the Tm of the untreated protein was considered to be statistically significant (Krishna et al., 2013).

One microliter of different concentrations of ABBA was mixed with 19 μL of recombinant protein in Eppendorf (EP) tubes; the blank control group was treated with dimethyl sulfoxide (DMSO) instead of ABBA. The tubes were incubated at room temperature for 10 min. Pronase E was prepared on ice and added into each tube. The blank control group was treated with TNC buffer. Tubes were placed at 4°C for 30 min. Finally, 3 μL of cold 20 × protease inhibitor solution was added into the tubes to stop the reaction. The results were analyzed by SDS-PAGE.

The X-ray crystallographic structure of the Coa-thrombin complex (PDB ID:1NU7) was used as a template for building the homology model of vWbp with Discovery Studio 2017 (Accelrys Inc., San Diego, CA, United States). The structure of ABBA was obtained from the PubChem database (CAS number: 12777-70-7). The parameters were at their default setting in the docking tab, and the grid maps were constructed to be large enough to include the binding sites of vWbp as well as significant regions of the surrounding surface. After molecular docking, the conformation with the highest consensus scoring pose was selected as the most likely binding conformation.

Molecular dynamics (MD) simulation was performed to simulate the interactions between vWbp and ABBA using Discovery Studio 2017. The system was centered in a cubic box of TIP3P water molecules (Jorgensen et al., 2001). The system was first minimized by 5000 steps of steepest and 5000 steps of conjugate gradient. Then, the system was heated to the target temperature of 300 K for a period of 20 ps in constant pressure, periodic boundary conditions (NPT). Then, the program was set to equilibrate the system by 5 ns of constant pressure and temperature (NPT) with a time step of 2 fs, which was followed by 6 ns of production simulation performed under the same conditions. A cut off of 14 Å was used for non-bonded interactions, and long-range electrostatic interactions were treated by means of the particle mesh Ewald (PME) method (Darden et al., 2001). The MD simulation results were analyzed using Discovery Studio 2017.

The previously described in vitro catheter infection model was used with some minor modifications (Vanassche et al., 2013). Sterile, polyurethane, triple-lumen, central venous catheters were cut into 2-mm fragments. These fragments were placed in a suspension of either wild-type S. aureus Newman or S. aureus Newman vWbp-knockout strains (ΔvWbp) at an OD₆₀₀ of 1.0 and incubated on a shaking platform at 37°C for 30 min. After being rinsed with sterile NaCl (0.9%), the catheters were placed in 1 ml of fresh rabbit plasma spiked with fibrinogen containing heparin with or without ABBA. After 24 h, catheters were rinsed with sterile NaCl (0.9%) and fixed overnight. Following a 2-h post-fixation period in 2% OsO₄, the samples were dehydrated with a series of ethanol concentrations. After overnight immersion in hexamethyldisilazane, the samples were coated with platina and visualized using a JEOL 7401F scanning electron microscope (JEOL Europe, Zaventem, Belgium) at 2.0 kV.

Mice were bred and maintained under specific-pathogen-free conditions. The animal experiments were approved by and conducted in accordance with the principles of the Basel Declaration and the guidelines of the Animal Care and Use Committee of Jilin University. Eight-week-old C57BL/6J mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd.

Female mice were divided into three groups: Newman, Newman + ABBA and ΔvWbp. After being anesthetized by ether, the mice were infected intranasally with 30 μL of a 6 × 10⁸ colony-forming units (CFU) S. aureus suspension. After 2 h, infected mice were subcutaneously injected with 100 mg/kg of ABBA or the same volume of DMSO with a 12-h interval. For survival research, mice were observed closely for signs of impending death at 12, 24, 36, 48, 60, 72, 84, and 96 h, if they appeared moribund (hunched posture, inability to move, dirty fur, labored breathing and unresponsive to external stimuli), mice were euthanized by carbon dioxide inhalation and were considered non-survivors. The isobole exponents were determined using the isobologram equation. For determination of CFU counts, mice were euthanized by carbon dioxide inhalation at 24 h post-infection. Left lungs were excised, weighed and homogenized for bacterial CFU counting by the serial dilution and plating method. Left lungs were fixed in 4% formalin and submitted for histopathological sectioning and haematoxylin-eosin staining, and the samples were then visualized by light microscopy.

