Galangin as a direct inhibitor of vWbp protects mice from Staphylococcus aureus‐induced pneumonia

Abstract The surge in multidrug resistance in Staphylococcus aureus (S. aureus) and the lag in antibiotic discovery necessitate the development of new anti‐infective strategies to reduce S. aureus infections. In S. aureus, von Willebrand factor‐binding protein (vWbp) is not only the main coagulase that triggers host prothrombin activation and formation of fibrin cables but also bridges the bacterial cell wall and von Willebrand factor, thereby allowing S. aureus to bind to platelets and endothelial cells, playing a vital role in pathogenesis of S. aureus infections. Here, we have identified that galangin, a bioactive compound found in honey and Alpinia officinarum Hance, is a potent and direct inhibitor of vWbp by coagulation activity inhibition assay, thermal shift assay and biolayer interferometry assay. Molecular dynamic simulations and verification experiments revealed that the Trp‐64 and Leu‐69 residues are necessary for the binding of galangin to vWbp. Significantly, galangin attenuated S. aureus virulence in a mouse S. aureus‐induced pneumonia model. In addition, we also identified that galangin can enhance the therapeutic effect of latamoxef on S. aureus‐induced pneumonia. Taken together, the results suggest that galangin may be used for the development of therapeutic drugs or utilized as adjuvants to combine with antibiotics to combat S. aureus‐related infections.

proteinaceous toxins. 6 Numerous experiments have confirmed that interfering with S. aureus virulence is a compelling approach for combating S. aureus-associated infections because it exerts less evolutionary pressure on bacteria than traditional strategies, thereby reducing the risk of development of resistance. 7,8 A distinctive feature of coagulase-positive S. aureus isolates is their ability to clot blood. This phenotype is caused by coagulase (Coa) or von Willebrand factor-binding protein (vWbp), both of which can bypass the physiological coagulation cascade and directly activate thrombin. 9 During host infection, either Coa or vWbp can bind to exosite I of prothrombin to form a coagulaseprothrombin complex referred to as staphylothrombin, in which Coa or vWbp activates prothrombin by changing its conformation. 10 Active staphylothrombin converts fibrinogen to insoluble fibrin, 11 forming a fibrin meshwork. Staphylothrombin-mediated fibrin generation contributes to colonization by S. aureus of the vascular wall, evasion of immune killing and spread of S. aureus via the bloodstream to all organ systems. 12 The inhibition or deletion of coagulases can significantly reduce the disease-causing potential of S. aureus and ameliorate disease progression in subcutaneous abscess sepsis, catheter infection and endocarditis in preclinical disease models, 13,14 indicating that S. aureus coagulases are appealing druggable targets for the treatment of S. aureus infections. Furthermore, the absence of Coa or vWbp does not affect the growth of S. aureus, and inhibition of these proteins does not exert selection pressure to promote the development of resistance. Therefore, inhibitors of coagulases have great research value for the treatment of S. aureus infections. 15 Although coagulase has been recognized as an important virulence factor of S. aureus, to date, few effective clinical inhibitors of coagulase have been reported. Our preliminary work of anticoagulation test identified galangin as an anti-Sa-vWbp molecule from 232 traditional Chinese medicines stored in our laboratory. In this study, we found that galangin (3,5,7-

| Bacterial strains, growth conditions and chemicals
The reference strains used in this study were S. aureus Newman D2C (ATCC 25904). The vWbp mutant strain (ΔvWbp) was constructed previously 16 and stored in the laboratory. Staphylococci were cultivated in brain-heart infusion (BHI) broth or on tryptone soya broth (TSB) agar plates at 37°C. E. coli strains DH5α and BL21 (DE3) were grown in Luria-Bertani (LB) broth or on LB agar plates at 37°C with agitation. When necessary, ampicillin (100 μg/ml) was added to the LB broth, while chloramphenicol (10 μg/ml) was added to the BHI broth. Galangin was acquired from Chengdu Ruifensi Biotech Company.

