Staphylococcus aureus strain USA 300 and S. aureus ΔSrtA strain USA 300 ΔSrtA, obtained from our lab, were used in the present study and cultured in brain-heart infusion (BHI) broth (Sigma) at 37°C. Chalcone was purchased from the Tianjin Yifang S&T Co., Ltd. (Tianjin, China). The fluorescent peptide Dabcyl-QALPETGEE-Edans was purchased from GL Biochem (Shanghai, China).

The DNA sequence encoding the WT-SrtA protein was amplified from S. aureus USA 300 genomic DNA and used as a template along with the corresponding primers (Table 1) to amplify the S. aureus SrtA sequence through PCR. The PCR products were digested with Nde1 and BamH1 and then cloned into the pGEX-6P-1 expression vector. The pGEX-6P-1-srtA plasmid encoding WT-SrtA was generated after the sequence was confirmed by DNA sequencing. Site-directed mutagenesis for V168A-SrtA, I182A-SrtA, and R197A-SrtA was carried out using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The mutagenic primer pairs employed to produce the three mutants are listed in Table 1.

WT-SrtA and all mutant constructs were transformed into Escherichia coli BL21 (DE3) cells. Subsequently, the transformants were grown and selected in Luria-Bertani (LB) media with ampicillin (100 mg/L) at 37°C. To induce protein expression, 1 mM IPTG (Sigma) was added into the bacteria suspension when the OD (600 nm) reached 0.6–0.8, followed by overnight growth at 16°C. The bacteria were harvested through centrifugation at 3,000 × g for 30 min at 4°C and resuspended in the reaction buffer (50 mM Tris-HCl, 5 mM CaCl₂ and 150 mM NaCl, pH 7.5). The cell debris was removed through centrifugation at 10,000 × g for 1 h at 4°C after sonication. The supernatant was applied to a self-packaged GST-affinity column (2 ml glutathione Sepharose 4B) (GE Amersham Biosciences, Piscataway, NJ, USA). First, the reaction buffer was used to remove the unbound contaminating proteins, then Precision Protease was used to digest the GST-tagged protein at 4°C overnight. Finally, the reaction buffer was used again to wash off the target protein. The point mutations V168A-SrtA, I182A-SrtA, and R197A-SrtA were expressed and purified similarly to WT-SrtA.

To measure the activity of WT and mutant SrtA, a FRET assay was used. As detailed earlier (Tonthat et al., 1999), 100 μl of a mixture consisting of the reaction buffer, purified proteins and different concentrations of chalcone were added in the appropriate order to 96-well plates and incubated for 30 min at 37°C. Subsequently, upon the addition of the fluorescent peptide substrate Dabcyl-QALPETGEE-Edans, the reaction was initiated, and after incubation for 1 h at 37°C, the fluorescence was read using emission and excitation wavelengths of 350 and 520 nm, respectively.

The minimum inhibitory concentration (MIC) of chalcone against S. aureus was determined by broth microdilution according to the NCCLS guideline M₃₁-A₂. For growth curve plotting, 1 ml of the overnight bacterial cultures was transferred (1: 50) to BHI broth containing different concentrations of chalcone. The absorbance reading was taken at OD (600 nm)_.

Overnight cultures of S. aureus wild-type strain (WT strain) were inoculated 1: 1000 into fresh BHI broth and grown to an OD (600 nm) of 1.0 with chalcone or dimethyl sulfoxide (DMSO, as the solvent control) at 37°C. Meanwhile, the USA300 ΔSrtA strain (WTΔSrtA strain) was used as the positive control. The bacteria were fixed with a 4% formaldehyde solution for 20 min after centrifugal collection (3,000 × g for 5 min) and washed twice with PBS. The bacteria were then resuspended in PBS containing a 1: 25 dilution of FITC-labeled goat anti-rabbit IgG (eBioscience) and incubated for 2.5 h at room temperature. Subsequently, the cells were washed three times with PBS and added to poly-L-lysine-coated glass slides. The SpA-related fluorescence was observed using a confocal laser-scanning microscope (Olympus, Shanghai, China).