The experimental data were performed using GraphPad Prism 5.0 (GraphPad Software) and were assessed using independent Student’s t-test with SPSS 22.0 statistical software. P values < 0.05 were considered statistically significant.

To determine the inhibitory effect of ABBA, the tube coagulation assay and the agarose plate assay were performed. The result of the tube coagulation assay showed the coagulation time increased in a dose-depend manner (Figure 1A), indicating that ABBA can inhibit the coagulase activity of vWbp. Serial dilutions of ABBA were added to tubes only conclude rabbit plasma. There were no coagulation in tubes after 24 h (Supplementary Figure 1A). In the agarose plate assay, fibrinogen is converted to fibrin to form turbid halos in the plates. The agarose plate assay was used to confirm the inhibitory activity of ABBA against vWbp. Figure 1Ba shows that the sizes of the coagulation zones increased with vWbp concentrations increasing from 0.625 to 10 mg/ml. The sizes of zones 2–5 were 83.24, 68.46, 54.68, and 38.02% of the size of well 1 (Figure 1Bb). The results indicate that vWbp converts fibrinogen to fibrin in a dose-dependent manner. Based on Figure 3A, 5 mg/ml vWbp was chosen for the subsequent experiment. Serial dilutions of ABBA (128, 64, 32, and 16 μg/ml) were added to wells 2–5 (Figure 1Bc). The sizes of zones of 2–5 were 30.39, 64.57, 88.67, and 92.02% of the size of well 1 (Figure 1Bd); 128 and 64 μg/ml of ABBA could inhibit the coagulase activity of vWbp significantly. Serial dilutions of ABBA were added to wells only conclude protein buffer. There were no turbid halos in the plates after 12 h (Supplementary Figure 1B).

The MICs of ABBA on S. aureus Newman were determined to be greater than 1024 μg/ml via the broth microdilution method. The growth curves of S. aureus Newman with or without ABBA (512 μg/ml) did not differ significantly. The growth curve of the vWbp mutant also did not differ significantly from that of S. aureus Newman (Figure 2B). These results indicate that ABBA could be a potential small-molecule inhibitor of vWbp that is effective at a concentration far lower than its MIC.

To examine whether the inhibitory effect of ABBA on S. aureus-induced coagulation by affecting the expression of vWbp, we extracted whole cell proteins of S. aureus cultures treated with 16, 32, 64, 128, and 256 μg/ml ABBA. The vWbp expression levels were determined by Western blotting. The gray values of blot of the different treated samples were further analyzed for confirmation of the expression levels. The results revealed that the expression levels were similar at different concentrations of ABBA (Figures 3A,B). The result shows that ABBA did not affect vWbp expression.

Binding with low-molecular-weight ligands can increase the thermal stability of a protein, as described by Koshland et al. (1958). Proteins unfold when heated. SYPRO orange binds to hydrophobic surfaces non-specifically, leading to an increase in fluorescence intensity. The fluorescence intensity starts to decrease after maximum binding is achieved. In general, a curve shift greater than 2°C is considered a statistically significant change in measured Tm when determining ligand-binding affinity. In Figure 4A, the blue curve is the melting curve of vWbp, and the red curve is the melting curve of vWbp incubated with ABBA (64 μg/ml). Comparison of the blue and red curves shows that Tm value changed from 39 to 50°C. This 11°C shift is statistically significant. The results showed that ABBA can improve the thermal stability of vWbp, indicating that ABBA can directly interact with vWbp.

Binding of drugs has been proposed to stabilize target proteins, either globally or locally. This stabilization can occur due to a specific conformation or simply via the masking of protease recognition sites, thereby reducing the protease sensitivity of the target protein. DARTS is a general methodology for identifying and studying protein-ligand interactions. The principle of this technique is that when a small molecule compound binds to a protein, the interaction make the protein becoming protease resistant by stabilizing the protein structure (Lomenick et al., 2009). DARTS was performed to further identify the direct binding of ABBA to the vWbp. As shown in Figure 4B, when the concentration of pronase E was 10 mg/ml, vWbp was digested completely. Addition of ABBA protected vWbp from digestion, and the digestibility was associated with the concentration of ABBA. The results demonstrate that ABBA can directly interact with vWbp and stabilize vWbp. Furthermore, with increasing drug concentration, the drug molecules binding to the vWbp protein also increased.