| Minimum inhibitory concentration and growth curve
The minimum inhibitory concentration (MIC) for galangin against S. aureus Newman was determined using the microdilution method in accordance with the standard M100-S15 proposed by the Clinical and Laboratory Standards Institute (CLSI). 17 For growth curve plotting, an overnight culture of S. aureus was inoculated into fresh BHI broth (1:100) containing 256 μg/ml galangin and incubated at 37°C for 24 h. The absorbance values were measured at 600 nm.

| Cloning, expression and purification of recombinant vWbp
The DNA sequence encoding vWbp was amplified by polymerase chain reaction (PCR) from genomic DNA from the S. aureus Newman strain using the primer pair vWbp-F/vWbp-R. The PCR product was digested with BamHI and XhoI and cloned into the pET15b expression vector via the same enzyme restriction sites, yielding pET15b-vWbp. After confirmation by DNA sequencing, pET15b-vWbp was transformed into the E. coli BL21 (DE3) strain.
Briefly, overnight cultures of BL21 (DE3) cells were diluted in LB broth to obtain an OD 600 of ~0.1 and cultured until an OD 600 between 0.6 and 0.8 was reached. The expression of recombinant vWbp was induced with 0.5 mM isopropylthioβ-D-galactoside (IPTG) for an additional 12 h at 16°C. The cells were harvested, and the bacterial sediment was resuspended in a buffer containing 0.1 M Tris-HCl (pH 7.5) and 0.5 M NaCl and lysed by sonication. The lysates were subjected to centrifugation at 12,000 g for 60 min. The recombinant His-tagged vWbp protein was purified by a 6 × His/Ni-NTA system (His Trap; GE Healthcare) as described previously. 18 In addition, site-directed mutagenesis to produce the substitutions D70A, W64A, L69A and M83A in vWbp was performed with a site-directed mutagenesis kit (TransGen Biotech).
All primers are shown in Table 1. The mutations were determined by DNA sequencing, and the detailed protein expression and purification process used for the mutant proteins was identical to that used for the wild-type (WT) protein.

| In vitro coagulase activity assays
To evaluate the coagulation activity of recombinant vWbp, plate and tube coagulase activity assays were conducted. For tube coagulase activity assays, 10 μl of purified vWbp in PBS (100 μM) was mixed with 190 μl of anticoagulated rabbit blood with heparin sodium, and 2.5 μl of different concentrations of galangin in glass tubes. The final concentrations of galangin were set as 0, 16, 32, 64, 128 and 256 μg/ ml. Blood clotting was observed by tilting the tubes after incubation at 37°C and recording the time required for coagulation. For the plate coagulation assay, sterile agarose plates containing 0.9% agarose, 0.4% PEG 8000, 3 mg/ml bovine fibrinogen and 1% rabbit blood were prepared, and wells that were approximately 1 mm in diameter were punched into the agarose plates with a gel puncher before the assay.

Different concentrations of recombinant protein were distributed into
the wells in the agarose plates. The precipitation of fibrin was measured after incubation of the plates at 37°C overnight.

| Preparation of a polyclonal antiserum against vWbp
Domestic rabbits were immunized with 0.4 mg of recombinant vWbp combined with Freund's complete adjuvant by multisite subcutaneous injection. Two boosters of 0.4 mg of vWbp with Freund's incomplete adjuvant were administered similarly at 2-week intervals. After completing the immunization procedure, the rabbits were anaesthetized, and the blood sera were collected by carotid intubation and stored in 0.1% sodium azide at -20°C. anti-rabbit antibody, and the protein bands were detected by a gel imaging system. Images were captured using a KODAK DCS 315 digital camera.

