In vitro activity and In vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD target.

Staphylococcus xylosus (S. xylosus) is a type of coagulase-negative Staphylococcus, which was previously considered as non-pathogenic. However, recent studies have linked it with cases of mastitis in cows. Isoliquiritigenin (ISL) is a bioactive compound with pharmacological functions including antibacterial activity. In this study, we evaluated the effect of ISL on S. xylosus in vitro and in vivo. The MIC of ISL against S. xylosus was 80 μg/mL. It was observed that sub-MICs of ISL (1/2MIC, 1/4MIC, 1/8MIC) significantly inhibited the formation of S. xylosus biofilm in vitro. Previous studies have observed that inhibiting imidazole glycerol phosphate dehydratase (IGPD) concomitantly inhibited biofilm formation in S. xylosus. So, we designed experiments to target the formation of IGPD or inhibits its activities in S. xylosus ATCC 700404. The results indicated that the activity of IGPD and its histidine content decreased significantly under 1/2 MIC (40 μg/mL) ISL, and the expression of IGPD gene (hisB) and IGPD protein was significantly down-regulated. Furthermore, Bio-layer interferometry experiments showed that ISL directly interacted with IGPD protein (with strong affinity; KD = 234 μM). In addition, molecular docking was used to predict the binding mode of ISL and IGPD. In vivo tests revealed that, ISL significantly reduced TNF-α and IL-6 levels, mitigated the destruction of the mammary glands and reversed the production of inflammatory cells in mice. The results of the study suggest that, ISL may inhibit S. xylosus growth by acting on IGPD, which can be used as a target protein to treat infections caused by S. xylosus.

Introduction

Bovine mastitis is globally recognized as the most common and costly disease affecting dairy herds [1]. To date, more than 50 Staphylococcus species and subspecies have been characterized as causal agents of staphylococcal mastitis. The genus is divided into coagulase positive staphylococci and coagulase negative staphylococci (CNS) based on their ability to coagulate plasma [2]. Remarkably, CNS infection is increasingly recognized as the leading cause of clinical and subclinical dairy cow mastitis worldwide. At the same time, CNS species tend to be more resistant to antimicrobials than Staphylococcus aureus (S. aureus), and they easily develop multi-resistance [2, 3]. Staphylococcus xylosus (S. xylosus) is a CNS that is widespread in the skin of animals and man, and generally considered as a non-pathogenic bacterium [4, 5]. However, recent studies have shown that it is the main bacterial isolate in cows with cases of mastitis caused by CNS [6, 7]. Moreover, clinical isolates of S. xylosus have multiple drug-resistant phenotypes, which has made the treatment of dairy cow mastitis very difficult [6]. Consequently, the need to develop new drugs with novel antibacterial mechanisms is very vital in the treatment of Staphylococcal mastitis in dairy cows.

In order to solve the problem of bacterial resistance and develop new antimicrobial agents, researchers are exploring new frontiers including the disruption or abrogation of bacterial metabolic pathways. The relationship between bacterial metabolism and drug resistance has been well documented [8, 9]. Recent studies have shown that histidine is the product of an important nitrogen metabolism pathway in bacteria and imidazole glycerol phosphate dehydratase (IGPD) has been described as one of the key enzymes involved in its biosynthesis including acting as the first specific synthase in L-histidine synthesis pathway [10]. IGPD only exists in bacteria and plants [11, 12]. Moreover, it is associated with bacterial growth and biofilm formation [13, 14]. On the other hand, clinical treatment failure frequently occurs with the use of antibiotics due to the development of drug resistance [15]. The presence of antibiotic residues in milk significantly reduces its quality and has undesirable effects on human health [16-18]. Thus, targeting IGPD by a better and more effective alternative therapy is an important strategy in correcting the public health problem of excessive use of antibiotics and the menace of multi-drug resistant bacteria.

It has been reported that flavonoids such as baicalin can interfere with the formation of biofilm in S. xylosus by interfering with IGPD [19]. Flavonoids are a large class of natural compounds and have been extensively studied over the past decade for their antibacterial activity [20]. In some cases, flavonoids have showed up to six-fold stronger antibacterial activities than standard drugs in the market [21]. Isoliquiritigenin (ISL, Fig 1) is a flavonoid compound, which complies with the Lipinski‘s Rule of Five and has good anti-oxidation, anti-inflammatory, anti-tumor and antimicrobial activity [22-24]. Studies have shown that ISL has the potential to treat oral bacterial infections and inhibit the formation biofilms in S. aureus [25, 26]. At the same time, Gaur et al. observed that ISL has the potential to reverse the resistance of methicillin-resistant S. aureus [23]. Nevertheless, few studies have shown its effect on the growth of S. xylosus, the main bacterial isolates causing cow mastitis. As a natural Chinese medicine monomer, ISL is cheap, readily available, and has low toxicity [27, 28]. This study shows the antibacterial potential of Isoliquiritigenin against S. xylosus by regulating the expression of IGPD, and revealed the mechanism by which ISL attenuates the virulence of S.xylosus. In addition, the therapeutic effect of ISL against S.xylosus virulence was further determined in an animal mastitis model. This study will provide a scientific basis for the treatment of S. xylosus mastitis and the development of new drugs using Isoliquiritigenin in the future.

Fig 1

The chemical structure of isoliquiritigenin.

Materials and methods

Growth conditions and reagents

S. xylosus ATCC 700404 (from the American Type Culture Collection) and its IGPD mutant strain (obtained from the previous experiment in our laboratory by homologous recombination) were grown in Tryptic Soy Broth (TSB, Summus Ltd, Harbin, Heilongjiang, China) at 37°C with constant shaking [14]. For biofilm culture, the mid-exponential growth culture of S. xylosus ATCC 700404 (Wild-type strain and mutant strain) was diluted with TSB to an optical density of 0.1 at OD595, and 200 μL of the diluted growth culture was added to wells of a 96-well microplate (Corning Costar® 3599 Corning, NY, USA) at 37°C for 24 h. 98% pure ISL was purchased from Shanghai Sigma-Aldrich Trading Co., Ltd. and dissolved in DMSO. ISL was prepared to a concentration of 12,800 μg/mL and double dilutions was used to do inhibitory tests in vitro.

