Baohuoside I targets SaeR as an antivirulence strategy to disrupt MRSA biofilm formation and pathogenicity

AbstractThe emergence of methicillin-resistant Staphylococcus aureus (MRSA) represents a critical global health challenge, making the SaeRS two-component system (TCS), a key regulator of S. aureus virulence, an ideal target for novel therapeutic approaches. In this study, virtual screening and thermal shift assays identified Baohuoside I (BI), a flavonol glycoside, as a potent inhibitor of the SaeR response regulator. BI significantly attenuated S. aureus pathogenicity without bactericidal effects, suppressing the expression of key virulence factors, such as hemolysin A (Hla) and Panton-Valentine leukocidin (PVL), while modulating immune evasion pathways. Additionally, BI disrupted biofilm formation, promoting the development of porous, less structured biofilms. Biochemical assays, including EMSA, CETSA, fluorescence quenching, and SPR, confirmed strong binding interactions between SaeR and BI. In vivo, BI demonstrated therapeutic efficacy in Galleria mellonella and rat MRSA models. These findings establish BI as a promising lead for nonbactericidal therapies to combat MRSA infections and mitigate resistance.

IntroductionStaphylococcus aureus (S. aureus) is highly prevalent and well adapted to the human host, necessitating the continuous refinement of key strategies to combat its evolving resistance mechanisms1. Antibiotics remain the primary clinical intervention for S. aureus infections, exerting bacteriostatic or bactericidal effects. However, this approach places significant selective pressure on bacteria, driving the emergence of resistant strains such as MRSA. In the United States alone, MRSA causes more than 10,600 deaths and 323,700 infections annually2. By 2050, it is projected that 10 million people per year will die due to antimicrobial resistance3. The widespread outbreaks of healthcare-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), livestock-associated MRSA (LA-MRSA), and even vancomycin-resistant S. aureus (VRSA) indicate that we are facing a loss of battle against bacterial resistance4,5,6,7.S. aureus is a leading cause of septic arthritis, which can lead to systemic sepsis. Despite advancements in antimicrobial therapies and hospital care, the mortality rate of septic arthritis has not significantly improved over the past 25 years, ranging from 5% to 15%8. Unfortunately, the development of novel antibiotics has lagged behind the rapid rise in resistance; no new antibiotic classes have been developed in over 20 years9. Clinicians are forced to rely on combinations of existing antibiotics, such as triple β-lactam regimens, but these approaches still stimulate the emergence of resistant strains10. Consequently, there is an urgent need for anti-infective agents with novel mechanisms of action.The pathogenesis of S. aureus is governed by a complex array of virulence factors, including surface adhesion proteins and secreted toxins11. In S. aureus-induced septic arthritis, these virulence factors enable S. aureus to attach to host cells or extracellular matrix (ECM) components, inhibit complement activity, reduce antibody function, destroy host cells, and evade immune clearance, playing key roles in every stage of infection12,13. Importantly, these virulence factors are nonessential for bacterial growth, making them ideal targets for antivirulence therapies14. Additionally, biofilm-associated infections in septic arthritis are notoriously difficult to treat owing to their inherent resistance to host immune responses and poor antibiotic penetration15. Clinical antibiotics often show limited efficacy against biofilms, underscoring the urgent need for effective antibiofilm strategies to reduce the incidence of biofilm-related bacterial infections16. Targeting virulence factors aims to inhibit their activity, aiding the host immune system in pathogen clearance and exerting minimal selective pressure for resistance. This approach could offer a promising strategy to control S. aureus infections without promoting antibiotic resistance.The SaeRS two-component system (TCS) is a critical downstream regulator in S. aureus that orchestrates the expression of numerous virulence factors and plays a key role in biofilm formation17,18,19. The SaeRS TCS comprises the sensor histidine kinase SaeS, the response regulator SaeR, and two accessory proteins, SaeP and SaeQ20. SaeS senses environmental signals and undergoes autophosphorylation; phosphorylated SaeR (SaeR-P) then binds to the SaeR binding site (SBS), activating the transcription of SaeP, SaeQ, and more than 20 virulence genes, including hla, hlb (encoding β-hemolysin), coa (encoding coagulase), map (encoding a membrane component-binding protein), and lukE (encoding leukotoxin)21. Mutants of SaeRS exhibit reduced expression of exotoxins (hemolysin A (Hla), Panton-Valentine leukocidin (PVL)), and the production of Hla is completely abolished, resulting in decreased hemolytic activity22. In a murine pyelonephritis model, the absence of SaeRS significantly inhibited hla expression both in vitro and in vivo, whereas mutations in agr or sarA had only marginal effects on hla expression in vivo23. Previous studies have demonstrated that flavonoid derivatives, such as yellow Sterone B1 and PM-56, can bind to SaeS, inhibiting its histidine kinase activity. However, targeting SaeS alone may be unreliable and incomplete, as SaeR can still be phosphorylated by other unknown signals, thereby activating the expression of downstream virulence genes21.Medicinal plants have long been rich sources of antimicrobial compounds used to prevent and treat bacterial infections, and many bioactive products from traditional Chinese medicine (TCM) have shown promising antivirulence and antibiofilm properties24,25. Flavonol glycosides such as kaempferol, flavonoid disaccharides, and icariin have been proven to exert potent antibiofilm effects26,27. Epimedium koreanum Nakai is a widely used TCM in the treatment of fractures and other bone diseases, and it is also employed in formulations for dementia, erectile dysfunction, and cardiovascular diseases28,29. Baohuoside I (BI), also known as Icariin II—a flavonol glycoside with potent therapeutic potential—has been extracted from E. koreanum. BI has shown anti-inflammatory, vasodilatory, anticancer, and antiosteoporotic activities30,31, yet its antimicrobial properties remain unexplored.In this study, we identified BI as a flavonoid capable of disrupting the biological function of SaeR. We systematically investigated its anti-S. aureus and antibiofilm activities and explored its underlying mechanism of action. More importantly, we confirmed the safety and efficacy of BI in both in vitro and in vivo models, establishing it as a promising antivirulence agent for the control of S. aureus infections.ResultsInhibition of S. aureus SaeR by BIOur preliminary investigation and existing research revealed that flavonoids exhibit a notable propensity to mitigate S. aureus virulence. This observation guided the selection of a small-molecule library, which was virtually screened specifically against SaeR via Discovery Studio, with the selection process illustrated in Fig. 1A. We subsequently identified 17 candidate compounds through virtual screening, all of which presented CDOCKER absolute scores exceeding 120 (Supplementary Table 1). This