| Thermal shift assays
The

| Fluorescence quenching assay
The binding constant (K A ) values for galangin with the various proteins were detected using fluorescence quenching assays as previously described. 24 The excitation wavelength was set at 280 nm, and the fluorescence spectra were scanned in the wavelength interval of 280-400 nm with excitation and emission slits of 5 nm. The fluorescence intensity value at 292 nm was recorded, and the K A was calculated.

| Mouse model of S. aureusinduced pneumonia
Animal experiments were carried out according to the ethical standards and approved protocols of the Animal Welfare and Use Committee of Jilin University. Inbred C57BL/6J mice aged 7 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd.
The S. aureus-induced pneumonia model was established as described previously. 25

| Statistical analysis
GraphPad Prism 5.0 was used for analysis of the experimental data.
Experimental data were evaluated using a one-way ANOVO in SPSS 22.0 statistical software. A value of p < 0.05 was considered statistically significant.

| Galangin is an inhibitor of vWbp
To investigate the inhibitory effect of galangin on the clotting ability of vWbp, a tube coagulase activity assay was first used to detect the effect of galangin against vWbp, as shown in Figure 1B

| Galangin does not affect the growth of S. aureus
To test whether galangin influences the survival of S. aureus, the MIC of galangin against S. aureus was determined. The MIC value of galangin against the tested strains was greater than 512 μg/ml.
Furthermore, the growth curve indicates that the growth status of S. aureus was not affected when 256 μg/ml galangin was present in the medium. No difference in the growth rate was observed between the S. aureus ΔvWbp strain and the WT strain ( Figure 2A).
Hence, galangin at a concentration lower than 256 μg/ml had no antibacterial effect on S. aureus in vitro.

| Determination of the binding of galangin to vWbp
The fluorescence-based thermal shift assay (TSA) is a general method used for investigating protein-ligand interactions based on the principle that the binding of small molecular compounds influences the thermal stability of proteins. Ligand binding usually induces a change in the conformational stability of proteins, which is reflected by a shift in the melting temperature (T m ). A shift in the T m greater than 2°C indicates significant binding. 26 To confirm the direct interaction between galangin and vWbp, the thermal stability of vWbp in the absence and presence of galangin was determined using TSA. As shown in Figure 3A, the addition of galangin shifted the T m of vWbp by 2.5°C, indicating that galangin directly binds to vWbp.
To further investigate the kinetic aspects of the interaction between galangin and vWbp, a BLI assay was performed. BLI is a reliable optical technique that investigates the interactions of proteins with other proteins or small molecules by analysing the interference pattern of the light reflected off the protein binding surface. 19 In our study, we used a Ni-NTA sensor to monitor the change in the

| Binding mode of galangin with vWbp
Using a molecular modelling approach, we studied the binding mode of galangin with vWbp. The flexibility of the residues of the vWbpgalangin system and those of free vWbp was studied according to the root mean square fluctuation (RMSF). As shown in Figure 4A,  did not affect the clotting activity of vWbp ( Figure 4D). However, the inhibitory effect of galangin on all mutants was significantly lower than its effect on WT vWbp. In particular, the sensitivity of W64A-vWbp and L69A-vWbp to galangin inhibition decreased most obviously ( Figure 4E).
We studied the binding affinity of galangin to WT vWbp and its mutants by a fluorescence quenching assay. The binding constant (K A ) between galangin and WT vWbp and the mutants was calculated. The results showed that the values of K A between galangin and the vWbp mutants all decreased, and the K A for W64A-vWbp and L69A-vWbp decreased most significantly ( Table 2). We further performed the thermal shift assay for the four mutant proteins with or without galangin ( Table 3). The addition of galangin increased the Tm of mutant proteins but at a lower level as compared with WT. L69A-VWbp (±) galangin and W64A (±) galangin showed less Tm shift than that of D70A-VWbp (±) galangin and M83A (±) galangin (Table 3), which was consistent with the results of the fluorescence quenching assay. These results indicated that L69 and W64 were the potential key sites for the binding of galangin to vWbp.