Minimum inhibitory concentration (MIC) determination

The MIC of ISL was determined in TSB using broth dilution micro method in a 96-well polystyrene microtiter plates [29]. 20 μL of ISL was obtained from the stock solution (12,800 μg/mL) and diluted to give a final concentration range from 1,280 μg/mL to 0.625 μg/mL. These were sequentially mixed with 180 μL of bacteria-containing TSB in a 96-well polystyrene microplate. Treated S. xylosus were incubated at 37°C in TSB and observed for turbidity. The MIC was considered as the lowest concentration of each ISL with no visible microbial growth after 24 h of incubation [30].

Time-dependent killing assay

The time-kill curves test was performed based on the method provided by Wang [31], with little modifications. Briefly, the S. xylosus suspensions were grown to an OD595 value of 0.5 (1 × 105 CFU/mL) in TSB. The ISL was added to the cultures to obtain final concentrations of 1/2 MIC, MIC, 2 MIC, 4 MIC and 8 MIC, respectively. A similar experiment without ISL treatment was set up to serve as the control. Cell survival was determined by plating duplicate 10-fold serial dilutions for each sample at appropriate time points and counting the colonies after incubation for 24 h at 37°C. The detection limit was 1.0 log10 CFU/mL.

Determining the effects of ISL on biofilm formation

The procedure was done in line with an earlier procedure described by Zhou [14]. S. xylosus ATCC 700404 and its IGPD mutant [14] strain were cultured at the mid-exponential growth phase to an optical density of 0.1 at OD595. Cultures and sub-MICs ISL were added to each well of a 96-well microplate and a 6-well microplate (Corning Costar® 3599 Corning, NY, USA), respectively. In addition, a negative control group (bacterial culture without ISL) was setup. After incubation for 24 h at 37°C, crystal violet staining was performed and followed by scanning electron microscopy analysis. The biofilms were obtained from bacterial cells and prepared for analysis as described by Zhou [14].

Quantitative RT-PCR analysis

In order to investigate the effect of ISL sub-MICs on the expression of the IGPD gene (hisB) in S. xylosus, bacterial culture (mid-log growth phase) supplemented with 1/2 MIC (40μg/mL) ISL was incubated for 24 h at 37°C with continuous shaking. Cells without ISL were used as control. Cultures were centrifuged at 10,000×g for 5 min and stored for 24 h. Thereafter, the bacterial cultures were treated with an RNase REMOVER I (Huayueyang Ltd, Beijing, China). An E.Z.N.A. ™ Bacterial RNA isolating kit was used to determine the total RNA levels. Subsequently, cDNA synthesis was performed according to PrimeScript TM RT reagent kit protocol (Takara Biomedical Technology Co., Ltd. Beijing, China). The specific primers (hisB gene: Forward: , Reverse: . house-keeping gene (16sRNA): Forward: , Reverse: ) used for the quantitative RT-PCR (Takara Biomedical Technology Co., Ltd. Beijing, China) were purchased from Takara. The quantitative RT-PCR procedure was performed as described by Yang [32].

Expression and purification of IGPD protein and preparation of its polyclonal antibody

Purified IGPD protein and anti-IGPD polyclonal antibody were obtained from our previous studies (S1 Fig). In a nutshell, the gene (hisB) encoding for IGPD from S. xylosus ATCC700404 was designed and cloned into pET30a expression vectors (Novagen). Plasmids encoding for IGPD were transformed into Escherichia coli BL21 (DE3) cells (Novagen) and protein expression was induced for 5 h with 1 mM Isopropyl β-D-1-Thiogalactopyranoside at 37°C in Luria broth. Cells were harvested and lysed by sonication. For IGPD, insoluble material was removed by centrifugation and the cell-free extracts were purified in a process involving affinity chromatography and molecular sieve chromatography. For the preparation of polyclonal antibodies, BALB/c mice (from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University) were injected four times subcutaneously with 1 mg of purified IGPD in Montanide ISA 206 (SEP PIC) adjuvant each time. The injections were given repeatedly at one-week interval from the beginning to the end of the experiment. Blood samples were collected from the caudal vein before the injection, and anti-sera were prepared to test the ELISA titers. The mice were euthanized 7 days after the fourth injection, and highly specific antiserum was collected.

Western blotting

The effect of ISL on IGPD protein of S. xylosus was analyzed by western blotting. Firstly, the S. xylosus culture media (mid-log growth phase) supplemented with 1/2 MIC ISL was incubated for 24 h at 37°C with continuous shaking. Cells without ISL served as control. Secondly, after the above cells were centrifuged, the supernatant was discarded and the cells were washed twice with PBS. Cells were then suspended in 100 μL PBS and sonicated for 3 min repeatedly for five times. Finally, 5 × loading buffer was added to the sample and boiled for 20 min. Subsequently, western blot analysis was performed according to the standard procedure [33].

Determination of histidine content

Wild-type and mutant strains (mid-log growth phase) were inoculated into sterile TSB medium, and ISL (1/2-MIC) was added with continuous shaking in an incubator for 24 h at 37°C. S. xylosus (wild-type and mutant strains) without ISL served as the control. After the above cells were centrifuged, the supernatant was discarded and the pellets were washed twice with PBS. The pellets were then suspended in sterile double-distilled water and the bacterial culture was sonicated to release histidine. The above liquid was filtered using a 0.45 μm filter, and the histidine content in the bacteria was determined by high performance liquid chromatography (HPLC) on a Waters Alliance HPLC system (Waters e2695, United States). A standard solution of 15 mg histidine (99% pure histidine—Beijing Solarbio Technology Co., Ltd.) was prepared by dissolving it in 250 mL (0.1 mol/L) hydrochloric acid. The determination of the histidine content was performed on a 5 μm Diamosil C18 column (4.6 mm × 150 mm, Japan). The chromatographic separation was carried out on a 5 μm Diamosil C18 column (4.6 mm × 250 mm, Japan) with a gradient solvent A (10 mmol/L diammonium hydrogen phosphate buffer (containing 10 mmol/L sodium 1-octanesulfonate and obtaining pH 2.0 by phosphoric acid) and solvent B (acetonitrile) as mobile phase at a flow rate of 1 mL/min. The gradient conditions were 0 to 5 min, 95% solvent A; 5 to 6 min, 95% to 86% solvent A; 6 to 15 min, 86% solvent A; 15 to 16 min, 86% to 87% solvent A; 16 to 25 min, 87% solvent A. The detection wavelength was set at 205 nm, and the injection volume was 100 μL. The column temperature was set at 8°C. Quantification of histidine in the sonicated-treated bacterial culture was done using HPLC at 205 nm against concentration using the external standard method.

