Wogonin attenuates the pathogenicity of Streptococcus pneumoniae by double‐target inhibition of Pneumolysin and Sortase A

Abstract Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of respiratory disease in patients and can cause respiratory distress and other symptoms in severe cases. Pneumolysin (PLY) is a pore‐forming toxin that induces host tissue injury and inflammatory responses. Sortase A (SrtA), a catalytic enzyme that anchors surface‐associated virulence factors, is critical for S. pneumoniae virulence. Here, we found that the active ingredient of the Chinese herb Scutellaria baicalensis, wogonin, simultaneously inhibited the haemolytic activity of PLY and SrtA activity. Consequently, wogonin decreased PLY‐mediated cell damage and reduced SrtA‐mediated biofilm formation by S. pneumoniae. Furthermore, our data indicated that wogonin did not affect PLY expression but directly altered its oligomerization, leading to reduced activity. Furthermore, the analysis of a mouse pneumonia model further revealed that wogonin reduced mortality in mice infected with S. pneumoniae laboratory strain D39 and S. pneumoniae clinical isolate E1, reduced the number of colony‐forming units in infected mice and decreased the W/D ratio and levels of the inflammatory factors TNF‐α, IL‐6 and IL‐1β in the lungs of infected mice. Thus, wogonin reduces S. pneumoniae pathogenicity by inhibiting the dual targets PLY and SrtA, providing a treatment option for S. pneumoniae infection.

or new strategies to treat diseases such as pneumonia caused by S. pneumoniae infection has become a hot topic.
In the past, only capsular polysaccharides 5,6 were considered the main virulence factor of S. pneumoniae. Further studies have shown that the virulence factors [7][8][9][10][11] of S. pneumoniae also include hyaluronate lyase (Hyl), pneumolysin (PLY), choline-binding protein A (CbpA) and sortase A (Sortase A). Among them, pneumolysin, a cytolytic toxin belonging to the cholesterol-dependent cytolysin (CDC) family, 12 Streptococcus pneumoniae sortase A (SrtA) 17 is a cysteine transpeptidase whose main role is to cleave between the threonine (T) and glycine (G) residues of the LPXTG motif in the surface protein 18 and anchor the resulting protein to the cell wall. Sortase A (SrtA) is a group of membrane-bound transpeptidases that are widely distributed in Gram-positive bacteria and covalently bind surface proteins to the peptidoglycan of the corresponding cell wall, which play a key role in bacterial survival and pathogenicity. Chang and colleagues reported that SrtA-deficient mutants (ΔsrtA) 19 formed fewer biofilms and were less able to immobilize fibronectin, fibrinogen and vitronectin. Song and colleagues found that EGCG 20 inhibits inflammatory responses in S. pneumoniae-infected mice by inhibiting PLY and SrtA activity. Jianfeng Wang found that a natural compound, quercetin, 21 a SrtA inhibitor, reduces S. pneumoniae virulence by reducing biofilm formation.
Currently, the antivirulence activity of natural compounds is receiving increasing attention. Wogonin is a flavonoid that is widely found in the traditional Chinese medicines Scutellaria baicalensis Georgi and Scutellaria barbata. According to a recent study, 22 flavonoids regulate the accumulation of reactive oxygen species and possess anti-inflammatory and antimicrobial properties. Zheng and colleagues 23 proposed that wogonin may ameliorate renal inflammation in subjects with diabetic nephropathy by inhibiting the NF-κB and TGF-β1/Smad3 signalling pathways. Other researchers have investigated whether wogonin is able to treat acute lung injury, 24,25 asthma, 26,27 leukaemia, 28,29 lung cancers 30 and other diseases.
Nevertheless, the potential effects of wogonin on S. pneumoniae have not been reported.
In this article, we found that wogonin is a dual-target inhibitor of PLY and SrtA in S. pneumoniae. Wogonin treatment effectively inhibited PLY activity and alleviated the SrtA-mediated adhesion of S. pneumoniae to host cells, and we further investigated the potential therapeutic effects and mechanisms of wogonin in cells and mouse models.