threshold value was defined to maximize the probability of identifying compounds with significant binding affinity while maintaining a feasible number of lead candidates for subsequent evaluation.Fig. 1: Identification of Baohuoside I (BI) as a SaeR inhibitor.A Schematic of the virtual screening and thermal shift assay (TSA) used to identify SaeR inhibitors from a natural small-molecule library. B TSA analysis showing the effect of BI on SaeR thermal stability. C Chemical structure of BI. D Minimum inhibitory concentration (MIC) of BI against S. aureus USA300 (WT group). E Growth curves of S. aureus USA300 with and without BI treatment (128 μg/mL). F Cell viability assay using MTT to evaluate the effects of different BI concentrations (0 to 128 μg/mL) on ATDC5 cells. G In vivo toxicity of BI evaluated in G. mellonella larvae (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageThe thermal shift assay (TSA) is a widely utilized technique for identifying protein inhibitors on the basis of the principle that ligand binding induces changes in the thermal stability of the target protein. Changes in protein stability can be characterized by shifts in the melting temperature (Tm) of the protein. A Tm shift >2 °C suggests significant ligand binding to the target protein32. To further investigate this possibility, we purified the SaeR protein (Supplementary Fig. 1) and employed TSA to assess the binding affinities of the candidate compounds to SaeR. Among them, BI increased the Tm of the SaeR protein from 42.0 °C to 47.0 °C, indicating significant stabilization of SaeR and identifying BI as a promising compound for further study (Fig. 1B, C).BI does not affect the growth of S. aureus and has no apparent toxicityUnlike antibiotics, which exert bactericidal effects, antivirulence agents aim to reduce the production of virulence factors without inhibiting bacterial growth. To simulate the effects of antivirulence agents on SaeR, we constructed a SaeR-knockdown (kd-saeR) S. aureus strain (Supplementary Fig. 2A–D). We then monitored the growth kinetics of S. aureus USA300 in the presence of different concentrations of BI to determine whether it affected bacterial growth. As shown in Fig. 1D, the minimum inhibitory concentration (MIC) of BI against USA300 was determined to be greater than 256 μg/mL via broth microdilution. Notably, the MIC of BI remained stable across 20 serial passages of S. aureus cultured under drug-exposure conditions, suggesting that BI does not readily induce resistance in S. aureus (Supplementary Table 2). Furthermore, no significant differences in growth rate were observed between the BI-treated group (128 μg/mL), the kd-saeR group, and the DMSO control group (Fig. 1E). These findings indicate that BI, at this concentration, does not inhibit the growth of S. aureus USA300, which aligns with the expected properties of an antivirulence agent. In further cytotoxicity assays, BI had no adverse effects on the viability of ATDC5 chondrocytes at a concentration of 128 μg/mL (Fig. 1F). We extended toxicity evaluations in vivo using G. mellonella larvae. BI was injected into the last left proleg of the larvae at doses of 20 or 50 mg/kg. The condition and melanization of the larvae were assessed daily, with survival tracked over 5 days postinjection. The results indicated that BI was well tolerated at both doses, with no mortality observed (Fig. 1G). These findings provide a solid foundation for further exploration of BI as a potential antivirulence agent.BI inhibits S. aureus hemolytic activity and the expression of hla and PVL in vitroSaeR regulates multiple virulence factors and toxins that critically influence infection pathogenesis and the disruption of the host immune response. Of particular interest are pore-forming toxins, including Hla and PVL, which selectively target immune cell membranes, induce alterations in transmembrane ion gradients, disrupt membrane integrity, and lead to cell lysis—a hallmark of their mode of action. Western blot analysis revealed that BI inhibited the expression of Hla and PVL in S. aureus USA300 in a dose-dependent manner (Fig. 2A, B, Supplementary Fig. 4A, B). To assess the impact of BI on Hla activity, we performed hemolysis assays. BI inhibited Hla activity in S. aureus USA300 in a dose-dependent manner. At a concentration of 128 μg/mL, the hemolytic activity of the S. aureus supernatant was significantly reduced to 14.07 ± 1.78% (Fig. 2C). Subsequent real-time quantitative reverse transcription‒PCR (RT‒qPCR) analysis confirmed that BI downregulated the transcription of the hla and lukE genes in S. aureus USA300 (Fig. 2E).Fig. 2: Impact of BI on SaeR-regulated virulence factors.A Western blot analysis of the effect of BI on Hla expression in S. aureus USA300. B Western blot analysis of the effect of BI on PVL expression in S. aureus USA300. C Hemolysis assay assessing the effect of BI on the hemolytic activity of S. aureus USA300, which revealed a dose-dependent reduction in the number of lysed red blood cells. D Fibrinogen binding assay evaluating the effect of BI on the adhesion of S. aureus USA300. E RT‒qPCR analysis showing the changes in the transcription levels of S. aureus virulence genes at different BI concentrations. F Effects of different concentrations of BI on the internalization of S. aureus into ATDC5 cells (scale bar: 10 μm). G Plate count results of internalized S. aureus in ATDC5 cells, demonstrating the ability of BI to inhibit bacterial internalization. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageBI inhibits S. aureus adherence to fibrinogenThe widespread transmission of S. aureus in hospital settings and subsequent infections are closely associated with its high adherence ability33. We utilized crystal violet staining to evaluate whether BI could inhibit S. aureus adherence to fibrinogen. As shown in Fig. 2D, BI inhibited S. aureus adherence to fibrinogen in a dose-dependent manner. At a concentration of 64 μg/mL, the adherence rate was significantly reduced to 29.79 ± 2.04%. RT‒qPCR analysis further confirmed that BI reduced the expression of the map gene in S. aureus (Fig. 2E).BI inhibits SaeR-regulated virulence factor transcription in S. aureus in vitroTo explore the relationships between SaeR and key virulence factors involved in intravascular adhesion and biofilm formation, we assessed the effects of BI on the transcription of critical genes (hlgC, splA, chp, psm-α) during S. aureus infection via RT‒qPCR. As shown in Fig. 2E, BI (64 μg/mL) significantly reduced the transcription levels of hlgC, splA, chp, and psm-α by 2.02-fold, 7.15-fold, 4.71-fold and 26.29-fold, respectively, compared with those in the untreated group. Notably, the transcript levels of the aforementioned genes in the kd-saeR strain exhibited a similar trend. These results demonstrate that BI inhibits the transcription of SaeR-regulated virulence genes in vitro.BI reduces S. aureus internalization into host cellsDuring infections such as septic arthritis, S. aureus can evade host immunity and antibiotic treatment by invading cells and forming biofilms on joint surfaces. We evaluated the ability of S. aureus to invade ATDC5 cells in the presence or absence of BI. As shown in Fig. 2F, BI treatment significantly reduced the internalization of S. aureus into ATDC5 cells. Additionally, colony-forming unit (CFU) counts of intracellular S. aureus confirmed