Bio-layer interferometry (BLI)

BLI experiments were performed using an Octet system (Forte Bio) placed in PBS pH 7.4, 0.05% (v/v) Tween-20 and 1 mg/mL BSA running buffer at room temperature (25°C). The freshly prepared IGPD protein (23 μg/mL) was coupled to the tip of a Forte Bio Octet NTA instrument. A dilution series of ISL (125,000 nM to 3,906 nM) was used to measure a dose-response curve of association and dissociation. The dissociation period was set at 5 min.

Enzyme activity assays

S. xylosus cultures (mid-log growth phase) were supplemented with 1/2-MIC of ISL and incubated at 37°C for 24 h. Cells without ISL served as control. The bacterial culture was centrifuged at 11,000 × g for 5 min. The supernatant was collected and centrifuged at 600 × g for 5 min, then the cells were sonicated on ice for 15 min. The activity of the enzyme was determined in line with a previously described stopped-assay protocol [34] with minor modifications. The reaction mixture consisted of PBS buffer with a pH of 7.4 and supernatant. The reactions were carried out at 37°C using IGP (Santa Cruz Biotechnology, USA). The reaction was stopped by adding sodium hydroxide after an interval of 30 s. The reaction mixture was then incubated at 37°C for 20 min to convert the product imidazoleacetol-phosphate (IAP) into an enolized form, the absorbance was read at 280 nm against a blank in a Shimadzu UV spectrophotometer. The extinction coefficient of IAP formed under these conditions is as reported previously [34].

Molecular docking analysis of interactions between ISL and IGPD

Molecular docking method was used to verify the binding of ISL and IGPD. Homology modeling of IGPD had been done in an earlier study by Chen (S. aureus; PDB entry 2AE8.F) [19]. Furthermore, we also optimized the tertiary structure of IGPD as described by Ahangar [12]. The active region of the IGPD was set as a sphere (X:14.88, Y:116.26, Z:97.66, radius:15Å) by Discovery Studio 3.0 (DS 3.0). All the docking studies were performed by CDOCKER, which adopts the CHARMM forcefield in the DS 3.0 to dock flexible ligands into protein binding sites.

Animal experiments

Twenty-five (25) Pregnant BALB/c mice were obtained from the experimental animal center of the Second Affiliated Hospital of Harbin Medical University. The protocol for this experiment was approved by the ethics committee of Harbin Veterinary Research Institute at the Chinese Academy of Agricultural Sciences (SYXK (Hei) 2012–2067). The care and handling of the animals were in accordance with the ARRIVE (animals in research: reporting in vivo experiments) guidelines and CONSORT (Consolidated Standards of Reporting Trials) statement [35, 36]. After 3 days of feeding without specific pathogen, they were randomly divided into 5 groups, A, B, C, D and E groups (n = 5).

For the establishment of a mastitis model of infection, lactating BALB/c pregnant mice, 10–12 weeks of age and weighing 30–32 g, were anesthetized using sodium pentobarbital (50 mg/kg bodyweight) and infected by injecting the canal glands with 100 μL (1×109 CFU/mL) of S. xylosus (wild-type strain or mutant strain) suspension at the 4th breast on both sides of the abdomen counting from the head (L4 and R4). After infection for 24 h, each breast (L4 and R4) was injected with 100 μL (270 μg/kg) ISL. Animals in the control group received only 10%DMSO. All animals were monitored for 48 h to observe any pathological changes. At the end of this period (48 h), they were all euthanized by cervical dislocation. To evaluate the pathology of mastitis, mammary gland tissues isolated from the euthanized mice were fixed in 4% paraformaldehyde, stained with hematoxylin and eosin, and visualized by light microscopy. Furthermore, additional mammary gland tissues were homogenized in normal saline, after which they were partially diluted and inoculated onto TSB agar plates for the assessment of their bacterial load. The levels of the inflammatory cytokines IL-6 and TNF-α in the supernatant were measured using ELISA kits (Quanzhou Konuodi Biotechnology Co., Ltd.). Finally, total RNA was extracted from the mammary gland using RNAiso Plus and chloroform. The transcription levels of IL-6 and TNF-α were determined by real-time quantitative reverse-transcription PCR (RT-PCR). Primers for IL-6 and TNF-α and the GAPDH used as the housekeeping gene as well as conditions for RT-PCR have been described elsewhere [37, 38].

Statistical analysis

The experiments were all performed in triplicate. Data were subjected to statistical analysis and the Wilcoxon test was conducted by SPSS17.0 software. The values were reported as mean ± standard deviation (SD). Asterisks (*) indicated statistically significant different means compared with the control group (*p < 0.05, **p < 0.01, and ***p < 0.001).

Results

Antimicrobial activity of ISL

The broth dilution method was used to determine the MIC, which quantified the antibacterial effect of ISL on S. xylosus. The MIC was 80 μg/mL.

Time-kill curves are shown in Fig 2. Compared with the control group, 1/2 MIC group did not interfere with the growth of S. xylosus. After 8 hours of ISL treatment at MIC level, the number of living cells decreased by 38.12%. At the same time, S. xylosus was completely killed at 2 MIC ISL. At the beginning (0 h), ISL had a lower CFU than the control group (except for the 1/2 MIC group), since the pretreatment took approximately 5 to 10 minutes to be completed. Moreover, the bacterial colony forming unit and the ISL concentration were dose-dependent. This may explain the decline in the initial viability of the bacteria and also demonstrates the rapid antimicrobial activity of the ISL. The results indicate that ISL may be a promising candidate for the treatment of S. xylosus infection.