| Haemolysis experiment
Ten microliters of PLY(3 mg/mL, 1:10-1:1000 for activity detection, prepared as previous described) were mixed with wogonin (0, 16, 32, 64 and 128 μg/mL respectively) in 195 μL of PBS, and incubated with 5 μL of sheep blood erythrocytes (purchased from Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 10 min. 20,31 Next, the supernatant was removed by centrifugation at 3000 rpm for 5 min, and the absorbance at OD 543 nm was measured. The PBS and 1% Triton X-100 treatment groups were used as negative controls and positive controls respectively. The OD 543 nm value of the positive control culture was regarded as 100%, the negative control culture was regarded as 0% and the percent hae- Additionally, the supernatants of S. pneumoniae D39 and S. pneumoniae E1 with an OD 600 nm = 0.8 were collected by centrifugation (3000 × g) at 4°C for 5 min. And the influence of wogonin on the haemolytic activity of supernatants was examined as described above.

| MIC determination
Streptococcus pneumoniae D39 or S. pneumoniae E1 was incubated in a 37°C incubator until reaching an OD 600 nm of approximately 0.5. THB was used to dilute the bacterial broth and dispense it such that the OD 600 nm was 0.1. Different concentrations of wogonin or antibiotics (chloramphenicol, amikacin, kanamycin, tetracycline, polymyxin E, gentamycin and vancomycin purchased from Shanghai Yuanye Bio-Technology Co., Ltd.) were mixed according to the broth microdilution method prior to an incubation for 12 h at 37°C with 5% CO 2 . The MIC indicates the lowest concentration of the tested antibiotics at which the microorganism did not show visible growth.

| Determination of S. pneumoniae growth
Streptococcus pneumoniae D39 or S. pneumoniae E1 was inoculated into THY medium at 37°C and grown until reaching an OD 600 nm of 0.3. Then, the bacteria were further cultured with different concentrations of wogonin. The OD 600 nm was measured every hour for 6 h.
Finally, all the data were plotted as a curve.

| Analysis of the inhibition of SrtA peptidase activity
Evaluation of the inhibitory effect of wogonin on Sortase A (prepared by Song and colleagues previous described) was performed

| Biofilm formation assay
Streptococcus pneumoniae D39 was inoculated into 2 mL of fresh THY medium and incubated overnight at 37°C and with 5% CO 2 .
Different concentrations of wogonin (0, 16, 32 and 64 μg/mL) were added once the culture reached logarithmic growth (OD 600 nm = 0.4, 1:100), and the culture (500 μL) was transferred to 24-well plates following an 12 h incubation and washed three times with PBS. One well was supplemented with 400 μL of 0.1% crystal violet staining solution 33 and incubated for 1 h. Then, the crystal violet was dissolved by adding 200 μL of 33% glacial acetic acid (v/v). And the OD value at 570 nm was measured.
After washing the biofilm three times with PBS, 200 μL of trypsin were added to each well, and the samples were incubated at 37°C for 5 min. Then, 800 μL of sterile water was added to each well, and the mixture was then transferred to a 2 mL centrifuge tube and shaken for 5 min with a microshaker. Ten microliters of the sample were diluted in a 10-fold gradient, applied to a blood agar plate and incubated at 37°C with 5% CO 2 for 24 h. The number of plated bacteria was counted.
The bacterial culture supernatant was collected by centrifugation (5000 × g, 10 min). Following an incubation with 5× loading buffer at 100°C for 20 min, the proteins in the supernatants were separated on 10% SDS-PAGE gels and transferred to PVDF membranes with a semidry transfer instrument. Then, the membranes were blocked with 5% skim milk powder at room temperature for 2 h. After an incubation with a mouse-derived anti-PLY monoclonal antibody

| Oligomerization analysis
The recombinant PLY protein was mixed with different concentrations of wogonin (0, 16, 32 and 64 μg/mL) in a constant temperature water bath at 37°C for 1 h. Then, 5 × β-mercaptoethanol-free loading buffer was added to each sample prior to another 10 min of incubation at 55°C. Following separation on 10% SDS-PAGE gels, PLY oligomerization was detected as described above.

| Reverse transcription PCR (RT-PCR) analysis
An RT-PCR assay was performed to determine whether the expression of the ply gene was affected by wogonin. First, S. pneumoniae D39 was cultured until reaching OD 600 nm = 0.3 and then different concentrations of wogonin were added and cells were grown to the postexponential growth phase. Next, total RNA was prepared and reverse transcribed into cDNAs using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China).
The S. pneumoniae 16S rRNA housekeeping gene was chosen as an internal control to quantify the expression level of the ply gene.