that BI limited bacterial invasion in a dose-dependent manner (Fig. 2G).BI enhances neutrophil chemotaxis and mitigates S. aureus-induced damage to ATDC5 cellsS. aureus secretes a range of immune-modulating factors, such as PVL, CHIP, and PSM, that impair neutrophil function and induce neutrophil lysis, enabling evasion of host immunity. Consistent with the RT‒qPCR results, BI significantly reduced the transcription levels of lukE, chp, and psm-α in a dose-dependent manner (Fig. 2E). Neutrophil recruitment is a critical step in controlling infections. Transwell migration assays34 demonstrated that BI significantly enhanced the recruitment of rat neutrophils in response to S. aureus compared with that in the PBS control group (Fig. 3A, B).Fig. 3: BI enhances neutrophil chemotaxis and mitigates S. aureus-induced ATDC5 cell damage.A Schematic of the Transwell migration assay. Neutrophils were seeded in the upper chamber, and the supernatants of S. aureus cultures treated with various concentrations of BI were placed in the lower chamber. B Effects of S. aureus culture supernatants on blocking neutrophil chemotaxis. C, D Live/dead cell assay showing the protective effect of BI against S. aureus infection in ATDC5 cells (scale bar: 100 μm). E LDH release assay in ATDC5 cells. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageTo further evaluate the protective effects of BI against S. aureus-induced cytotoxicity in ATDC5 cells, we exposed infected ATDC5 cells to various doses of BI and conducted calcein-AM/PI double staining. Fluorescence microscopy revealed prominent green fluorescence (live cells) with minimal red fluorescence (dead cells) (Fig. 3C, D). The kd-saeR group presented a similar trend. These observations underscore the potential of BI to alleviate S. aureus-induced cytotoxic effects in ATDC5 cells, particularly at 64 μg/mL. This conclusion was further supported by the lactate dehydrogenase (LDH) release assay, in which BI treatment significantly reduced LDH release from damaged ATDC5 cells (Fig. 3E).BI inhibits biofilm maturation and reduces eDNA levels in the biofilm matrix, altering the biofilm structureWe next assessed the impact of BI on S. aureus biofilm formation. Crystal violet staining demonstrated that BI inhibited biofilm formation in a dose-dependent manner (Fig. 4A), with an IC50 of 95.16 μg/mL (Fig. 4B). Additionally, we evaluated its effect on preformed biofilms and revealed that BI was effective against biofilms in their early maturation stages (Fig. 4C).Fig. 4: BI affects S. aureus biofilm formation and matrix composition.A Crystal violet staining showing the effect of BI on biofilm formation by S. aureus. B The IC50 of BI for biofilm formation inhibition was determined to be 95.16 μg/mL. C Crystal violet staining showing the impact of BI on biofilm development at different stages. The time on the x-axis represents the moment at which the compound was introduced. D Effects of BI on S. aureus motility. E BI’s effect on Triton X-100-induced autolysis. F, G Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) images showing the impact of BI on the S. aureus biofilm structure. H Agarose gel electrophoresis and quantification of eDNA in the biofilm matrix. I Congo red agar plate showing differences in PIA content. J Protein quantification in the biofilm matrix. K Schematic representation of the impact of BI on biofilm formation: BI inhibits biofilm development and alters biofilm structure by affecting the eDNA content in the biofilm matrix. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageSince motility plays a critical role in initial bacterial adhesion and subsequent biofilm formation35, we examined the preliminary effects of BI on S. aureus motility. S. aureus, a nonflagellated bacterium, exhibits motility characterized by sliding or colony spreading. Compared with the wild-type (WT) group, BI (64 μg/mL) significantly inhibited the sliding motility of S. aureus (Fig. 4D). During biofilm formation, autolysis leads to the release of extracellular DNA (eDNA), which further promotes biofilm development. Triton X-100-induced autolysis was significantly lower in BI-treated S. aureus than in untreated S. aureus (Fig. 4E).Confocal laser scanning microscopy (CLSM) revealed that, compared with SaeR-silenced (kd-saeR) S. aureus, untreated WT S. aureus formed denser biofilms, whereas BI-treated WT strains produced thinner biofilms. Notably, with increasing concentrations of BI, the biofilms formed by the WT strains became looser and more porous (Fig. 4F, Supplementary Fig. 5). Scanning electron microscopy (SEM) images confirmed similar trends, revealing compact biofilm structures in the WT group (Fig. 4G). Collectively, these findings suggest that BI restricts biofilm formation and disrupts the biofilm architecture of S. aureus.To preliminarily explore the mechanism by which BI inhibits biofilm formation, we quantified eDNA levels in biofilms formed by BI-treated and untreated S. aureus. The results revealed a reduction in eDNA levels in BI-treated biofilms (Fig. 4H). However, there were no significant differences in the levels of polysaccharides (PIAs) or proteins in the biofilms between the groups (Fig. 4I, J). Thus, the inhibition of biofilm formation by BI is mediated through its effects on initial bacterial adhesion and its ability to reduce eDNA levels within the biofilm matrix (Fig. 4K).Interaction between BI and SaeRWe treated S. aureus with BI for 24 h and found that the transcription of the SaeRS TCS was suppressed, as shown by RT‒qPCR (Fig. 5A). Electrophoretic mobility shift assays (EMSAs) further demonstrated that SaeR exhibited specific binding affinity for the P1 promoter fragment. BI inhibited SaeR-P1 binding in a dose-dependent manner, confirming its regulatory effect (Fig. 5B, Supplementary Fig. 6). Additionally, cellular thermal shift assay (CETSA) experiments revealed significant differences between the BI-treated and dimethyl sulfoxide (DMSO)-treated groups as the temperature increased, indicating a direct interaction between BI and SaeR (Fig. 5C, Supplementary Fig. 7). These findings suggest a strong interaction between BI and SaeR.Fig. 5: Direct interaction between BI and SaeR.A Inhibition efficiency of BI on the SaeRS TCS. B Dose-dependent inhibition of the interaction between SaeR and the P1 promoter by BI and relative band intensity analysis. C Effect of BI on SaeR thermal stability as determined by a cellular thermal shift assay (CETSA). D Fluorescence quenching assay to determine the binding affinity between BI and SaeR. E Surface plasmon resonance (SPR) analysis of the binding kinetics between BI and SaeR. The colored lines represent dynamic signal responses. F Molecular docking simulation identifying key amino-acid residues involved in the interaction between BI and SaeR. G The effects of BI on the activity of the SaeR mutants K156A, W182A, and R199A were explored by fluorescence quenching. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageWe further analyzed the kinetics of the binding of BI to SaeR via fluorescence quenching assays. By measuring the intrinsic fluorescence intensity of SaeR before and after the addition of BI, we evaluated the interaction and conformational changes of SaeR. Upon titration of BI into recombinant SaeR, the fluorescence intensity decreased in a concentration-dependent manner at the maximum emission wavelength of 320 nm, indicating static quenching. The Stern‒Volmer plot (Fig. 5D) derived from these measurements revealed that the binding constant (KA) of BI to SaeR was 1.1116 × 10⁴ L/mol, suggesting a binding interaction.Surface plasmon resonance (SPR) analysis was employed to further verify the binding kinetics between BI and SaeR (Fig. 5E). The association rate constant (Ka) and dissociation rate constant (Kd) were determined to be 1.88 × 104 M−1 s−1 and 3.70 × 10−3 s−1, respectively. The equilibrium dissociation constant (KD = 1.97 × 10⁻⁷ M) indicated a strong binding affinity between SaeR and BI.Investigation of the interaction mode between SaeR and BITo confirm the binding of BI to the functional domains of SaeR, we performed molecular docking. The docking analysis (Fig. 5F) predicted key amino-acid residues involved in the binding of BI to SaeR, supporting its potential as a candidate for further investigation. LYS156, TRP182, and ARG199 were identified as critical residues involved in BI-SaeR binding. Using the MMGBSA method, the binding free energy (ΔGbind) of the SaeR-BI complex was calculated to be −7.3 kcal/mol (Supplementary Table 3).On the basis of the binding sites predicted by molecular modeling, we generated four SaeR mutants through site-directed mutagenesis: K121A-SaeR, K156A-SaeR, W182A-SaeR, and R199A-SaeR, with K121A-SaeR serving as a nonrecognition site control. Further fluorescence quenching assays revealed that the affinity constants of K156A-SaeR, W182A-SaeR, and R199A-SaeR for BI were significantly lower than those of the wild-type SaeR, whereas the affinity of K121A-SaeR for BI was not significantly different from that of the wild-type protein (Fig. 5G, Supplementary Fig. 8). These findings indicate that the key amino acids LYS156, TRP182, and ARG199 are critical for the interaction between BI and SaeR.BI protects G. mellonella from S. aureus infectionThe G. mellonella larval model, which has an immune system highly similar to that of vertebrates, was used to assess the therapeutic efficacy of BI. By monitoring larval activity, cocoon formation, melanization, and survival, we evaluated the treatment effect of BI. Following infection with USA300 and the administration of BI, larval survival was tracked for 5 days (Fig. 6A).Fig. 6: BI protects G. mellonella larvae from MRSA infection.A Schematic representation of the G. mellonella MRSA infection model and observation timeline. B Effects of various treatments on the appearance of G. mellonella larvae, including the control group, MRSA infection group, vancomycin treatment group (50 mg/kg), low-dose BI treatment group (20 mg/kg), high-dose BI treatment group (50 mg/kg) and kd-saeR strain infection group (n = 10). C Survival rates of G. mellonella larvae over 120 h across different treatment groups. D Colony-forming unit (CFU) counts from infected larvae (n = 5) via the agar plate dilution method. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageThe kd-saeR strain displayed a marked reduction in both infectivity and pathogenicity, indicating a significant decline in its ability to induce disease. After day 5, images revealed that high-dose BI treatment significantly improved survival (Fig. 6B). Over time, we observed a concentration-dependent increase in larval survival, indicating that BI effectively mitigated MRSA infection. By the end of the study, the survival rate had reached 60% (Fig. 6C). On the basis of the health evaluation scores, BI demonstrated significant efficacy in improving the health score in the G. mellonella MRSA infection model (Supplementary Fig. 9A, B). Sterile homogenization of the larvae followed by colony counting at 48 h post infection revealed that BI reduced the bacterial load from 9.02 log10 CFU to 6.49 log10 CFU (Fig. 6D).BI reduces damage in an S. aureus-induced septic arthritis rat modelGiven the in vitro effectiveness of BI in inhibiting SaeR activity, we sought to further evaluate its potential in treating S. aureus-induced septic arthritis in vivo. The rats were intra-articularly infected with S. aureus and treated with 50 mg/kg BI (Fig. 7A). Knee joint bioluminescence was measured daily and gradually increased in all groups from day 1 to day 3, peaking on day 3. Notably, the bioluminescence intensity decreased in both the vancomycin and BI treatment groups compared with that in the untreated WT group from day 4 to day 7, indicating reduced infection severity (Fig. 7B, C). Knee joint diameter measurements and arthritis scores revealed that BI or vancomycin treatment significantly reduced knee swelling compared with that in infected control rats (Fig. 7D, E; Supplementary Fig. 10).Fig. 7: Effect of BI on septic arthritis caused by S. aureus infection.A Schematic of the septic arthritis rat model induced by S. aureus. B, C Representative bioluminescence images and quantification of bioluminescence intensity in the septic arthritis model (n = 5) on days 1, 2, 3, 4, 5, 6, and 7. D, E Representative images of rat knees and comparisons of knee joint diameters on day 7. F–H Three-dimensional reconstructions of the knee joint via micro-CT and two-dimensional images in the coronal (COR), sagittal (SAG), and axial (AX) planes (n = 3). The bone volume/total volume (BV/TV) and bone mineral density (BMD) of the femoral and tibial condyles were measured via micro-CT. I Hematoxylin and eosin (H&E) staining of paraffin-embedded tissue sections. J Quantification of MRSA bacteria in synovial fluid (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group.Full size imageMicrocomputed tomography (micro-CT) was used to assess joint structure during infection. Three-dimensional (3D) imaging demonstrated a significant reduction in joint damage in the SaeR-knockdown strain. In contrast, the MRSA-infected group exhibited significant cortical and trabecular bone erosion at the distal femur and proximal tibia, with an increase in involucra (markers of reactive bone formation) (Fig. 7F). MRSA infection led to reduced bone volume/total volume (BV/TV) and tissue mineral density (TMD), which were improved by treatment with BI or vancomycin (Fig. 7G, H). Histological analysis of knee joint tissue collected on day 7 posttreatment via Hematoxylin‒eosin (H&E) staining revealed severe inflammatory exudation, necrotic regions, and neutrophil infiltration in the no-treatment group (Fig. 7I). In contrast, the inflammatory response was markedly reduced in both the vancomycin- and BI-treated groups. The SaeR-knockdown strain also presented reduced signs of joint pathological damage (Fig. 7I). These findings highlight the protective effects of BI in cases of bacterial synovial joint infection via the inhibition of SaeR.On day 7 posttreatment, bacterial colony counts in synovial fluid from rat knee joints were significantly lower in the BI-treated group than in the control group (Fig. 7J). These findings demonstrate that BI has therapeutic potential against S. aureus-induced septic arthritis, further corroborating its beneficial effects, which are attributed to its antivirulence properties.DiscussionAntibiotics have long been regarded as the cornerstone in the fight against bacterial infections, including conditions such as septic arthritis. Over the past 15 years, the use of antibiotics has increased dramatically. However, the therapeutic efficacy of these critical drugs is increasingly undermined by the relentless development of bacterial