Fig 2

Time-kill curves of ISL against S. xylosus cells.

From the results, we observed that ISL sub-MICs significantly interfered with S. xylosus biofilm formation when compared with the control group after treatment. It is noteworthy that there was no significant interference with the formation of biofilm in the mutant strains by ISL sub-MICs (Fig 3).

Fig 3

Determination of biofilm formation ability (A and B) and scanning electron microscope observation (C, D, E and F) of Effect of sub-MICs of ISL on biofilm formation by wild-type S. xylosus strain. Data are expressed as means ± SDs. Controls refer to the absence of ISL. Significantly different means are indicated with asterisks (*) (*p < 0.05, **p < 0.01, and ***p < 0.001) compared to the untreated control group. (B) Effect of sub-MICs of ISL on biofilm formation by mutant S. xylosus ATCC700404 strain. Data are expressed as means ± SDs. Controls refer to the absence of ISL. Means without asterisks (*) have no significant difference compared to the untreated control group. (C) Untreated wild-type strains; (D) Wild-type strains treated with 1/2 MIC of ISL (40μg/mL); (E) Untreated mutant strains; (F) Mutant strains treated with 1/2 MIC of ISL (20μg/mL).

Inhibitory effect of 1/2 MIC ISL on IGPD expression in S. xylosus

The effect of 1/2 MIC ISL on S. xylosus IGPD was evaluated by quantitative RT-PCR and western blotting. The results showed that the expressions of IGPD synthesis gene (hisB) and IGPD protein were significantly down-regulated compared with the control group (p < 0.05) (Fig 4A and 4B). We speculated that ISL inhibits the hisB-related genes expression, thereby inhibiting the expression of IGPD proteins.

Fig 4

Regulation of S. xylosus IGPD by ISL.

(A) Effect of 1/2MIC of ISL on mRNA expression of hisB gene in S. xylosus. Data are expressed as means ± SDs. Significantly different means are indicated with asterisks (*) compared to the untreated control group (*p < 0.05, **p < 0.01, and ***p < 0.001). (B) Effect of 1/2MIC of ISL on the expression of IGPD protein in S. xylosus. (C) Determination of IGPD activity. Wild-type strain grown in the presence of 1/2-MICs of ISL. S. xylosus ATCC700404 served as control. Data are expressed as means ± SDs. Significantly different means are indicated with asterisks (*) compared to the untreated control group (*p < 0.05, **p < 0.01, and ***p < 0.001).

This experiment tested the ability of the supernatant to bind to its substrate IGP and analyzed the activity of IGPD in S. xylosus. In comparison with the control group, the activity of IGPD was inhibited at 1/2 MIC ISL (p < 0.05) (Fig 4C).

HPLC chromatograms and histidine standard solution are shown in Fig 5A. The retention time of histidine in the bacterial extract was same as that of the standard (14.753 min) (Fig 5A I). Using the external standard method, the histidine content of S. xylosus (wild-type strain and mutant strain) with and without 1/2 MIC ISL were analyzed. It was observed that the histidine content of the wild-type strain was significantly reduced compared to the control (p < 0.05). Furthermore, the histidine content of the mutant strain was not significantly affected by ISL (p > 0.05) (Fig 5B).

Fig 5

Determination of S. xylosus histidine content.

(A) The HPLC chromatograms of bacterial extract and histidine standard solution. I) The HPLC chromatograms of bacterial extract (black, retention time 14.781 min) and histidine standard solution (red, retention time 14.753 min). III and V) S. xylosus (wild-type strain or mutant strain) grown in the presence of 1/2 MIC of ISL. II and IV) Untreated wild-type strain and mutant strain served as a control. (B) Peak area analysis of S. xylosus histidine content by HPLC. S. xylosus (wild-type strain or mutant strain) grown in the presence of 1/2 MIC of ISL. Untreated wild-type strain and mutant strain were used as control. Data are expressed as means ± SDs. Significant difference compared to the untreated control group are represented with asterisk (*) (*p < 0.05, **p < 0.01, and ***p < 0.001).

BLI analysis of ISL binding to IGPD protein

We used BLI to detect the interaction between IGPD protein and ISL. The IGPD protein was first immobilized on the surface of the tip of the NTA sensor and reacted with the ISL solution. If the ISL binds to the IGPD protein, it causes a change in the surface light interference, and information on the intermolecular interaction can be obtained by analyzing the change in the light interference. Looking at Fig 6, the binding of ISL to IGPD protein reached equilibrium within 300 seconds, and it was dose-dependent as the concentration of ISL increased. The results showed that ISL interacted with IGPD protein and the dissociation equilibrium constant was given as KD = 234 μM.

Fig 6

Kinetic analysis by BLI of the binding of ISL to IGPD.

Molecular docking analysis

Previous studies have shown that IGPD is a homo 24mer with two Mn2+ and binds at each of the catalytic centers [12]. Therefore, the tertiary structure model of the constructed S. xylosus IGPD should contain three identical subunits containing two Mn2+ (Fig 7A). In order to further understand the binding mode of IGPD and ISL, the molecular docking was conducted by CDOCKER. It is clear that the ISL is located at the catalytic center of the IGPD (Fig 7B). In the process of simulation, it was observed that the ISL bound to IGPD via hydrophobic, hydrogen bonds and electrostatic interactions with the amino acid residues ILE70, MET96, ASN142, and GLU162, respectively. In addition, ISL formed stronger metallic bonds with Mn2+ (Fig 7C).

Fig 7

Computer methodological analysis.

(A) Tertiary structure model of S. xylosus IGPD. (B) Determination of the 3D structure of the IGPD-ISL complex using a molecular modeling method. (C) The predicted binding mode of IGPD with the ISL is shown, with labeling of key residues in the binding sites.

