A potential bio‐control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O

Abstract Listeria monocytogenes (LM) is a classical model intracellular pathogen and the leading cause of listeriosis, which has long been a global public health issue. The successful infection of LM is related to a series of virulence factors, such as the transpeptidase enzyme sortase A (SrtA) and listeriolysin O (LLO), which are crucial for bacterial internalization and escape from phagosomes respectively. It is speculated that targeting multiple virulence factors may be due to a synergistic effect on listeriosis therapy. In this study, an active flavonoids component of Scutellaria baicalensis Georgi, baicalein, was found to potently block both listerial SrtA catalyzed activity and LLO hemolytic activity within 16 μg/mL. After pretreatment with baicalein, 86.30 (±11.35) % of LM failed to associate with Caco‐2 cells compared to the LM without preincubation (regarded as 100% internalization). Furthermore, baicalein addition may aid in bacterial degradation and clearance in macrophagocytes. During a 5 h observation, LM in cells incubated with baicalein showed significantly decreased vacuole escapes and sluggish endocellular growth. In addition, baicalein directly prevented LM‐induced cells injury and mice fatality (survival rate from 10.00% to 54.55% in 4 days post‐intraperitoneal injection). Taken together, as an antagonist against SrtA and LLO, baicalein can be further developed into a biotherapeutic agent for listeriosis.

widely as a model intracellular organism in infection biology and immunology, the study of listeriosis may be given to improve treatment of other gram-positive and intracellular bacterial infections. 1,9 LM employs an intracellular lifecycle and is ingested by professional and non-professional phagocytes (such as epithelial cells). The internalization of LM into epithelial cells is mainly based on the enzymatic activity of sortase A (SrtA). By using listeriolysin O (LLO), LM can lyse vacuoles and escape to reach the host cytosol and replicate. Finally, LM will move towards the membrane, form membrane protrusions and invade the adjacent cells. 10 LM can assemble adherence and internalization-related surface biotin to achieve internalization. The catalysing activity of SrtA, a kind of conservative cysteine transpeptidase enzyme widely existing in LM and other gram-positive pathogens, is involved in the process of surface proteins covalently attaching onto bacterial peptidoglycan. 11 SrtA is a target for anti-listeriosis drugs, which has been investigated to contribute greatly to bacterial pathogenicity, as the mutation of SrtA resulted in a remarkably decreased virulence in cell models and in mouse models compared to wild type or single mutant internalin strains. [12][13][14] Listeriolysin O (LLO), encoded by the Listeria hly gene, is a pore-forming toxin which is called "the Swiss army knife of Listeria. 15 " LLO is a cholesterol-dependent cytolysin (CDC) family member, is essential for lysing membrane-bound compartments in a pH-dependent manner and blocking phagosome-lysosome fusion. 16 Soluble LLO monomers bind to host membrane cholesterol, oligomerize and form large pores. 17,18 Strains with lower LLO expression failed to escape from phagosomes and had very slow replication in the internalization vacuole. However, hly mutants also displayed significantly attenuated phenotypes in in vivo infections. 18 The root of Scutellaria baicalensis Georgi, Skullcap root, is a popular and multipurpose Chinese medicinal plant with a long use history in China and other East Asian countries as a therapeutic agent for fever, allergic reaction, or bacterial/viral infections. 19,20 As Skullcap root was traditionally utilized by oral administration, it is possible to discover a potential biocontrol agent against food-born pathogen infection from Skullcap root exacts. Baicalein (5, 6, 7-trihydroxyflavone) is one of the major flavone constituents, which was reported to attenuate inflammation and cerebral cortex apoptosis, and prevent γ-induced neurogenesis impairment in recent researches. 21,22 The multifarious bioactivities of baicalein may be associated with the anti-infectious mechanisms of Skullcap root.
In this study, we investigated the impact of baicalein on LM infection both in vitro and in vivo. We first determined the anti-listerial SrtA peptidase activity of baicalein. The inhibition on SrtA significantly decreased colonization into epithelial cells. Simultaneously, baicalein decreased bacterial vacuole escape and multiplication owing to its efficient blockage of LLO. The inhibition of SrtA and LLO seems to be combined for cellular protection. Baicalein administration also was an effectual therapy in mouse models of peritoneal infection. Therefore, our results suggested that baicalein is a potential anti-LM infection compound.