The WT strain was grown in BHI broth to an OD (600 nm) of 0.5 with chalcone or DMSO in a shaking incubator with a shaker rate of 190 rpm at 37°C; the WTΔSrtA strain was used as a positive control. The bacteria were collected centrifugally (3,000 × g for 5 min), and after washing with PBS three times, the cells were resuspended using PBS and adjusted to an OD (600 nm) of 1.0. Fibronectin from bovine plasma with a concentration of 2 μg/ml was added into 96-well flat-bottom polystyrene microtiter plates, 100 μl in every well, and incubated overnight at 4°C. After washing three times with PBS, 100 μl of bovine serum albumin (BSA) at a concentration of 5% was added into the plate and incubated for 2 h at 37°C. The plates were washed three times afterwards, then 100 μl of the bacterial suspension was added and incubated for 2 h at 37°C. Subsequently, after removing the suspension and washing three times with PBS, 100 μl of crystal violet with a concentration of 0.4% was added and incubated for 30 min at 37°C. The absorbance of the plates was then read at 570 nm with a microplate reader (Tecan, Austria) after the plates were washed again and dried.

The WT strain was grown in BHI broth to an OD (600 nm) of 0.6 with chalcone or DMSO with a shaker rate of 190 rpm at 37°C (the WTΔSrtA strain was used as the positive control), and then, 10 μl of the bacterial solution was added into the 96-well flat-bottom polystyrene microtiter plates containing 290 ml BHI of broth and 3% (w/v) sucrose with or without chalcone. The mixture was incubated anaerobically under still culture conditions for 18 h at 37°C. After incubation, the liquid containing the bacteria and medium was removed, followed by the addition of 100 μl of 10% formaldehyde solution, which was then left overnight at room temperature to fix the biofilm. Subsequently, the formaldehyde was removed, and each well was stained with 100 μl of 0.1% v/v crystal violet for 30 min at room temperature. After rinsing with double distilled water and drying, 200 μl of 33% acetic acid was added to each well and all of the contents were mixed manually. The absorbance of the plates was subsequently read at 490 nm.

J774 cells were suspended in DMEM / HIGH GLUCOSE supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Invitrogen), and approximately 3 × 10⁵ cells were seeded into each well of 24-well flat-bottom polystyrene microtiter plates containing 12 mm diameter coverslips overnight (37°C, 5% CO₂). The WT strain was grown in BHI broth to an OD (600 nm) of 1.0 with chalcone or DMSO in a shaking incubator with a shaker rate of 190 rpm at 37°C; the WTΔSrtA strain was used as the positive control. The bacteria were then adjusted to an OD (600 nm) of 1.0, and 1 ml of the bacterial solution was added into each well and incubated for 60 min at 37°C. After each well was washed three times with PBS, 1 ml of DMEM / HIGH GLUCOSE supplemented with 300 μg / ml gentamicin was added and incubated for 30 min at 37°C. The J774 cells on the coverslips were then lysed in the sterile distilled water and plated onto BHI broth agar plates for CFU after washing three times with PBS. The plates were placed overnight at 37°C.

In this work, the initial structure of SrtA was obtained from the X-ray crystallography 3D structure (PDB code: 3CI5). To obtain the starting structure of the ligand/SrtA complex for a molecular dynamics (MD) simulation, a standard docking procedure for a rigid protein and a flexible ligand was performed with AutoDock 4 (Morris et al., 2009; Hu et al., 2010). Subsequently, the molecular dynamics simulation of the complex's systems was performed, and the details of the processes of the computational biology method were described in a previous report (Dong et al., 2013; Lv et al., 2013; Niu et al., 2013).

In our paper, the fluorescence-quenching method was used to measure the binding constants (K_A) of ligands with proteins. A 280-nm excitation wavelength with a 5-nm bandpass and a 345-nm emission wavelength with a 10-nm bandpass were used for the measurements. Details of the measurements were described previously (Bandyopadhyay et al., 2002; Jurasekova et al., 2009).

Overnight cultures of the WT strain were inoculated 1: 100 into fresh BHI broth and grown to an OD (600 nm) of 0.3 at 37°C. DMSO or different concentrations of chalcone were then added for further culture. To determine the effect of chalcone on the hemolysis activity of S. aureus, 1 ml of supernatant was harvested (3,000 × g, 5 min), and 100 μl of the bacterial culture supernatants were incubated with rabbit erythrocytes whose final concentration was 2.5% in PBS at 37°C for 20 min, after which the samples were centrifuged (10,000 × g, 1 min). Finally, the release of hemoglobin was measured at OD (543 nm).