ISL inhibits S. xylosus in murine mastitis model of infection

In addition to the critical role of ISL against the activity of IGPD in S. xylosus, we also postulated that it can also inhibit the growth of S. xylosus in vivo. To verify this, the therapeutic effect of ISL on mastitis in mice was evaluated, and the histopathological analysis of mammary gland tissues at 48 h post-infection was performed. No inflammatory response or pathological change was detected in the negative control group (Fig 8A). While significant inflammatory responses were observed in the mice infected with S. xylosus (wild-type strains and mutant strains were given 10%DMSO to neutralize the effect of the solvent); responses observed included destroyed or abnormal mammary glands, dense inflammatory cells, and depletion of epithelial cells in the tissues of the mammary gland (Fig 8B and 8D). However, the ISL treatment effectively alleviated the S. xylosus-induced inflammatory responses and pathological changes in the infected mice, which exhibited only mild tissue injury, demonstrating a potential prophylactic effect of the ISL against S. xylosus-induced mastitis (Fig 8C). It is important to note that ISL did not reduce the inflammatory response in mice with mastitis induced by S. xylosus-mutant strain (Fig 8E). Furthermore, the bacterial counts in the mammary gland tissues indicated a significant decrease in the bacterial load in the ISL-treated mice compared with the untreated mice (wild-type strain). The bacterial load of the ISL-treated mice was not significantly different from those of the mice infected with the mutant strain (Fig 8H). In addition, the levels of mRNA and cytokines including IL-6 and TNF-α in the S. xylosus-infected (wild-type strains and mutant strains) mice were markedly increased compared with negative control group (10% DMSO only), whereas ISL significantly reduced the increased levels of mRNA and cytokine in the group infected with (wild-type strain) infected mice (Fig 8F and 8G and S2 Fig), there was no significant effect on the group infected with the mutant strain.

Fig 8

ISL provides protection against S. xylosus mastitis in a mouse model.

Lactating BALB/c mice were infected by injecting the canal glands with 1 × 109 CFU bacterial cells. (A-E) The gross pathological changes (I) and histopathological analysis (II) of the mammary gland tissues at 48 h post-infection (n = 5) (Magnification: 600×). The infection group showed a large number of inflammatory factors and breast tissue destruction (arrowheads). Pathological abnormalities were significantly reduced by 250 μg/kg ISL in infected mice compared with DMSO treated mice. (F, G) ISL reduces the inflammatory response in infected mice. The levels of cytokines, including IL-6, and TNF-α, in the mammary gland tissues of infected mice were evaluated by ELISA (n = 5) (*p < 0.05, **p < 0.01, and ***p < 0.001). (H) The effect of the ISL on the bacterial burden in infected mice. Mammary gland tissues were collected, homogenized, and plated on TSB agar plates for the assessment of the bacterial burden (n = 5) (*p < 0.05, **p < 0.01, and ***p < 0.001). Results are reported as mean ± SD.

Discussion

Cow mastitis is considered to be the most infectious disease affecting the dairy industry [39, 40]. In recent years, S. xylosus has become the most common CNS bacteria isolated from clinical mastitis [2, 6, 7]. Therefore, urgent attention should be given to the treatment of cow mastitis caused by S. xylosus. Currently, studies have demonstrated that flavonoids have anti-bacterial activity and can interfere with the formation of biofilms [30, 41]. ISL is a natural compound with low molecular weight and high drug-forming property. It conforms to the Lipinski's Rule of Five and is a kind of flavanone compound with an excellent [22, 25, 42], and higher antimicrobial activity in comparison with other flavonoids [20]. Nevertheless, few studies have reported its effect on S. xylosus. In this study, we found that ISL had potent biological activity against S. xylosus (MIC was 80 μg/mL). A time-kill curve further confirmed the effect of the concentration and time-dependent killing of ISL on S. xylosus. In addition, TCP and SEM assays revealed that sub-MICs of ISL significantly interfered with S. xylosus biofilm formation in vitro.

Several studies have shown that IGPD is an essential enzyme in plants and microorganisms, and thus it is an attractive target for the development of herbicides and antibacterial agents, for which there are presently a limited number of biological targets [43]. Our previous studies have indicated that cefquinome and baicalin can interfere with S. xylosus biofilm formation by interfering with IGPD. At the same time, the interaction with IGPD will lead to a decrease in S. xylosus histidine content and its enzyme activity [14, 19]. Since ISL can inhibit the growth of S. xylosus, the sub-MIC of ISL can also interfere with its biofilm formation. So, we postulated that ISL can also affect S. xylosus by regulating the synthesis of IGPD or directly targeting it. First, we evaluated the relationship between ISL and IGPD gene (hisB) by RT-PCR. From the results, it was observed that 1/2MIC of ISL, significantly down-regulated the expression of hisB in the wild-type strains. We speculated that ISL may inhibit the hisB gene or its upstream and downstream genes, resulting in the down-regulated expression of the hisB gene. Subsequently, in order to verify the results, we performed western blot analysis. Similarly, 1/2 MIC ISL significantly interfered with S.xyosus IGPD protein expression. We speculated that ISL inhibiteds the hisB-related genes expression, thereby inhibiting the expression of IGPD proteins. On the other hand, histidine is the product of an important nitrogen metabolism pathway in bacteria [10], and the synthesis of histidine has been reported to indirectly affect the growth of Corynebacterium, Saccharomyces cerevisiae and E. coli [13, 44]. When IGPD, a key enzyme for microbial histidine biosynthesis is inhibited, its histidine content will also decrease [13, 14]. In this study, the histidine content of S. xylosus (wild-type strain) was significantly lowered by 1/2 MIC ISL compared with untreated group. No effect was observed in the mutant strain group treated with ISL. This may be due to the inability of the mutant strain to express IGPD, thus, not providing a target for ISL.