Previous research has demonstrated the interaction between vWbp and ABBA. To investigate the mechanism of this interaction, the vWbp-ABBA complex was equilibrated after 20 ns of MD simulation. The root-mean-square deviation (RMSD) values of vWbp were plotted as a curve (Figure 5A). The results showed that the vWbp structures of all the systems were stabilized during the simulations. The theoretical binding mode of ABBA and vWbp is shown in Figure 5B. ABBA adopted a compact conformation, binding at the “central cavity” in the elbow of vWbp. Specifically, as shown in Figures 5C,D, the hydroxyl group at the 2-position in the diene of ABBA formed conventional hydrogen bonds with the residues His-71 and Ala-72. The hydroxyl group at the 2-position in the phenyl group of ABBA formed conventional hydrogen bonds with the residue Gly-73. The phenyl group of ABBA exhibited a pi-anion interaction with residue Glu-75. Another phenyl group of ABBA exhibited pi–pi stacking with residue Tyr-83. Moreover, the residues Arg-70, Tyr-74, and Gln-87 can form van der Waals interactions with ABBA. In brief, the above MD simulations provided a rational prediction of the interactions between ABBA and vWbp.

The results of scanning electron microscopy showed the presence of abundant adherent material on catheter fragments inoculated with S. aureus Newman WT as well as heparin-spiked plasma for 24 h (Figure 6, top row). The adherent biomaterial consisted of fibrin and dispersed cocci. The amount of adherent material was significantly reduced in the presence of ABBA (128 μg/ml). Only a small number of cocci were observed to be directly adhered to the catheter surface without any surrounding matrix (Figure 6, bottom row). Similarly, the ΔvWbp strain was unable to induce this rapid deposition of adherent biomaterial (Figure 6, middle row).

Von Willebrand factor-binding protein has been reported to be an important virulence factor in mouse models of S. aureus pneumonia (Sawai et al., 1997). Based on the in vitro observation that ABBA can inhibit the blood coagulation activity of the vWbp, a mouse model of S. aureus pneumonia was established to investigate the in vivo protective effects of ABBA. Mice were infected with 30 μL of S. aureus Newman (2 × 10⁸ CFU/10 μL) by intranasal administration. Then, the mice were subcutaneously injected with either DMSO or 100 mg/kg ABBA every 12 h. The mice infected with ΔvWbp strain were used as negative controls. The survival was monitored every 12 h. And the result shows that mice treated with ABBA were resulted a significant increase in survival (P < 0.01) than mice treated with DMSO (Figure 7A). 70% of mice in ABBA group were survived over the 96 h experiment. And there were no death in ΔvWbp group and ΔvWbp + ABBA group.

The numbers of CFU in the lungs were counted to evaluate the effects of ABBA in the treatment of pneumonia. As shown in Figure 7B, the CFU counts in the lungs infected mice treated with ABBA (6.62 ± 1.01 log CFU/g) were significantly lower than those in the lungs of mice treated with DMSO (9.38 ± 1.24 log CFU/g) (P < 0.01). As a comparison, the CFU counts in the lungs of mice infected with the ΔvWbp strain were also low (6.21 ± 0.36 log CFU/g). The CFU counts in lungs infected with the ΔvWbp strain and treated with ABBA (5.97 ± 0.65 log CFU/g) were also significantly lower than Newman group, which indicate that there were no off-target effects. In addition, the lung tissues of mice treated with ABBA were pink, soft and elastic, while those of mice treated with DMSO were dark red, swollen and inelastic (Figure 7C). Further histopathological research showed that more alveolar destruction, pulmonary hyperaemia and greater inflammatory cell infiltration occurred in infected mice treated with DMSO than in those treated with ABBA (Figure 7D). The results revealed that ABBA can reduce infection and prevent mice from contracting S. aureus pneumonia.