resistance. Among the most concerning pathogens is S. aureus, including MRSA, which has developed resistance to nearly all known antibiotics. Given the remarkable adaptability of pathogens, the sole reliance on antibiotics often results in unsatisfactory outcomes. This highlights the urgent need to identify novel therapeutic targets and develop new strategies to combat infections caused by this formidable pathogen36.S. aureus virulence is largely attributed to its repertoire of surface-associated virulence factors, exotoxins, enterotoxins, and superantigens37. These toxins, including Hla, Hlb, δ-toxin, PVL, and phenol-soluble modulins (PSMs), play key roles in leukocyte lysis38, with Hla and PSMs also contributing to biofilm formation. Although upstream regulators control the expression of multiple virulence factors, targeting these regulators alone may not fully suppress virulence gene expression and, in some cases, may even increase the expression of certain factors. Therefore, targeting downstream regulators—those that strictly control virulence factor expression—is a more promising approach to effectively inhibit S. aureus virulence and identify potential therapeutic candidates.The SaeRS TCS is composed of the sensor histidine kinase SaeS and the response regulator SaeR20. In the presence of external stimuli, such as β-lactam antibiotics or α-defensins, SaeS undergoes autophosphorylation and subsequently transfers the phosphate group to SaeR. SaeR-P binds to the promoter regions of target genes, including the P1 promoter of the sae locus, where it recruits RNA polymerase to initiate transcription39. Given the critical role of SaeR-regulated genes in S. aureus pathogenesis, SaeR represents a viable target for the development of novel anti-infective agents. Moreover, the highly conserved nature of the saeR sequence across S. aureus strains underscores its importance in virulence regulation, suggesting that SaeR-targeted antivirulence therapies could be effective against a wide range of S. aureus strains.In this study, we utilized structure-based virtual screening to identify BI as a selective inhibitor of SaeR. BI was shown to block the promoter-binding region of the SaeR protein, thereby inhibiting its function, reducing the expression of SaeR-dependent virulence factors, and mitigating the pathological damage associated with septic arthritis (Fig. 8). This unique approach provides compelling evidence for the development of antivirulence therapies targeting MRSA.Fig. 8: Schematic diagram of the effects of BI in the treatment of S. aureus-infected septic arthritis rats.In septic arthritis, BI interferes with the disruption of SaeR-mediated virulence factors. SaeRS TCS regulation model. BI-mediated perturbation obstructs the SaeR‒DNA interaction, which manifests as reduced biogenesis of the Hla heptamer complex and reduced hemolysis of red blood cells. The decreased release of S. aureus PVL weakens the immune escape ability of S. aureus. Notably, BI also regulates SaeR-dependent bacterial adhesion and biofilm generation to further mitigate pathological damage in septic arthritis.Full size imagePrevious studies using SaeR-deficient S. aureus strains have demonstrated reduced pathogenicity in various infection models40,41. However, since SaeR is critical for the initial stages of infection, S. aureus lacking SaeR is often unable to establish infection in animal models, complicating efforts to determine whether SaeR-targeted antivirulence therapies could effectively reduce bacterial virulence. Additionally, owing to the nonantibiotic nature of antivirulence molecules, comparisons of the intracellular survival of wild-type, compound-treated, and SaeR-deficient strains do not provide a clear indication of the potential of SaeR as a therapeutic target. To overcome these limitations, we employed a CRISPR-dCas9-based knockdown plasmid to mimic antivirulence therapy targeting SaeR. This enabled us to evaluate the contribution of SaeR to S. aureus pathogenesis, further validating it as a target for antivirulence drug development.Our molecular biology and mutagenesis studies confirmed the specificity of BI for SaeR. SPR and EMSA demonstrated the strong binding affinity of BI for SaeR, competitively inhibiting the ability of SaeR to function as a transcriptional regulator and suppress the transcription and expression of downstream virulence factors. Molecular docking revealed that BI stably interacts with the promoter-binding region of SaeR via hydrogen bonds. Site-directed mutagenesis and fluorescence quenching assays demonstrated that the affinity of BaI for the mutated SaeR proteins was lower than that for the wild-type protein, suggesting that the key amino acids LYS156, TRP182, and ARG199 are critical for the interaction between BI and SaeR.S. aureus is the most common bacterial pathogen associated with septic arthritis. Despite prolonged intravenous antibiotic therapy, septic arthritis often recurs, complicating treatment42. In addition to secreting a wide array of virulence factors that cause severe infections, S. aureus can be internalized by host cells and form biofilms on cartilage surfaces, a likely mechanism underlying the recurrence and antibiotic resistance of septic arthritis43,44. Biofilms formed by S. aureus serve as both physical and chemical barriers to antibiotic penetration and immune system attack, particularly in the context of septic arthritis. The internalization of S. aureus into host phagocytic and nonphagocytic cells allows it to persist as a reservoir for recurrent septic arthritis45,46. Prior to this study, it was unclear whether S. aureus could directly infect chondrocytes. Our results indicate that MRSA can penetrate chondrocytes, suggesting that, in addition to systemic vancomycin treatment, cell-penetrating antibiotics may be necessary to effectively target intracellular infections. Consistent with clinical observations47, systemic vancomycin treatment reduces the overall MRSA burden in patients with septic arthritis but fails to eradicate MRSA from immune cells and joint tissues. This phenomenon suggests that intracellular and interstitial bacterial infections may contribute to the persistence and recurrence of septic arthritis.Biofilms are structured bacterial communities encased within a self-produced ECM, formed through a complex and dynamic process regulated by multiple factors. This process can be divided into four major stages: initial attachment, irreversible attachment, maturation, and dispersion. In the early stages of biofilm formation, S. aureus synthesizes various surface-anchoring proteins that mediate initial attachment and promote adhesion development. The aggregation of bacteria is subsequently driven by components of the biofilm ECM, including polysaccharide intercellular adhesin (PIA or PNAG), biofilm-associated proteins, and eDNA48. During the dispersion stage, bacteria embedded within the mature biofilm can revert to a planktonic state, spreading to secondary sites and exacerbating primary infections. The release of bacteria from biofilms also facilitates the colonization of new sites, leading to persistent or recurrent infections49. Owing to this problem, the biofilm matrix acts as a barrier to antibiotic penetration, while metabolically inactive bacterial phenotypes further diminish the effectiveness of antibiotics50, particularly in pyogenic joint infections.The biofilm