To determine the effect of chalcone on hemolysis induced by the bacterial culture supernatants, the bacterial supernatants were pre-cultured with different concentrations of chalcone as described above.

An equal volume of the bacterial culture supernatants that were harvested in the hemolysis assay was resolved using SDS-PAGE, and the proteins were transferred onto PVDF membranes. After blocking in 5% non-fat milk for 2 h, the membranes were incubated with a primary rabbit anti-Hla antibody (Sigma-Aldrich) diluted 1: 8,000 for 2 h and a horseradish peroxidase-conjugated secondary antibody (Proteintech) diluted 1: 4,000 for 2 h. The signals were visualized on a Tanon-4200 imager using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK).

The WT strain with DMSO or various concentrations of chalcone was cultured and grown to an OD (600 nm) of 2.5 at 37°C. As described previously (Qiu et al., 2011), the total RNA from the cultured bacteria was isolated and then reverse transcribed into cDNA using the Takara RNA PCR kit (AMV), ver. 3.0 (Takara, Kyoto, Japan). According to the manufacturer's instructions, the PCR reactions were performed in 25-μl volumes using SYBR Premix Ex Taq TM (Takara). The 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France) was used to assess the PCR amplification. All primer pairs used for this assay conformed with a previous study (Qiu et al., 2011). It is worth noting that the housekeeping gene, gyrBRNA, was used as an endogenous control to normalize the expressional levels between the samples.

Hla, as a vital factor, has been shown to participate in mediating A549 cell injury and death (Bubeck and Olaf, 2008). For this reason, A549 cells were cultured and transferred into 96-well flat-bottom polystyrene microtiter plates at a density of 2 × 10⁴ cells per well in 200 μl of culture medium and incubated with the bacterial suspensions harvested above. After a 5-h incubation at 37°C, the Live/Dead (green/red) reagent (Roche) and the Cytotoxicity Detection kit (LDH, Roche) were used to assess the therapeutic effect of chalcone on A549 cells. The images of the Live/Dead cells were acquired using a confocal laser-scanning microscope, and the release of LDH was determined on a microplate reader (Tecan, Austria) at 490 nm.

The mice (6- to 8-week-old-female C57BL/6J) used for the animal experiments were obtained from the Experimental Animal Center of Jilin University. The animal experiments were approved by and conducted in accordance with the guidelines of the Animal Care and Use Committee of Jilin University. Overnight cultures of S. aureus were inoculated 1: 100 into fresh BHI broth and grown to an OD (600 nm) of 0.6 at 37°C. After centrifugal collection (3,000 × g for 5 min) and washing three times with PBS, the bacteria were resuspended in PBS to the required concentration for survival studies. Bacteria (4 × 10⁸ CFUs) were dropped into the left nare of the mice. To investigate the therapeutic effect of chalcone, the mice were treated with a subcutaneous injection of chalcone of 150 mg/kg after infection with S. aureus and then at 12-h intervals thereafter. The control mice were treated with equal volumes of DMSO simultaneously. The mortality analysis was monitored after 96 h, with ten mice contained in each experimental group.

The statistical significance of the treated and control group were assessed using the log-rank tests for the survival curves, and for other assays, a Student's t-test was used. The differences were analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) statistical software, and the differences were considered statistically significant when P < 0.05. The data are presented as the mean ± SD.

Chalcone (Figure 1A), one of the effective components of herbal plants, has been identified to be a remarkable inhibitor against bacteria virulence factors (Wallockrichards et al., 2015; Li et al., 2016). In this study, chalcone was shown to be a potential inhibitor against S. aureus SrtA and Hla. A FRET assay was used to determine the inhibitory activity of chalcone against SrtA, and the result indicated that the inhibition of SrtA activity was significant in the presence of various concentrations of chalcone (Figure 1A). We determined that the half maximal inhibitory concentration (IC₅₀) of chalcone was 53.15 μM. It is worth noting that the minimum inhibitory concentration (MIC) of chalcone against the tested S. aureus strain was >4,864 μM. Meanwhile, the growth of S. aureus USA 300 was not visibly affected by chalcone at concentrations sufficient to inhibit SrtA (Figure 1B).

