Quercetin protects rats from catheter‐related Staphylococcus aureus infections by inhibiting coagulase activity

Abstract Coagulase (Coa) activity is essential for the virulence of Staphylococcus aureus (S aureus), one of the most important pathogenic bacteria leading to catheter‐related bloodstream infections (CRBSI). We have demonstrated that the mutation of coagulase improved outcomes in disease models of S aureus CRBSI, suggesting that targeting Coa may represent a novel antiinfective strategy for CRBSI. Here, we found that quercetin, a natural compound that does not affect S aureus viability, could inhibit Coa activity. Chemical biological analysis revealed that the direct engagement of quercetin with the active site (residues Tyr187, Leu221 and His228) of Coa inhibited its activity. Furthermore, treatment with quercetin reduced the retention of bacteria on catheter surfaces, decreased the bacterial load in the kidneys and alleviated kidney abscesses in vivo. These data suggest that antiinfective therapy targeting Coa with quercetin may represent a novel strategy and provide a new leading compound with which to combat bacterial infections.

In previous studies, methicillin-resistant S aureus (MRSA) was reported to be resistant to vancomycin after the application of methicillin for the treatment of penicillin-resistant S aureus, 7 emphasizing the essentiality of developing new strategies to control S aureus infections.
S aureus can express a wide array of surface and secreted proteins, one of the most important of which is coagulase (Coa). Coagulase, which is secreted by S aureus, has the ability to form clots when inoculated into blood containing heparin. 8 The cleavage of fibrinogen to fibrin induced by Coa is critical for the establishment of S aureus infection. 9 When a catheter is inserted into a blood vessel, the catheter surface is rapidly coated with fibrinogen. Then, Coa coverts fibrinogen into fibrin fibrils to protect bacteria from opsonophagocytic clearance. This results in pathogens adhering to and remaining on the surface of intravascular catheters, and it is vital to the pathogenicity of CRBSI. 10 Coagulase is not essential for the growth of S aureus; therefore, targeting Coa would reduce the possibility of the development of resistance. In recent years, it has been reported that Coa activity can be inhibited by the thrombin inhibitors dabigatran 11 and argatroban. 12 As physiological thrombin inhibitors, they may have side effects, such as the blood not properly coagulating.
In this study, a blood coagulation assay was used to screen for anti-Coa molecules among approximately 200 natural compounds, and quercetin was found to have effective inhibitory activity against Coa. Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a five hydroxyl antioxidant flavonoid, processes anti-inflammatory, anticancer and pro-metabolic pharmacological functions. 13 Here, the role of Coa in S aureus infection and the potential therapeutic effect of quercetin by inhibiting Coa in CRSBI were further determined.

| Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are described in Table 1. Staphylococcus aureus strains were grown in a brain-heart infusion medium that was supplemented with chloramphenicol (10 μg/mL) when required. Escherichia coli strains were grown in Lysogeny Broth medium that was supplemented with ampicillin (100 μg/mL) when required.

| Preparation of recombinant Coa
The full-length coding sequence of mature Coa was cloned into the pET15b vector using the primers Coa_foward_XhoI (GAACTCGAGTCTAGCTTATTTACATGG) and Coa_reverse_BamHI (GTAGGATCCTGGGATAGAGTTACAAAC) to obtain His 6 -Coa.
The Tyr187Ala, Leu221Ala and His228Ala of Coa were generated by site-directed mutagenesis using the Coa-pET15b plasmid as Escherichia coli BL21 (DE3) harboring the expression vectors were grown at 37°C and induced with 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG). Following their induction, the cells were centrifuged at 4000 rpm for 30 minutes, suspended in 1× column buffer (0.1 mol/L Tris-HCl pH 7.5, 0.5 mol/L NaCl) and lysed by an ultrasonic disrupter.
The lysates were centrifuged at 12 000 rpm for 1 hour, and the supernatant was subjected to Ni-NTA affinity chromatography, washed with column buffer with 40 μmol/L imidazole and eluted with 500 μmol/L imidazole. The protein was concentrated and stored at −80°C.

| Construction of a Coa deletion mutant of the newman strain
The coa gene in the S aureus Newman strain was inactivated by allelic exchange as previously described. 14  and Spc-r (GCGGAATTCGTTTTCTAAAATCTGAT) from the plasmid pSET2s. These three fragments were mixed, digested with EcoRI and NcoI and ligated at 4°C for 1 hour. Using the primers Up-srtA-f and Down-srtA-r, a 2.0-kb fragment of the ligation product was amplified by PCR, digested with BamHI and SalI, inserted into pBT2 and used for allele replacement as previously described. 14 The mutation was confirmed by PCR sequence analysis and Western blotting analysis based on the Newman strain and its Coa mutant. The coa knockout strain showed a normal growth rate in Brain Heart Infusion (BHI) broth.