structure is crucial for its function, as dense biofilms prevent leukocyte infiltration, shielding bacteria from immune clearance51. Numerous studies have shown that inhibiting the expression of virulence factors such as hla, psm, and other toxins can impair biofilm formation52,53,54. In our in vitro studies, we observed that BI significantly altered the biofilm structure of S. aureus. Notably, compared with untreated biofilms, biofilms treated with BI presented a much looser and more porous structure. More importantly, BI effectively inhibited biofilm formation in its early stages, disrupting the natural development trajectory of the biofilm and preventing the cascade of detrimental consequences associated with biofilm maturation. Biofilms are composed primarily of eDNA, polysaccharides, and extracellular proteins. Further analysis revealed that BI inhibits biofilm formation mainly by reducing eDNA and simultaneously suppressing bacterial autolysis. Since eDNA is crucial for the stability and formation of biofilms, these findings highlight its key role in the antibiofilm activity of BI. Moreover, the formation of biofilms and the invasion of S. aureus are tightly regulated by multiple factors, and the exact mechanisms through which BI affects biofilm formation require further exploration.Plant-derived small molecules, known as phytotoxins or phytochemicals, offer a range of antimicrobial mechanisms distinct from those of traditional antibiotics. Although their antimicrobial effects may be subtler, these compounds provide a multifaceted approach to targeting pathogens. Our RT‒qPCR results demonstrated that BI suppressed the secretion of several S. aureus virulence factors. For example, BI downregulated hlgC expression, suggesting that SaeR inhibitors can reduce bacterial survival by inhibiting the production of γ-hemolysin, a key contributor to bacterial persistence in the bloodstream55. S. aureus secretes six serine protease-like proteins (SplA-SplF), with SplA, in particular, promoting bacterial invasion and dissemination by cleaving glycosylated surface proteins56. Therefore, the inhibition of SaeR by BI, which leads to reduced SplA expression, may alleviate chronic infections and limit bacterial invasion. Similarly, Map (MHC class II analog protein) is a secreted protein that binds to various ECM components, including fibronectin, fibrinogen, and thrombospondin37, facilitating bacterial adhesion to host tissues during infection. The downregulation of map expression by BI may inhibit bacterial attachment to host tissues during infection. The chemotaxis inhibitory protein of S. aureus (CHIPS), encoded by the chp gene, is an extracellular protein that effectively blocks neutrophil and monocyte chemotaxis, enabling S. aureus to evade host innate immune responses57. The inhibition of chp by BI could increase bacterial susceptibility to immune clearance, thus reducing the bacterial load during infection. Our in vitro data confirmed that BI mitigates S. aureus virulence by targeting and inhibiting SaeR. Moreover, our study highlights the protective effects of BI in MRSA-induced septic arthritis, suggesting a novel therapeutic development pathway.In conclusion, this study successfully identified BI as a promising antivirulence agent with potential clinical applications. The binding of BI to SaeR underscores the attractiveness of SaeR as a druggable target for S. aureus infections. Our findings also provide a framework for developing novel nonantibiotic therapies to treat S. aureus infections, highlighting a new direction for combating this persistent pathogen.MethodsBacterial strains, growth conditions, and chemical reagentsThe S. aureus strain used in this study was ATCC®BAA-1717™ (USA300-HOU-MR). The SaeR-knockdown strain (kd-SaeR)58 and bioluminescent strain Xen2959 were constructed following established protocols. All S. aureus strains were cultured in brain heart infusion (BHI) broth or on BHI agar plates supplemented with chloramphenicol (10 µg/mL). Escherichia coli (E. coli) strains were grown in Luria–Bertani (LB) broth at 37 °C, with kanamycin (50 µg/mL) added when appropriate. Unless otherwise specified, all the cultures were grown at 37 °C in a shaking incubator. The strains involved are listed in Supplementary Table 4. BI, a natural product with a purity of 99.43%, was purchased from Letian Mei Biotech Co., Ltd. (Chengdu, China), and a quality inspection report is provided in Supplementary Fig. 3. The general reagents used were obtained from Sangon Biotech (Shanghai, China).Expression and purification of recombinant SaeR and its mutantsThe primers used were designed on the basis of the genome sequence of S. aureus MW2 (NC-003923) published by the National Center for Biotechnology Information (NCBI). For recombinant 6-His-tagged SaeR protein expression, the full-length SaeR gene was amplified from USA300 genomic DNA. The PCR product was digested with BamHI and XhoI and inserted into the pET28a plasmid, which was then transformed into E. coli DH5α. The recombinant plasmid pET28a-saeR was further transformed into E. coli BL21 (DE3) for expression of the recombinant BL21-pET28a-saeR strain, followed by overnight culture at 37 °C. Site-directed mutagenesis of K121A-saeR, K156A-saeR, W182A-saeR, and R199A-saeR was performed via a site-directed mutagenesis kit (TianGen®, China), and the mutations were verified via DNA sequencing. The mutant plasmids were subsequently transformed into E. coli BL21 (DE3) and cultured overnight at 37 °C.The cultures were diluted 1:100 into 1000 mL of LB medium containing kanamycin (50 µg/mL) and grown until the OD600 nm reached 0.6–0.8. Isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 0.1 mmol/L to induce protein expression at 16 °C for 16 h. The bacteria were harvested via centrifugation (4000 rpm, 30 min), washed twice with phosphate-buffered saline (PBS), and resuspended in 25 mL of non-denaturing lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl). The cells were lysed on ice via an ultrasonic homogenizer (Xiaomei ultrasonic instrument Co., Ltd., China. 200 W; 5 s pulses with 10 s intervals for 2–3 h), followed by centrifugation at 12,000 rpm for 1 h at 4 °C. The supernatant was added to Ni-NTA His-tag purification agarose (MedChemExpress, China), which had been preequilibrated with a nondenaturing binding buffer. The contaminating proteins were washed with a buffer containing 50 mmol/L imidazole, and the target protein was eluted with 400 mmol/L imidazole. SDS‒PAGE was performed to verify protein purity, and the purified SaeR protein was stored at −80 °C for further experiments.Virtual screening with discovery studioThe 3D structure of SaeR (PDB ID: 4QWQ) was obtained from the Protein Data Bank (www.rcsb.org). For virtual screening, a small-molecule library was assembled using data from the ZINC website (https://zinc.docking.org/) and additional compounds from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The binding site for SaeR was predicted on the basis of the shape of the receptor via Discovery Studio60. CDOCKER, a CHARMM-based docking tool, was used for rigid receptor docking. Ligand conformations were first generated via high-temperature molecular dynamics with various random seeds, and the final conformations were minimized via a rigid receptor model. For each conformation, the CHARMM energy and interaction energy was calculated61, and the