A549 cells (human lung epithelial cells, purchased from ATCC, USA)
were cultured in complete medium consisting of DMEM supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 after inoculation in 24-well plates with 5 × 10 4 cells per well for an overnight culture. S. pneumoniae D39 was inoculated into 2 mL of fresh THY culture medium, incubated overnight at 37°C with 5% CO 2 in the presence of different concentrations of wogonin, washed three times with PBS (pH 7.4) by centrifugation (5000 × g, 10 min) and resuspended in PBS for subsequent analysis.
Next, A549 cells were cocultured with the above S. pneumoniae D39 suspension at an MOI of 30. After coculture for 2 h at 37°C with 5% CO 2 , the culture supernatant was extracted from each well. Then, the A549 culture system was flushed three times with PBS (pH 7.4).
Next, the A549 cells were treated with 200 μL of 0.25% trypsin (containing 0.02% EDTA) and lysed with 800 μL of 0.02% Triton X-100. The number of S. pneumoniae D39 bacteria was calculated using the serial dilution and plate counting method. S. pneumoniae D39-infected samples incubated without wogonin were used as positive controls, and cells without any treatment were used as negative controls.

| Statistical analysis
Experimental data are presented as the means ± SD and were analysed using SPSS 22.0 statistical software (Chicago, IL, USA) as well as GraphPad Prism 8.0.2 software. Independent Student's t-test was adopted to determine statistical significance. Three replicates were analysed per sample, with *p < 0.05 and **p < 0.01.

| Wogonin inhibits the haemolytic activity of PLY
PLY is a virulence factor of S. pneumoniae that causes erythrocyte haemolysis. 34 As shown in Figure 1B use in the next step of screening inhibitors. Wogonin ( Figure 1A) is a flavonoid with broad pharmacological activities. After adding different concentrations of wogonin to the haemolytic reaction system, wogonin significantly inhibited the haemolytic activity of PLY in a dose-dependent manner ( Figure 1C,D). The IC 50 was 52.8 μg/mL. Thus, these results suggest that wogonin is a potent PLY inhibitor.
Furthermore, we tested the resistance of the S. pneumoniae laboratory strain D39 and S. pneumoniae clinical isolate E1 to different antibiotics. As shown in Figure 2A The haemolytic activity of PLY in the supernatants of S. pneumoniae D39 ( Figure 2C) and S. pneumoniae E1 ( Figure 2D) was also inhibited in a gradient-dependent manner after incubation with wogonin. Briefly, our results revealed that wogonin represents an effective inhibitor of PLY in S. pneumoniae, whose antibiotic resistance is increasing in the clinical setting.

| Wogonin reduces the peptidase activity of SrtA without affecting S. pneumoniae growth
The suppressive effect of wogonin on SrtA peptidase activity was defined by performing a FRET assay. Different concentrations of wogonin (0-128 μg/mL) were added to the SrtA activity detection system, and the SrtA peptidase activity decreased in a gradient ( Figure 3A). In addition, after the coculture of wogonin and S. pneumoniae, the membrane permeability of S. pneumoniae was increased with increasing wogonin concentrations ( Figure 3B). Furthermore, treatment with wogonin at concentrations that efficiently suppressed PLY activity and SrtA activity resulted in no visible effect on S. pneumoniae growth ( Figure 3C,D).
SrtA inhibitors exert a certain inhibitory effect on the formation of S. pneumoniae biofilms. 12,13 We assessed the effect of wogonin on S. pneumoniae D39 and S. pneumoniae E1 biofilm formation by performing crystal violet staining and counting biofilm colonies. As the concentration of wogonin increased, the colour of crystal violetstained wells became lighter ( Figure 4A,B), and the absorbance of the biofilm after acetic acid dissolution also decreased ( Figure 4C,D).
Simultaneously, the number of bacteria in the biofilm was also significantly reduced ( Figure 4E,F). In general, the findings revealed that wogonin is an effective SrtA inhibitor that does not affect S. pneumoniae growth.