| Determination of the minimum inhibitory concentration and growth curves
The minimum inhibitory concentration (MIC) of quercetin against S aureus investigated by broth microdilution. 15 To plot the growth curves of S aureus, 1 mL of S aureus cultured overnight was added to 50 mL of sterile BHI broth with or without quercetin (256 μg/mL).
The absorbance was measured at 600 nm via Infinite ® F200 PRO.

| Blood coagulation
To evaluate whether quercetin can inhibit the blood coagulation activity of the Coa from S aureus, a tube coagulation assay based on fresh rabbit blood containing 1% heparin was performed. Ten In the plate coagulation assay, 0.9% blood agarose plates were prepared containing 0.4% PEG8000, 3 mg/mL bovine fibrinogen and and a negative control was performed using 2 μL of DMSO. The coagulation areas were measured after incubation at 37 C for 18 hours.

| Thermal shift assay
To verify the interaction between the protein and quercetin, a thermal shift assay was performed. First adequate Cypro-Orange (5000×) was diluted 100 times and mixed with the target protein on ice. Then, 4 μL of the above sample was mixed with 2 μL of quercetin and 14 μL of protein buffer in PCR tubes.

| Catheter fibrin deposition in scanning electron microscopy
The previously described catheter fibrin deposition model was used with some minor modifications. 10

| In vivo catheter infection model
Rats were bred and maintained under specific pathogen-free conditions. All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University. Briefly, female Wistar rats (200-220 g) were divided into the following three groups: Newman, Newman + quercetin and Δcoa, with six rats in each group. These rats were anaesthetized with 10% Nembutal given intraperitoneally. Using cutaneous antisepsis, an incision was made to expose the left external jugular vein.

| Statistical analysis
All experimental data were analysed using GraphPad Prism 5.0 (GraphPad Software). Values are expressed as the mean ± standard error of the mean (SEM, n = 3). The statistical significance was assessed with a one-way ANOVA (Dunnett's t test) and a two-tailed Student's t test. P values <0.05 were considered statistically significant.

| Confirmation of the Coa mutant
To determine the importance of Coa in S aureus pathogenesis, homologous recombination was used to generate the Coa mutant in S aureus Newman. The Coa mutant was first confirmed by PCR ( Figure 1A). Western blotting analysis with an anti-Coa antibody revealed that the parent Newman stain but not the coa mutant expressed Coa ( Figure 1B).

| Quercetin has no effect on the growth of S aureus
The MICs of quercetin (Chengdu Herbpurify Co., Ltd., purity > 98%) against both S aureus strains (S aureus Newman and the Coa mutant) were greater than 1024 µg/mL, suggesting that this compound has no effect on bacterial viability. Furthermore, growth curves of S aureus Newman and the Coa mutant showed the same growth state with or without 256 μg/mL quercetin (Figure 2). Taken together, our results revealed that bacterial growth was undisturbed by quercetin even at a concentration of 256 μg/mL.

| Quercetin inhibits S aureus Coa activity
A test tube coagulation assay was performed to investigate whether quercetin can inhibit Coa activity. Recombinant His 6 -Coa was purified by affinity chromatography on Ni-NTA. When Coa (100 nmol/L) was added to rabbit blood with heparin, the blood coagulated within 10 minutes ( Figure 3A). However, when recombinant protein was added to rabbit blood mixed with heparin and quercetin, quercetin interfered with the coagulation of the blood. When the concentration of quercetin was 64 μg/mL, the coagulation was prolonged to 6 hours. When the concentration of quercetin was 128 μg/mL, the blood remained without clots for more than 12 hours ( Figure 3A).
The plate coagulation assay was further employed to measure the ability of quercetin to inhibit Coa. A turbid halo was seen in the plate due to the change in fibrinogen into a network of fibrin. As shown in the left panel of Figure 3B,

| The inhibitor quercetin thermally stabilizes Coa
To identify whether quercetin can thermally stabilize Coa, the apparent melting curve was plotted to show the potential binding of fluorescent dye Cypro-Orange and Coa. The protein starts to unfold when warmed, the dye binds to exposed hydrophobic parts of the protein, and a fluorescent signal is released. The fluorescence F I G U R E 1 Construction of coagulase (Coa) mutant in the Newman strain. A, PCR products of the parent Newman strain and its mutant stain Δcoa. B, Western blotting with anti-Coa antibody showed the presence of Coa in the wild-type Newman strain but not in the mutant strain Δcoa intensity reaches a maximum and then starts to decrease. The plotted curve shifts right when the protein becomes more stable. As shown in Figure 4, the blue curve indicates the melting curve of Coa, and the red curve is the melting curve of Coa mixed with quercetin (64 μg/mL). It is notable that the value of Tm changed from 39 to 45°C, representing a significant shift of 6°C ( Figure 4). In this study, a shift in ΔTm value greater than 2°C was considered to be significant. Taken together, our results revealed that quercetin could thermally stabilize Coa, suggesting that direct binding occurs between quercetin and Coa.