In order to verify whether ISL can bind to IGPD protein directly, we performed a BLI assay. BLI is an optical analysis technology which can monitor the interaction between two different molecules in real time [45]. The results showed clearly that ISL bound to IGPD protein directly with strong affinity (KD = 234 μM). Consequently, we speculated that ISL may act on the IGPD in S. xylosus, thus affecting the growth of the bacteria. In order to verify this conjecture, we carried out an enzyme activity experiment. The results indicated that the activity of IGPD in S. xylosus was significantly lower than that of the control group at 1/2 MIC of ISL. The results of BLI and enzyme activity assays revealed that ISL occupied the catalytic center of IGPD enzyme, and prevented the binding of IGPD with its substrate, then affecting S. xylosus (wild-type strain) enzyme activity. In order to further understand the binding mode of IGPD and ISL, we used CDOCKER for molecular docking. Previous studies have shown that IGPD is a manganese-dependent metalloenzyme composed of 24 identical subunits. Each active site consists of three subunits and two Mn2+, the two Mn2+ are located in the catalytic center of the active region [12, 46]. From our results, ISL bound to the amino acid residues ILE70, MET96, ASN142 and GLU162 in the IGPD active region by hydrophobic, hydrogen bonding and electrostatic interaction, respectively. In addition, ISL and the Mn2+ forms stronger metallic bonds. It was clear that the ISL occupied the catalytic center of IGPD. Thus, results from molecular docking further confirmed our hypothesis that ISL occupied the catalytic center of the S. xylosus IGPD enzyme and prevented the binding of IGPD to its substrate, which in turn affected the activity of S. xylosus.

Mastitis is an inflammatory disease of the breast tissue common to lactating animals and CNS has been identified as the most important group of pathogen responsible for most of the reported cases of mastitis in lactating dairy cows [2, 47–49]. Moreover, it can lead to severe breast tissue destruction and inflammation, and in most cases, treatment is not effective [6, 50]. In order to prove that ISL can effectively treat mastitis in vivo, we induced S. xylosus mastitis in mice. Histopathological examination revealed multiple inflammatory cell infiltration and mammary gland damage in the breast tissues. This was similar to what was observed in previous studies [51, 52]. In addition, cows with subclinical mastitis caused by CNS produce a local immune response in the mammary gland [5, 6], similar to a variety of inflammatory immune factors including TNF-α and IL-6 produced by Th1 and Th2-mediated immune responses in the presence of exogenous infection [53]. TNF-α is an early cytokine that plays a key role in the cascade of other pro-inflammatory cytokines and inflammatory mediators [54]. IL-6 is a pleiotropic cytokine involved in the physiology of almost every organ in an organism system [55]. Besides, TNF-α and IL-6 are crucial markers of inflammation in breast tissues [37, 56]. Therefore, the concentration of TNF-α and IL-6 in breast tissue was used as markers to indicate the occurrence of inflammation. From the results, the TNF-α and IL-6 mRNA and protein levels in the S. xylosus group were significantly increased. While significant inflammatory responses were observed in mice infected with S. xylosus, there was no observable inflammatory response or pathological change in the negative control group. Obviously, S. xylosus successfully induced mastitis in mice.

After S. xylosus infection, the production of inflammatory factors including TNF-α and IL-6 increased rapidly. Cytokines are well known to inhibit the growth of bacteria as part of host defense mechanism. In order to inhibit the growth of S. xylosus, Th1 and Th2 mediated immune responses produce large amounts of TNF-α and IL-6. But their overproduction can lead to systemic inflammation, which is destructive rather than protective to the host [5, 57, 58]. Therefore, inhibiting the production of TNF-α and IL-6 will concomitantly prevent inflammation. Recent studies have shown that ISL can inhibit the initial inflammatory changes on the vascular endothelium by eliminating the adhesion of monocytes [59]. In addition, ISL can down-regulate IL-6 expression in macrophages [60]. Our results revealed that ISL significantly reduced the cytokine and mRNA levels. On the other hand, ISL treatment effectively attenuated the S. xylosus-induced inflammatory responses and pathological changes in the infected mice which exhibited only mild tissue injury. It is worthy to note that treatment with ISL significantly reduced the bacterial load in the mammary gland tissue of the wild-type treatment group compared with the mutant strain treatment group. This indicates that ISL has an anti-inflammatory effect by regulating the expression of TNF-α and IL-6 and reversed the damaged mammary gland and mammary epithelial cells by targeting IGPD.

In conclusion, ISL down-regulated hisB gene expression, and thus interfered with the expression of IGPD protein in S. xylosus, leading to the decrease in its histidine content. Moreover, it competed with IGP in the catalytic center of IGPD, abrogating its ability to complete the dehydration reaction leading to the production of IAP and reduced the histidine content. Therefore, ISL inhibited the growth of S. xylosus and interfered with S. xylosus biofilm formation by targeting IGPD. Furthermore, in vivo studies confirmed that ISL effectively attenuated the damaged in mouse mammary gland tissues in the infected mice and reduced the levels of inflammatory factors produced by S. xylosus. To the best of our knowledge, this is the first paper reporting ISL as a potential competitive inhibitor to bind IGPD and interfere with the pathogenicity of S. xylosus. Therefore, ISL may be a candidate drug for the control of mastitis in dairy cows.

Preparation of IGPD.

(A) SDS-PAGE gel analysis of IGPD expression as induced by the presence or absence IPTG. Lane 1: Protein molecular mass marker; lane 2: The lysates of BL21 (DE3) cells containing pET30a-IGPD without IPTG; Lane 3: The lysates of BL21 (DE3) cells containing pET30a-IGPD with IPTG; Lane 4: The supernatants of BL21 (DE3) cells containing pET30a-IGPD with IPTG; Lane 5: The precipitates of BL21 (DE3) cells containing pET30a-IGPD with IPTG. (B) SDS-PAGE gel analysis of purified IGPD. The IGPD protein was purified by a Ni Sepharose 6 Fast Flow column. Lane 1: Protein molecular mass marker; lane 2: IGPD.

(TIF)

Click here for additional data file.

ISL reduces the expression of cytokines in infected mice.

(A, B) The expression levels of cytokines, including IL-6, and TNF-α, in the mammary gland tissues of infected mice were evaluated by qPCR (n = 5) (*p < 0.05, **p < 0.01, and ***p < 0.001).