| Galangin has a protective effect against S. aureus-induced pneumonia
Given that coagulase-positive S. aureus strains are highly pathogenic and can cause fatal suppurative pneumonia 27 and that vWbp is an important virulence determinant in S. aureus infections, 28 Figure 5B). To assess the pathological relevance of galangin protection, histopathological analysis of the lung specimens was performed. As shown in Figure 5C, the lungs of infected mice were red and hard; however, the lungs of uninfected mice and galangin-treated mice were pink and spongy.
Histopathological examination revealed severe alveolar destruction and large numbers of inflammatory cells in the lung tissues of DMSO-treated infected animals, while mice treated with galangin showed a reduction in inflammation of the lungs, which manifested as reduced accumulation of inflammatory cells. In addition, there were no significant differences between the ΔvWbp group and the galangin treatment group. Together, these data established that galangin was a potent therapeutic agent against S. aureus-induced pneumonia.

| The combination of galangin with latamoxef has an improved therapeutic effect on S. aureusinduced pneumonia
As an antivirulence agent, galangin disarms bacteria to reduce  Figure 6A). The CFU count ( Figure 6B) in the lungs of infected mice in the galangin treatment group (7.01 ± 0.59 log 10 CFU/g) and the latamoxef treatment group (6.64 ± 0.56 log 10 CFU/g) was observably lower than that in DMSO-treated mice (9.74 ± 0.45 log 10 CFU/g, p < 0.001). The combination treatment greatly reduced the number of bacteria in the lungs, and the CFU count in this group was the lowest (5.50 ± 0.46 log 10 CFU/g). As shown in Figure 6C, the combination therapy significantly reduced lung tissue damage and inflammation compared with monotherapy. Together, these data suggest that galangin can enhance the efficacy of latamoxef and that the combination therapy had a better treatment effect on S. aureus infection than monotherapy.

| DISCUSS ION
In this study, we attempted to screen novel vWbp inhibitors from a library of phytochemicals from Chinese herbal extracts. Galangin is a natural polyphenolic compound that possesses a variety of pharmacological activities, such as anti-inflammatory, 29 antioxidant 21 and antifibrotic properties. 30 Although galangin inhibited the clotting activity of vWbp, the addition of galangin to bacterial cultures (up to 256 μg/ml) did not affect the growth of S. aureus (Figure 2A) or the expression of vWbp ( Figure 2B and 2C), indicating that galangin selectively inhibited the coagulase activity of vWbp and did not act by inhibiting the growth of S. aureus or vWbp expression. Moreover, the results of the TSA and the BLI assay demonstrated that galangin directly binds to vWbp ( Figure 3). Molecular modelling revealed that the Asp-70,  Staphylococcus aureus is a common pathogen causing pneumonia. Among the major isolates obtained from pneumonia patients, MRSA accounts for approximately half of the cases initially diagnosed as pneumonia, 31 leading to a reported mortality as high as 56%. 32 It has been reported that the upregulation of key virulence factors may lead to increased toxicity and pathogenicity based on a model of pneumonia caused by MRSA. 33  S. aureus infections often show reduced sensitivity to antibiotic treatment. 34 Previous studies have shown that the combination of staphylothrombin inhibitors with antibiotics results in enhanced reduction in bacterial load in catheters and kidneys, 35 indicating that antivirulence adjuvants could be used to increase the therapeutic efficacy of antibiotics. In this study, we found that galangin significantly improved the therapeutic effect of latamoxef. Compared with the monotherapy group, treatment with galangin combined with latamoxef significantly reduced the bacterial load in the lungs, the pathological changes in the lungs and the survival rate were significantly improved (Figure 6), suggesting that inhibition of vWbp by galangin can improve the efficacy of latamoxef against S. aureusinduced pneumonia.

Proteins
In conclusion, our findings demonstrated that galangin is a novel inhibitor of S. aureus vWbp and can be used alone or in combination with antibiotics to combat S. aureus infections.