Furthermore, a hemolysis assay was used to determine the effect of chalcone on the hemolysis activity of the bacterial cultural supernatants. The results showed that chalcone at the concentrations tested in this study could significantly decrease the hemolysis activity of S. aureus in a dose-dependent manner when co-cultured with USA 300 strain (Figures 2A,B). Notably, minimal hemolysis was detected when the strain was cultured with 38 μM chalcone, and the IC₅₀ value for chalcone-induced inhibition of Hla was 17.63 μM. In addition, pre-incubation with chalcone had almost no effect on the hemolytic activities of the bacterial culture supernatants (Figures 2A,B), implying that chalcone-induced inhibition of Hla might act by means of blocking its expression. To verify this, a Western blotting assay with the bacterial culture supernatants harvested above and a real-time RT-PCR assay were performed. In line with our conjecture, the Hla levels in the supernatants were reduced in a dose-independent manner (Figure 2C), and under our experimental conditions, the transcription levels of the hla gene and the agrA gene in USA 300 were both down-regulated by chalcone in a dose-independent manner (Figure 2D). Taken together, our results established that chalcone was able to effectively inhibit SrtA activity and the expression of Hla while exerting little antimicrobial activity, implying that chalcone could represent an effective anti-infective agent for S. aureus infection.

Staphylococcus aureus SrtA anchors many different surface proteins in the cell wall, including SpA, a multifunctional molecule responsible for binding the Fcγ portion of host immunoglobulins (Falugi et al., 2013). Therefore, SpA is vital for immune evasion, and because of its function, a change in the amount of SpA in the cell wall after the addition of chalcone requires investigation. In this assay, S. aureus cultured with or without chalcone was stained with FITC-labeled goat anti-rabbit IgG, which made the cells display a green color whose strength was determined by the amount of SpA in the cell wall. With the aid of confocal laser-scanning microscope, the content variation of SpA in the cell wall was investigated. The result showed that the WTΔSrtA strain, used as a control, displayed much less SpA on its surface compared with the WT strain, and as expected, 76 μM chalcone present in the cell culture caused significant reductions in the SpA display (Figure 3). These consequences indicated that chalcone was capable of disturbing the assembly of sortase-mediated SpA in the cell wall.

In the previous study, we have drawn the conclusion that chalcone can visibly inhibit the SrtA-catalyzed transpeptidation at relatively low concentrations. The fibronectin-binding proteins FnBPA and FnBPB are LPXTG proteins expressed by S. aureus. Fibronectin (Fn) is regarded as a bridge between the bacterial adhesion FnBP and the mammalian cell integrin, accelerating the process of phagocytosis, thereby stimulating the internalization of bacteria; consequently, Fn and FnBPs play a vital role in the process of invading host cells (Foster and Höök, 1998; Roche et al., 2003). Because FnBPs anchored by S. aureus SrtA can make bacteria invasive, if their display in the cell wall is blocked by weakening SrtA activity, then bacterial invasion would be decreased. For this reason, we employed Fn-binding assays to determine if chalcone could reduce the adherence of S. aureus to Fn. As expected, the WTΔSrtA strain had a minimum adhesion rate to Fn owing to the damage to its Fn-binding function and no inhibition was observed in the samples treated with chalcone (Figure 4A). However, when the WT strain was treated with chalcone at different concentrations, the adhesion rates decreased in turn, respectively, indicating that the effect occurred in a dose-dependent manner. The variance between the 38 μM chalcone group and the WT group was a very significant difference, as was the 76 μM chalcone group (Figure 4A).

Bacteria enhance their ability to resist antibiotics and other antimicrobial agents by embedding in biofilms (Pugliese and Favero, 2002; Cerca et al., 2005; Høiby et al., 2010). Biofilm phenotypes are promoted by the FnBPs, the major cell wall autolysin or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) (O'Neill et al., 2007, 2008; Clarissa Pozzi et al., 2012). To investigate whether chalcone hindered biofilm formation, S. aureus biofilm formation was determined with or without chalcone. After dye treatment, the WTΔSrtA group was much more lightly stained than the WT group, and the visible difference between the biofilms formed under the conditions of untreated and treated with 76 μM chalcone was quite significant (Figure 4B). These results were in accordance with those of the quantitative analysis (Figure 4C). In summary, chalcone reduced biofilm formation, suggesting that the inhibitory effect of chalcone on S. aureus SrtA had occurred.