(TIF)

Click here for additional data file.

Original images for blots and gels.

(PDF)

Click here for additional data file.

2 Oct 2019

PONE-D-19-21816

In vitro activity and in vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD Target

PLOS ONE

Dear Dr. Yanhua Li,

We are please to inform you that your MS is acceptable for publication with some minor corrections as suggested by reviewers. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Nov 16 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Vivek Sharma, PhD

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of analgesia, and (3) efforts to alleviate suffering.

Additional Editor Comments (if provided):

Dr. Yanhua Li,

The MS PONE-D-19-21816 entitled "In vitro activity and in vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD Target" has been reviewed by three reviewers and they have suggested acceptance for same with some minor correction. Additionally, as per PLOS ONE guidelines, it is mandatory for authors to submit original blots. Therefore, MS is acceptable after these corrections.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors of the article have prepared an excellent work, giving the full information of mode of action of using isoliquiritigenin (ISL), a flavonoid compound, as an drug in mice model mastitis. The excellence of the work is greater because authors gave a model of examination for future drugs. The subject of the work is pioneer, very important in veterinary, so I recommend it to publish.

In my opinion there is lack of a special subtitle/or more detailed describtion both tested microorganisms; I feel there was not distincly pointed what served as controls during the experiments. It would be nice if authors could slightly improve the article in methodological parts, although this feeling do not make the article worse.

Reviewer #2: Line 16 and Line 43: delete (S. xylosus)

Line 37: Italics Staphylococcus

Line 54: add ‘and’ after natural compounds

Line 120: Add reference of the studies

Line 230-233: Little more explanation of results is required

Line 296: Italicise in vitro

Line 315: delete et al

Reviewer #3: 1. The authors have poorly written introduction. Likewise, in the first paragraph, the authors have given the importance of S. xylosus but in the next paragraph, they suddenly shifted to (IGPD) as the key enzymes of histidine biosynthesis and in next paragraph to flavonoids. Both these aspects are different and the authors must write in a way to easily understandable to the readers. The main aim of the study should be mentioned at the end in an elaborative manner.

2. The authors have also mentioned about the expression of IGPD in pET 30a and purification as well as preparation of its polyclonal antibody in methodology. The same should be a part of results and mentioned properly. The same results could also be a part as supplementary section.

3. The authors have reported the cytokine expression of TNF-α and IL-6. Why only two cytokines have been studied. The authors must relate the results to Th1 and Th2 type immune response based on cytokine expression. The authors must report comparative cytokine expression based on qPCR as well as ELISA as the respective results are reported based on ELISA only.

4. The entire discussion provides limited insight regarding structure-function and disease status. A more direct connection should be established by making predictions and experimental testing.

5. The authors should provide the PDB identification numbers of the structures used to generate the structural models shown and data used.

6. Throughout document (nearly every sentence) - There are numerous spelling, English grammar and syntax and typo errors. This manuscript could really benefit from English editing. For example:

7. Line 15, 34: Stapylococcus should be italics

8. Line 17: can causes

9. Line 59- main bacteria isolate

10. Line 102- meaning of ½ MIC should be mentioned

11. Line 146- Use appropriate word for “The determination was performed”

12. Line 257- it also could

13. Line 302- also can

14. Line 306-307- E. coil et al

15. Line 312- can be bind

16. Line 313- remove comma

17. Line 342-343- rewrite the sentence “There were almost no inflammatory responses or pathological changes were detected in the negative control group”

18. Line 350- remove comma

19. Line 353- has a therapeutic effective against

20. Line 22, 71, 104, 131, 297, 303 and many more - remove all sticky words

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Nov 2019

Reviewer #1:

Authors of the article have prepared an excellent work, giving the full information of mode of action of using isoliquiritigenin (ISL), a flavonoid compound, as an drug in mice model mastitis. The excellence of the work is greater because authors gave a model of examination for future drugs. The subject of the work is pioneer, very important in veterinary, so I recommend it to publish.

In my opinion there is lack of a special subtitle/or more detailed describtion both tested microorganisms; I feel there was not distincly pointed what served as controls during the experiments. It would be nice if authors could slightly improve the article in methodological parts, although this feeling do not make the article worse.

1.Thank you for your suggestion and careful review. I have changed the subtitle to “Isoliquiritigenin inhibits the growth of S.xylosus”.

2.Thank you for your advice, I have corrected the manuscript according to your comments. I have modified the experimental method by explaining the various control groups throughout the materials and methods section of the manuscript. These corrections have been indicated in lines 100 to 101, line 109, line 207.

3.Thank you for your valuable suggestion. I have expanded and improved the materials and methods section of the article. The modified portions are indicated in line 128, line 189, lines 214 to 218.

Revised and edited portions are written in red. I hope you will be satisfied with my answer. Thank you very much.

Thank you for giving me the chance to revise my article. I have sent this manuscript to a professional English language editor to improve the language. Thank you very much for your cautious correction. Please we will welcome any other suggestions and comments that will help to further improve the quality of the paper. Thank you once again for all your and valuable contributions.

Reviewer #2:

Line 16 and Line 43: delete (S. xylosus)

Line 37: Italics Staphylococcus

Line 54: add ‘and’ after natural compounds

Line 120: Add reference of the studies

Line 230-233: Little more explanation of results is required

Line 296: Italicise in vitro

Line 315: delete et al

Thank you for your valuable suggestion. I have corrected some of the grammatical errors in this article. I am very sorry for my negligence.

1. I have deleted S. xylosus in lines 18 and 44.

2. I have italicized the Staphylococcus in line 37.

3. I have added ‘and’ after natural compounds in line 63.

4. I have placed the reference picture (Fig. S1) in the supplementary material in lines 127 to 128.

5. I have added some explanations of the results in lines 243 to 245.

6. I have Italicized ‘in vitro’ in line 308.

7. I have deleted ‘et al’ from line 325.

Revised and edited portions are written in red. I hope you will be satisfied with my answer. Thank you very much.