| Peptidase activity assay
The inhibitory effect of baicalein on LM sortase A (SrtA) was determined using a fluorescence resonance energy transfer (FRET) assay.
Purified biologically active LM SrtA protein (stored at our lab) was

| Bacteria invasion analysis
For the invasion assay, Caco-2 cells were seeded and grown in 24-well-plates at a density of 3×10 5 per well and cultured overnight.  The non-adherent bacteria were moved by washing three times with PBS, and the remaining extracellular bacteria were killed with 20 μg/ mL gentamicin (dissolved in RPMI-1640 medium), followed by incubation for another 30 minutes. After three additional washes, the number of adherent and invasive bacteria was calculated by lysing Caco-2 cells with sterile water and streaking them onto TSB agar plates.

| Fluorescence labelling assay
Caco-2 cells were infected with LM as described in the invasion analysis assay at an MOI of 100:1 with or without baicalein addition.

| Haemolysis assay
Overnight ATCC 19115 cultures were expanded into fresh TSB at 1:100 with shaking for 2 h at 37°C, and then different concentrations of baicalein were added for further culture to an A600nm of 2.1. The supernatants were harvested by centrifugation (9 600 g, 2 min). Next, 200 μL supernatants or purified rLLO (20 ng/mL, stored in our lab) were first treated with different concentrations of baicalein at 37°C for 30 minutes in sterile acidic buffer (35 mmol/L Na 2 HPO 4 .12H 2 O, 0.125 mol/L NaCl, adjust pH value to 5.5 with acetic acid). Next, 2.5% rabbit erythrocytes (RBC) were added, followed by incubation for 15 minutes. The supernatant of the cocktail was harvested (10 000 rpm, 2 min) and measured. The A543nm of each sample was obtained after centrifugation (10 000 rpm, 1 min) to represent the haemolytic value. RBCs treated with ddH 2 O or acidic buffer served as a positive control or negative control, respectively. The percentage of lysed RBCs was determined by comparing each sample with the positive control, which was considered 100% hemolysis.

| Bacterial growth curve
An overnight LM culture was expanded into fresh TSB at 1:100 with shaking for 3 h at 37°C. And then the indicated concentrations of baicalein were added for further culture at an A600nm of 0.1. The A600nm value of each sample was analysed hourly with an ultraviolet spectrophotometer. This work continued for 6 h until the A600nm stopped increasing.

| Western blot assay
Bacterial culture was performed as described in the hemolysis assay.
After the A600nm reached 2.1, equal volumes of supernatants of different samples were harvested by centrifugation (2 400 g, 5 minutes). Next, 5×SDS-PAGE loading buffer containing β-mercaptoethanol(β-Me)was added, and the mixture was boiled for 10 minutes (100°C). Samples was separated by 12% SDS-PAGE, and the proteins were transferred onto polyvinylidene fluoride(PVDF) membranes. After blocking the membranes in a blocking buffer (5% BSA) for 2 h at room temperature, the membranes were first incubated with primary rabbit antibody against LLO (Abcam, diluted with blocking buffer at 1:1000) and then a peroxidase-conjugated secondary antibody (Proteintech, diluted with blocking buffer at 1:5000). The protein band signals were visualized with a Tanon-4200 imager, using ECL reagent (Pierce ™ ECL Western Blotting Substrate).

| Oligomerization analysis assay
Recombined LLO protein was preincubated with or without the indicated concentrations of baicalein at 37°C for 30 minutes with acidic buffer and high concentrations of chloridion. In vitro oligomerization of LLO was detected by Western blot assay, as previously described. 8,23 The optical density of high weight oligomers and LLO monomers was analysed with ImageJ software.

| Statistical analysis
All experimental data were presented as the mean ± SEM (n ≥ 3).  Figure 1A), was shown to significantly inhibit the activity of purified SrtA protein ( Figure 1B) in a concentration-dependent manner. To determine the potential influence of baicalein on LM internalization, an invasion assay was utilized. As expected, an obvious CFU decrease was observed in human intestinal Caco-2 cells infected with baicalein pretreated LM compared to cells infected with LM without preincubation (Figure 1C). After 1 hour of infection, extracellular and intracellular bacteria were visualized with immunofluorescence staining ( Figure 1D).
In the infected control group (without baicalein incubation), the minority of LM were stained both red and green, whereas most of the LM were stained with only red fluorescence, which indicated that the majority of cell-associated bacteria were intracellular. Consistent with the result of the invasion assay, samples infected with baicalein-pretreated LM were observed to have opposite staining, wherein the minority of cell-associated bacteria was stained red.
Consequently, these results suggest that baicalein is an LM SrtA inhibitor that efficiently attenuated LM internalization.