| Determination of the binding mode of Coa with quercetin
Based on the docking results, we performed a 200-ns molecular dynamics (MD) simulation of Coa-quercetin complex to determine the preferential binding mode of Coa with quercetin. To explore the dynamic stability of the models and to ensure the rationality of the sampling strategy, the root-mean-square deviation (RMSD) values of the protein backbone based on the starting structure throughout the simulation time were calculated and plotted in Figure 5A, indicating that all the protein structures were stabilized during the simulations.
Quercetin acted as a ligand to bind with Coa via intermolecular interactions, and localized to the active site of Coa, over the time course of the simulation. Figure 5B illustrates the predicted binding mode of quercetin to Coa, and the electrostatic potential of the residues surrounding the binding site was plotted using apbs software. 20 In detail, the binding model of quercetin with the active site of Coa ( Figure 5B) revealed that the 4H-chromen-4-onemoietyof quercetin formed strong π-π interactions with both the benzene ring of Tyr187 and the side chain of Pro227. Moreover, the hydroxyl group of His228 could form a hydrogen bond with quercetin, which was confirmed by energy decomposition analysis ( Figure 5B).

The above information indicated that the stabilization of the
Coa binding pocket in the complex was mainly due to the residues Tyr187, Pro227 and His228 ( Figure 5B).

| Identification of the binding site in the Coaquercetin complex
To gain more information about the residues surrounding the binding site and determine their contribution to the whole system, we  Figure 6A and B).
The total binding free energies of the Coa-quercetin complex according to the MM-GBSA method were summarized in Table 2.
For quercetin, the ΔG bind was estimated to be −7.93 kcal/mol, in- Furthermore, the activity of WT Coa and its mutants was almost identical, as evidenced by coagulation was observed for the WT Coa and its mutants ( Figure 6C). However, the sensitivity of inhibition of the coagulation of Coa mutants was much lower than the WT Coa ( Figure 6D). Thus, consistent with prediction derived from our molecular modelling, quercetin inhibited Coa coagulation by interacting with residues H228, Y187 and L221.

| Quercetin reduces adherent biomaterial deposition on catheters in vitro
A catheter fibrin deposition model was used to study whether

| Quercetin inhibits catheter colonization and metastatic infectious complications in rats
To investigate whether quercetin-mediated protection against cath-  Figure 8A).
Consistent with the bacterial load on the catheters, the bacterial load in the kidneys from the rats treated with quercetin was significantly lower than that in the kidneys from the rats injected with DMSO only (mean load [±SD], 5.76 ± 0.20 log CFU/g vs 7.13 ± 0.16 log CFU/g; P < 0.01, Figure 8B). Although a statistically significant reduction was observed for the bacterial load on the catheters between the Newman-infected rats that received quercetin and the rats that received the mutant Δcoa (mean load [±SD], 4.33 ± 0.59 log CFU /cm vs 3.70 ± 0.34 log CFU/cm; P < 0.01, Figure 8A), the difference in the bacterial load in the kidneys was not significant (mean load [±SD], 5.76 ± 0.20 log CFU/g vs 5.61 ± 0.28 log CFU/g; P > 0.05, Figure 8B).
In agreement with the in vitro results, the kidneys of Newmaninfected rats showed many visible surface abscesses with destruction of the renal unit and obstruction of the renal tubules ( Figure 8A).
However, significant alleviation of that pathological damage was observed in the infected rats that received quercetin or those infected with the Δcoa mutant ( Figure 8C). Taken together, our results established that quercetin protected rats from catheter-related infections by targeting Coa.

| D ISCUSS I ON
Coagulase is characterized as a virulence factor in S aureus, one of the important pathogenic bacteria leading to CRBSI. 22  Quercetin, a flavonoid found in plants such as fruits, vegetables and cereals, possesses pharmacological functions including those that are anti-inflammatory, anticancer and pro-metabolism. 13 Here, quercetin was also proven to possess the ability to inhibit S aureus virulence by targeting Coa. However, the bioavailability of this flavonoid limits its clinical application due to its relatively low solubility in water. 27 Thus, the modification of quercetin into increase its solubility is a prerequisite for the clinical application of this flavonoid.
Here, a novel strategy against S aureus CRBSI targeting Coa with quercetin was revealed, and this antiinfective therapy would supplement antibiotic therapy without increasing selective pressure on S aureus because Coa is not vital factor for bacterial viability.