Thank you for the time spent in correcting my article. This has given me the opportunity to modify the work. I am grateful for all your remarks concerning the grammatical errors in the article and subsequently, the manuscript has sent to a professional English language editor for correction. I have modified the paper in line with to all your comments. Please I will be very grateful if other portions that need further improvement is brought to my notice for further correction. Thank you once again for your time.

Reviewer #3:

1. The authors have poorly written introduction. Likewise, in the first paragraph, the authors have given the importance of S. xylosus but in the next paragraph, they suddenly shifted to (IGPD) as the key enzymes of histidine biosynthesis and in next paragraph to flavonoids. Both these aspects are different and the authors must write in a way to easily understandable to the readers. The main aim of the study should be mentioned at the end in an elaborative manner.

2. The authors have also mentioned about the expression of IGPD in pET 30a and purification as well as preparation of its polyclonal antibody in methodology. The same should be a part of results and mentioned properly. The same results could also be a part as supplementary section.

3. The authors have reported the cytokine expression of TNF-α and IL-6. Why only two cytokines have been studied. The authors must relate the results to Th1 and Th2 type immune response based on cytokine expression. The authors must report comparative cytokine expression based on qPCR as well as ELISA as the respective results are reported based on ELISA only.

4. The entire discussion provides limited insight regarding structure-function and disease status. A more direct connection should be established by making predictions and experimental testing.

5. The authors should provide the PDB identification numbers of the structures used to generate the structural models shown and data used.

6. Throughout document (nearly every sentence) - There are numerous spelling, English grammar and syntax and typo errors. This manuscript could really benefit from English editing. For example:

7. Line 15, 34: Stapylococcus should be italics

8. Line 17: can causes

9. Line 59- main bacteria isolate

10. Line 102- meaning of ½ MIC should be mentioned

11. Line 146- Use appropriate word for “The determination was performed”

12. Line 257- it also could

13. Line 302- also can

14. Line 306-307- E. coil et al

15. Line 312- can be bind

16. Line 313- remove comma

17. Line 342-343- rewrite the sentence “There were almost no inflammatory responses or pathological changes were detected in the negative control group”

18. Line 350- remove comma

19. Line 353- has a therapeutic effective against

20. Line 22, 71, 104, 131, 297, 303 and many more - remove all sticky words

1.Thank you for your suggestion and careful review. I have expanded and improved the introduction of this article to strengthen the connection between paragraphs. This is indicated in lines 46 to 63. I have also mentioned the main purpose of the study in a relatively detailed manner in lines 72 to 77.

2.Thank you for your advice, I have corrected the manuscript according to your suggestion. I have reported the protein expression and purification results as one of the supplemental materials (Fig. S1).

3.Thank you for such a careful review.

a. I have explained the critical roles of TNF-α and IL-6 as markers of inflammation in breast tissue in lines 355 to 357.

b. In lines 366 to 371, I have linked Th1 and Th2 type immune responses to cytokine expression and conducted in-depth analysis to buttress this claim.

c. I have edited the qPCR test in lines 214 to 218, and the results are shown in the supplementary materials section (Fig. S2).

4.Thank you for your suggestion and careful review. In lines 339 to 344, I have improved my discussion of structure-function and disease status with a more detailed explanation, linking postulations with confirmatory tests.

5. I have added the PDB identification number to line 189.

6.Thank you for your valuable suggestion. I am very sorry for my negligence. I have corrected some of the syntax errors in this article.

7. I have Italicized ‘Staphylococcus’ in lines 17 and 37.

8., I have edited ‘can causes’ and changed it to ‘linked it with cases of’ in line 19.

9. In line 71, I have replaced ‘main bacteria isolate’ to ‘main bacterial isolates’.

10. I have replaced ‘1/2 MIC’ with ‘sub-MICs’ in line 113.

11. ‘The determination was performed’ with ‘The experiment to determine the histidine content’ in line 159.

12. I have replaced ‘it also could’ with ‘it can also ‘in line 276.

13. I have replaced ‘also can’ with ‘interfered’ in line 321.

14. I have deleted et al from line 325.

15. I have changed ‘can be bind’ to ‘can bind’ in line 330.

16. I have removed the comma from line 331.

17. I have deleted ‘There were almost no inflammatory responses or pathological changes were detected in the negative control group’ and replaced it with ‘There were no observable inflammatory responses or pathological changes in the negative control group’ in lines 363 to 365.

18. I have deleted the comma from line 376.

19. I have deleted ‘has a therapeutic effective against’ from lines 378 to 380 and replaced it with ‘This indicates that ISL has an anti-inflammatory effect by regulating the expression of TNF-α and IL-6 and reversed the damaged mammary gland and mammary epithelial cells by targeting IGPD’.

20. I have removed all sticky words including lines 24, 82, 115, 144, 316 and 322.

Revised and edited portions are written in red. I hope I have been able to correct all the issues you raised in the original manuscript satisfactorily. Thank you once again for your valuable contributions.

Thank you for giving me the opportunity to revise my article. I was particularly concerned about the final decision of this article. There may be some deficiencies in my paper, but I will be very careful to modify and improve it. I am grateful for all your remarks concerning the grammatical errors in the article. I have sent this manuscript to a professional English language editor to improve the language. I have made cautious effort to modify the paper according to all your comments. Please feel free to inform me of any portion that need further correction. Thank you once again for your time.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Nov 2019

In vitro activity and in vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD Target

PONE-D-19-21816R1

Dear Dr. Yanhua Li,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Vivek Sharma, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Dr. Yanhua Li,

I am pleased to inform you that MS PONE-D-19-21816R1 entitled "In vitro activity and in vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD has been accepted for publication.

Thank you for submitting your work to PLOS One. We hope you consider us again for future submissions.

Kind regards

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The manuscript is revised and modified now. All the comments are addressed point wise and the present form of manuscript is acceptable.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

12 Dec 2019

PONE-D-19-21816R1

In vitro activity and in vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD target

Dear Dr. Li:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Vivek Sharma

Academic Editor

PLOS ONE