SrtA function is normally important for the display of sortase-mediated surface proteins, thus enabling bacteria to invade the host cells. To examine how chalcone affects S. aureus invasion, J774 cells were cultured with bacteria and chalcone at different concentrations. Not surprisingly, the numbers of bacteria entering the cells in the WTΔSrtA group showed a significant decrease compared with the WT group (Figure 4D). Consistent with the hypothesis, the addition of a dose of chalcone to the experimental system significantly decreased the quantity of bacteria, indicating that chalcone weakened the S. aureus invasion by inhibiting SrtA activity (Figure 4D).

Through the computational biology method, the potential binding mode of chalcone with SrtA in the active site was explored in this study. The chalcone was bound to SrtA, and according to the binding mode given (Figures 5A,B), it was clear that chalcone could bind to SrtA via Van der Waals and electrostatic interactions. During the time course of the simulation, chalcone could localize to the catalytic pocket of SrtA (residue 160–200). In detail, the binding model of chalcone with SrtA revealed that chalcone could form strong interactions with Val166, Gly167, Val168, Ile182, Val193, and Arg197, respectively. The complex was found to reach equilibrium at 100 ns based on the analysis of the root-mean-square deviations (RMSD) of the backbone C_α atoms (Figure 5C), which indicated that the complex system has reached equilibrium.

To explore the energy contributions from the residues of the binding sites in the SrtA- chalcone complex, the energy decomposition was calculated for the SrtA-chalcone complex system. Val168, Ile182 and Arg197 had a strong total binding energy contribution, with a ΔE_total of < -1.0 kcal/mol (Figure 5D). In addition, residues Gly167 and Trp194 also had the appreciable total binding energy contribution, with a ΔE_total of < -0.8 kcal/mol. These results suggested that these five residues were key residues for chalcone.

To confirm these theoretical results, the total binding free energy for the SrtA- chalcone complex and their detailed energy contributions calculated according to the MM-PBSA approach are summarized in Table 2. According to the calculation results, the binding free energy, ΔG_bind, of the interaction between chalcone and the protein decreased in the following order: WT-SrtA > V168A-SrtA > R197A-SrtA > I182A-SrtA, which means that WT-SrtA had the strongest ability to bind to chalcone. Using fluorescence spectroscopy quenching, we measured the ΔG_bind and the number of binding sites between chalcone and the three mutants, and these results were highly consistent with those obtained by computational methods (Table 2). To further validate the simulation results, three mutants, V168A-SrtA, I182A-SrtA, and R197A-SrtA, were constructed for the FRET assays. As expected, when compared with WT-SrtA, chalcone was significantly less sensitive for these three mutants (Figure 5E). These results indicated that the information generated by the MD simulation on the SrtA-Chalcone complex was reliable. Due to the binding of inhibitor, chalcone, with the activity region (residues of Gly167, Val168, Ile182, Cys184, Trp194, and Arg197), the biology activity of SrtA was inhibited.

In our co-culture system, the potential protective effect of chalcone on the Hla-induced injury of A549 cells was determined. In the Live/Dead assay, uninfected cells retained green fluorescence (Figure 6A), and inversely, injured cells retained red fluorescence. As expected, 38 μM chalcone showed an obvious reduction in cell injury, with almost no cytotoxic effect observed in A549 cells (Figures 6B–E). Simultaneously, the LDH assay showed that treatment with chalcone could significantly decrease the release of LDH into the supernatants in a dose-independent manner compared with the control group, indicating less cell death (Figure 6E). Above all, the results showed that chalcone had a protective effect on Hla–mediated A549 cell injury.

To investigate the effect of chalcone on the survival rate of mice inoculated with S. aureus, we performed survival experiments. Ninety-six hours after infection with 4 × 10⁸ CFUs of bacteria, only 20% of the WT-infected mice survived, in contrast to the WTΔSrtA group in which the survival rate was 100% (Figure 7). In the chalcone-treated group, the survival rate was significantly higher than in the group without treatment (Figure 7). The death of mice in the WT+chalcone group occurred only at 36, 48, and 72 h in the whole process of infection (Figure 7). These findings suggested that chalcone could prolong survival of the mice in S. aureus–induced infection.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.