top-ranked conformations on the basis of binding energy were selected for further evaluation.Thermal shift assayTSA was conducted as previously described, and SYPRO Orange dye (Thermo Fisher Scientific, China) was used to monitor the interaction between SaeR and small molecules. A 100× SYPRO Orange solution was mixed with SaeR protein (final concentration of 2 μM) on ice. Then, 6 μL of the mixture was combined with 6 μL of the small-molecule mixture and 8 μL of buffer (150 mM NaCl, 10 mM HEPES, pH 7.5), and the samples were added to a 96-well PCR plate. Fluorescence was measured via the IQ5 Real-Time PCR Detection System (Bio-Rad, USA) as the temperature increased from 25 °C to 95 °C at a rate of 1 °C/min, with Ex/Em wavelengths of 490 nm/530 nm. Wells containing the same buffer composition but without small molecules were used as controls.MIC assay and growth curve analysisThe MIC, defined as the lowest drug concentration that inhibits bacterial growth (OD600 nm < 0.01), was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines62. A 96-well plate containing S. aureus (1 × 105 CFU) and various concentrations of BI (1‒256 µg/mL) was incubated at 37 °C for 18 h. To plot the growth curve, S. aureus was incubated with 128 μg/mL BI at 37 °C, and the OD600 values were measured at various time points.Resistance induction assayThe resistance of S. aureus to BI was assessed via a twofold dilution method. Briefly, 180 μL of S. aureus (1 × 105 CFU/mL) was incubated with 20 μL of BI (5.12 mg/mL) or Oxacillin (5.12 mg/mL) in a 96-well plate. The subsequent wells contained serial dilutions of BI and Oxacillin at concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg/mL. The plate was incubated at 37 °C for 24 h, after which the MIC was determined. Following the determination of the MIC, 50 μL of bacterial suspension from wells containing sub-MIC concentrations (1/2 MIC) of BI or Oxacillin was transferred to 10 mL of BHI medium and incubated at 37 °C with shaking for 8 h. This process was repeated for 20 generations, with MIC measurements taken every 5 generations to monitor the development of bacterial resistance.Cytotoxicity assayThe ATDC5 cell line, derived from mouse teratocarcinoma cells, is a continuously cultured line widely used as an in vitro model for chondrocyte research. The cytotoxic effects of BI on ATDC5 cells were evaluated via the MTT assay. ATDC5 cells were seeded at a density of 2 × 10⁴ cells/well in a 96-well plate and incubated at 37 °C in a 5% CO2 incubator for 24 h. The cells were then treated with various concentrations of BI (8‒128 µg/mL) for 24 h. After the medium was removed, 5 mg/mL MTT solution was added to each well, and the mixture was incubated for 4 h. The formazan crystals were dissolved in 100 µL of DMSO, and the absorbance was measured at 490 nm.Western blot analysisS. aureus was cultured to an OD600 of 0.3, after which various concentrations of BI (8‒128 µg/mL) were added, and the cultures were grown until the late logarithmic phase (OD600 of 2.5). The bacteria were harvested, and the total protein was extracted via lysis buffer. Protein concentrations were quantified via a BCA protein assay kit (Beyotime, China). Equal amounts of protein (20 μg) were separated by SDS‒PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with an anti-Hla antibody (1:2000, Sigma, USA) or anti-PVL polyclonal antibody (1:2000, Abcam, England). After washing, the membranes were incubated with an HRP-conjugated secondary antibody (1:5000, Abcam, England) for 1 h at room temperature (RT). Visualization was achieved via an enhanced chemiluminescence kit (Servicebio, China), and images were captured via a chemiluminescence detection system.Hemolysis assayS. aureus was cultured to an OD600 of 0.3, and BI (8–128 µg/mL) was added. The cultures were incubated until the late logarithmic phase (OD600 of 2.5) and centrifuged (5500 rpm, 4 °C, 1 min), and the supernatant was filtered for further analysis. For the hemolysis assay, 100 µL of supernatant was mixed with 25 µL of defibrinated rabbit blood and 775 µL of PBS in a 1 mL reaction volume. Triton X-100 served as the negative control. The samples were incubated at 37 °C until complete hemolysis was observed in the untreated group. After centrifugation (5500 rpm, RT, 1 min), the OD543 nm was measured via a microplate reader.Adhesion of S. aureus to immobilized fibrinogenTo assess the effect of BI on S. aureus adherence to fibronectin, a 96-well plate was coated with bovine fibronectin (50 µg/mL; Source Leaf, China) overnight at 4 °C. After the fibronectin solution was removed, the wells were blocked with 3% bovine serum albumin (BSA; Sigma, USA) for 2 h to prevent nonspecific binding. S. aureus was grown to the logarithmic phase in the presence of various concentrations of BI (8–128 µg/mL). The 96-well plate was then washed and incubated with BI-treated S. aureus (5 × 10³ CFU/well) at 37 °C for 1 h. After incubation, the wells were washed with PBS, and the adherent cells were fixed with 4% paraformaldehyde for 30 min. The wells were then stained with 0.1% crystal violet for 20 min and washed, and the absorbance was measured at 600 nm via a microplate reader.RNA extraction and real-time quantitative reverse transcription PCR (RT‒qPCR)To examine the expression of virulence factors in S. aureus USA300, the bacteria were coincubated with BI (64 µg/mL) or the kd-saeR strain at 37 °C and 200 rpm for 24 h. Total RNA was extracted via TRIzol reagent, and reverse transcription was performed via a commercial kit (Servicebio, China). The synthesized cDNA was stored at −80 °C. RT‒qPCR was carried out via SYBR Green mix (Servicebio, China) to quantify the expression levels of target genes. The cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 30 s. Primers for key virulence genes were designed on the basis of the nucleotide sequences provided in Supplementary Table 5. The relative RNA levels of each gene were calculated via the 2-ΔΔCt method, with gyrb as the internal control.Fluorescence Staining of Intracellular bacteriaThe kd-SaeR strain was cultured on BHI agar plates supplemented with 10 μg/mL chloramphenicol. Wild-type S. aureus strains were incubated with BI (32 and 64 μg/mL) at 37 °C for 24 h, with DMSO (0.1%) added to the control groups. The bacterial cells were labeled with 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (MedChemExpress, China). ATDC5 cells were cultured in DMEM/high-glucose medium (Servicebio, China) and seeded at a density of 1 × 105 cells/well in a 24-well plate. After 24 h of incubation, ATDC5 cells were infected with S. aureus at a multiplicity of infection (MOI) of 5 for 1 h. The extracellular bacteria were then killed by treatment with 50 μg/mL gentamicin for 1 h, after which the cells were washed three times with sterile PBS (pH 7.4). The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS containing 0.1% Triton X-100 for 10 min, and stained with TRITC-conjugated phalloidin (Yeasen, China) and DAPI (Invitrogen, USA). Confocal laser scanning microscopy (Leica TCS DMI8, Germany) was used to capture images.Quantification of internalized bacteria via CFU countingATDC5 cells were seeded at a density of 1 × 105 cells/well in a 24-well plate and incubated for 24 h. The cells were infected with the S. aureus kd-saeR strain or BI-treated/untreated S. aureus strains (1 × 105 CFU/mL) for 1 h. After 1 h, the cells were treated with