| Baicalein strongly inhibits LLO pore-forming activity by targeting oligomerization
The LM hemolysin, LLO, plays a special role in LM pathogenicity. To determine if baicalein targeted LLO, a haemolysis assay was performed to detect the effect of baicalein on the pore-forming activity of secreted LLO. Baicalein-incubated bacterial culture supernatant had significantly reduced the hemolytic activity, which occurred in a dose-dependent manner (Figure 2A). Bacterial growth was detected to investigate whether the decreased hemolytic activity was associated with the inhibition of bacterial survival ( Figure 2B). The results indicated that active concentrations on LLO hemolytic activity showed no significant influence on bacteria growth. A Western blot assay was then performed to investigate whether the effect of baicalein on LLO was due to an influence on LLO production. The LLO content in the LM culture supernatant was not significantly different between the baicalein-treated group and control group ( Figure 2C), which suggested that Baicalein may directly target LLO. Then, we performed a hemolysis assay with purified LLO protein. Baicalein incubated with LLO attenuated hemolytic activity ( Figure 2D). In addition, we performed an oligomerization assay to visual LLO oligomers and monomers by Western blot assays ( Figure 2E). When analysing the bands of oligomers and monomers, the baicalein-treated samples had increased monomers and decreased oligomers, indicating a reduced oligomerization ( Figure 2F,G)

| Baicalein incubation inhibits LM-induced cell injury
Based on the previous study, we assumed that baicalein may protect  A, The higher picture shows a visual image. The lower picture shows the inhibition of haemolytic activity of the bacterial culture supernatant by the indicated concentrations of baicalein. B, The growth curve of LM co-cultured with various concentrations of baicalein. C, Western blotting analysis of LLO expression in culture supernatant. Bacteria were cultivated as described above. The same volume of culture supernatant was harvested at an A600nm of 2.1 and treated as described in Western blot assay. D, The hemolysis assay was performed with purified LLO protein, as described, in an acidic buffer. E, The impact of Baicalein on LLO oligomerization visualized with a Western blot image. F and G, The optical density of LLO oligomers and monomers analyzed with ImageJ software. **, P < 0.01 Furthermore, preincubated LM showed less pathogenicity in Caco-2 cells than bacteria without baicalein pretreatment ( Figure 5C).
Together, baicalein efficiently protected cells from LM-induced injury.  25 Baicalein has been reported to be an inhibitor of Staphylococcus aureus biofilm formation, and we assumed that baicalein can also influence listerial virulence. 26 In this study, baicalein was determined to efficiently block listerial sortase A (SrtA) cleavage activity and listeriolysin O (LLO) haemolytic activity with a pre-incubation in two absolutely distinct assays, which suggested a potential influence of baicalein towards proteins, such as  Baicalein is an acknowledged flavonoid with multiple biological activities. Studies have suggested that baicalein was more suitable for oral administration in rat owing to the well and rapid absorption in stomach and small intestine. 28 In our study, baicalein was given to LM-infected mice with an oral dose of 200 mg/kg body weight in one day, significantly reduced bacterial colonization in livers and spleens, and protected mice from death with the dose showed no sign of toxicity. In fact, baicalein has been proved to be a safe agent with maximum tolerated dose of 15 400 mg/kg in mice, and be tolerated with single oral doses of 100-2800 mg by human subjects. 29,30 In conclusion, baicalein significantly inhibited bacterial colonization and multiplication by targeting both listerial virulence factor SrtA and LLO (Figure 7), attenuated LM-induced infection both in vitro and in vivo at an innoxious dose. All the results together demonstrated that baicalein might be a safe and potential efficient therapeutic for listeriosis.

CONFLI CT OF INTEREST
The authors have no conflict of interest to declare. F I G U R E 6 Baicalein protects mice from LM infection. 6-8-week-old mice were infected with LM intraperitoneally and orally treated twice a day with baicalein or with CMC as a control. A, Analysis of bacterial burdens in the liver and spleen. The mice were infected with sublethal doses of bacteria and were mercy-killed at 60 h pi (B) Survival analysis of infected or uninfected mice treated with baicalein. Mice were infected with lethal doses of bacteria and were observed every 8 h. **, P < 0.01