| The dual-target inhibitory mechanism of wogonin attenuates S. pneumoniae-mediated cell damage
The dual inhibition of PLY activity and SrtA activity by wogonin prompted us to further determine the potential effect of wogonin on S. pneumoniae infection in vitro. We added different concentrations of wogonin to A549 cells ( Figure 5A) and J774 cells ( Figure 5B).
When the concentration reached 128 μg/mL, wogonin had little effect on cell viability. Wogonin treatment for 6 h had no obvious cytotoxic effect on A549 and J774 cells.
Then, the effect of wogonin on S. pneumoniae adhesion to A549 cells was detected by counting colonies. After counting colonies at 3 h and 6 h, we found that the number of S. pneumoniae D39 ( Figure 6A) and S. pneumoniae E1 ( Figure 6B) adhering to A549 cells decreased with wogonin treatment. Therefore, wogonin attenuates the ability of S. pneumoniae to adhere to and colonize A549 cells.
PLY not only destroys red blood cells but also directly damages epithelial cells. 35  wogonin to the culture system to evaluate whether it exerted a protective effect on A549 cells against PLY. Next, A549 cells were stained with live/dead (green/red staining) reagents. As shown in Figure 6C, almost all A549 cells died after coculture with PLY for 5 h. In contrast, 8 and 16 μg/mL wogonin showed weak protection against cell damage ( Figure 6D,E). When wogonin was added at 32 μg/mL ( Figure 6F) and 64 μg/mL ( Figure 6G), the survival rate of A549 cells was significantly increased in a concentration-dependent manner. The protective effect of wogonin on A549 cells was further determined by performing a lactate dehydrogenase (LDH) assay. Consistent with the above results, wogonin treatment exerted a similar protective effect on PLYmediated cytotoxicity in A549 cells ( Figure 6H). Thus, these findings established that PLY and SrtA are the potential targets as wogonin treatment for S. pneumoniae infection.

| Wogonin reduces PLY oligomerization
PLY is a pore-forming toxin belonging to the CDC family, whose members bind cholesterol in cell membranes and lyse cells through the oligomerization of soluble monomers to form relatively large pores. Western blot analysis showed that PLY oligomerization was significantly reduced after wogonin incubation in a dose-dependent manner ( Figure 7A). Furthermore, the expression of PLY in the bacterial supernatant and cells was not visibly affected by wogonin treatment ( Figure 7B-D). At the transcript level, the addition of wogonin had no effect on the transcription of ply the gene encoding PLY ( Figure 7E). These discoveries indicated that wogonin suppressed the haemolytic activity of PLY by inhibiting its oligomerization without affecting its transcription and expression.

| Wogonin protects mice from S. pneumoniae infection
To further validate the protective function of wogonin in vivo, we established the Streptococcus pneumoniae D39/E1 infection mouse pneumonia model. The survival of S. pneumoniae-infected mice was observed for 120 h. The 120 h survival rate of S. pneumoniae D39infected mice was 26.67%, which was increased to 73.33% after wogonin treatment ( Figure 8A). Similarly, the 120 h survival rate of S. pneumoniae E1-infected mice was only 33.33%, while the survival rate after wogonin treatment was 53.33% ( Figure 8B), suggesting that wogonin improved the survival rate of S. pneumoniae-infected mice. We then examined the extent of lung tissue damage in the target organ. The lungs of the mice in the infected group were darker in colour and obviously congested, while those in the uninfected group were pale pink with no congestion or oedema ( Figure 8C,D).
The lung tissue damage in the wogonin treatment group was significantly relieved ( Figure 8E). As shown in Figure 8F-H, we found that alveolar interstitial oedema, capillary congestion and adhesion

| DISCUSS ION
The ability of S. pneumoniae to spread and colonize in a host is a key aspect of pneumococcal population biology and a prerequisite for invasion. 10 traditional Chinese medicine extracts and main compounds 14,37,38 can effectively inhibit the synthesis of the PLY protein. In this experiment, we found that wogonin could significantly decrease the haemolytic activity of PLY. Furthermore, we also found that wogonin inhibited the haemolysis activity of PLY in the culture supernatant. Based on this finding, wogonin is a potent PLY inhibitor.

Furthermore, our results and other studies all found that clinical
Streptococcus pneumoniae isolate had higher resistance to common antibiotics such as amikacin and kanamycin than laboratory strains (such as S. pneumoniae D39). 39,40 Therefore, we provided a novel strategy to find dual-target inhibitors of PLY and SrtA to reduce pathogenicity without influencing the growth of S. pneumoniae. By In this study, we also found that wogonin improved the survival rate of PLY-treated A549 cells, with little toxicity to the cells. A sim-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no competing financial interests to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.