gentamicin (50 µg/mL) for 1 h to kill the extracellular bacteria. The cells were washed three times with sterile PBS and lysed with 0.1% Triton X-100 to release intracellular bacteria. Serial dilutions of cell lysates were plated on blood agar and incubated overnight at 37 °C, after which the CFU counts were determined. The relative number of internalized bacteria was calculated by dividing the CFU by the total number of ATDC5 cells.Neutrophil isolation and transwell migration assayPeripheral blood samples were collected from the rats, and neutrophils were isolated via a rat neutrophil isolation kit (Tianjin Haoyang Huake Biotechnology, China) according to the manufacturer’s instructions. The isolated neutrophils were resuspended in Hank’s balanced salt solution (HBSS) containing 1% BSA. S. aureus USA300 was incubated with various concentrations of BI (16–64 µg/mL) at 37 °C and 220 rpm for 4 h. The supernatant was collected and heat-inactivated at 60 °C for 30 min. A 24-well plate with 3.0 µm transwell inserts was used to evaluate neutrophil migration. Heat-inactivated S. aureus (3 × 108 cells/insert) was added to the inserts, and 600 µL of HBSS containing 1% BSA was added to the lower chamber. Neutrophils (1.5 × 104 cells) were added to the upper chamber and incubated at 37 °C for 1 h. After incubation, nonmigrating neutrophils were removed, and the remaining cells were stained with Alexa Fluor 647-conjugated Ly6G. The cells were washed three times with sterile PBS. The fluorescence was measured at 535 nm and 488 nm.Infection of ATDC5 cells and LDH assayATDC5 cells were seeded at a density of 2 × 104 cells/well in a 24-well plate and incubated for 12 h. The culture medium was replaced with a fresh RPMI 1640 medium containing 10% serum. S. aureus USA300 was added at an MOI of 50, and the cells were treated with various concentrations of BI (32 and 64 µg/mL) for 6 h. Cell viability was assessed via calcein-AM/PI staining to distinguish between live and dead ATDC5 cells, and images were captured via fluorescence microscopy. The control group consisted of untreated cells, and ImageJ software (ImageJ 1.52a) was used to quantify the ratio of live to dead cells. To measure LDH release, the supernatants were collected after 6 h of incubation, and LDH levels were measured via an LDH assay kit (Beyotime, China).Biofilm formation and development assaysS. aureus was cultured in a 96-well plate (1 × 106 CFU/mL) with or without BI treatment (128 µg/mL) at 37 °C overnight. After 24 h, the biofilms were gently washed three times with sterile PBS, fixed with 100 µL of methanol for 15 min, and stained with 0.1% crystal violet ethanol solution for 15 min. After the plate was washed, images were captured, and biofilm formation was quantified by adding 33% acetic acid to each well and measuring the absorbance at 570 nm.The impact of BI on various stages of biofilm formation was analyzed as previously described63, with slight modifications. More specifically, rabbit plasma-coated 96-well plates were inoculated with S. aureus. The control wells contained no compounds. BI was added sequentially to the wells at various times to a final concentration of 128 µg/mL. After coincubation for 24 h, the biofilms were stained with 1% crystal violet, and the absorbance was measured at 570 nm.Cell motility assayTo assess sliding motility, plates containing 1% tryptone, 0.25% NaCl, and 0.3% agar were prepared. A 15-µL aliquot of a standard S. aureus cell suspension (1 × 10⁸ CFU/mL) was inoculated at the center of the plates, either in the absence or presence of BI (64 µg/mL). After 24 h of incubation at 37 °C, the motility of S. aureus was observed.Triton X-100-induced autolysis assayOvernight cultures of S. aureus, either treated or not treated with BI (64 µg/mL), were centrifuged to collect the bacterial pellets. The pellets were washed and resuspended in 0.05 M Tris-HCl buffer (pH 7.0) containing 0.05% Triton X-100. The suspensions were incubated at 30 °C with shaking at 150 rpm. The OD600 nm was measured hourly to assess autolysis.CLSM and SEM for biofilm observationGlass coverslips were coated with lyophilized rabbit plasma at 4 °C overnight in a 24-well plate. S. aureus suspensions (1 × 105 CFU/mL) containing varying concentrations of BI (64 and 128 µg/mL) were added, and biofilms were allowed to form by incubating the plates at 37 °C for 24 h. The samples were gently washed with precooled PBS and fixed with glutaraldehyde for 15 min. FITC (0.001%) was added, and the mixture was incubated at 37 °C with shaking for 5 min. The samples were then stained in the dark at 4 °C for 15 min to 1 h. After washing with PBS, the coverslips were mounted on slides, and biofilm formation was observed via a confocal laser scanning microscope (Leica TCS DMI8, Germany). Live bacteria were visualized as green fluorescence.For SEM, glass coverslips coated with rabbit plasma were treated similarly to the CLSM setup. After incubation with S. aureus and BI, the samples were washed with sterile PBS to remove nonadherent bacteria and fixed overnight with glutaraldehyde at 4 °C. The samples were dehydrated in a series of ethanol concentrations, freeze-dried, and sputter-coated with platinum before the biofilm structure was imaged via SEM (HITACHI Regulus 8100, Japan).Quantification of eDNA, protein, and PIA in biofilmsS. aureus was cultured overnight at 37 °C and diluted in BHI to 1 × 106 CFU/mL. Bacterial suspensions (1 mL) were added to a 6-well plate, either treated or not treated with BI (64 µg/mL), and incubated at 37 °C for 24 h. The biofilms were collected by washing with PBS, filtered through 0.22 µm filters, and suspended in PBS. The concentrations of eDNA and proteins in the biofilm matrix were quantified via a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). The PIA content was evaluated by color changes on Congo red agar plates.Electrophoretic mobility shift assayPurified SaeR (20 μM) and SaeSC (1 μM) were mixed in phosphorylation buffer (10 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, and 10% glycerol), followed by the addition of 1 mM ATP and incubation at RT for 5 min. EMSA was performed with 80 ng of fluorescein (FAM)-labeled DNA probes mixed with varying amounts of SaeR in 25 µL of gel mobility shift buffer (10 mM Tris-HCl, pH 7.4; 50 mM KCl; 5 mM MgCl2; 10% glycerol; and 3 μg/mL sheared salmon sperm DNA). After 30 min of incubation at RT, the samples were subjected to electrophoresis on an 8% polyacrylamide gel (100 V for prerun, 85 V for 30 min of separation), and the gels were imaged.Cellular thermal shift assaySaeR proteins were expressed via the same method, specifically through E. coli BL21(DE3) pET28a-SaeR64. At the outset, the bacterial lysates were centrifuged to collect the supernatants. One portion of the supernatant was treated with BI (32 μg/mL), and the other portion was treated with DMSO as a control. After incubation at 37 °C for 1 h, the supernatants were centrifuged and distributed into PCR tubes. The samples were heated at specific temperatures for 5 min, followed by immersion in a quick 3-minute ice bath. The supernatant was collected by centrifugation and mixed with protein loading buffer. The samples were subjected to 12% SDS‒PAGE for separation. Protein band intensities related to stability were quantified via ImageJ software.Fluorescence quenching assayThe binding constant (KA) of BI to SaeR and the SaeR mutant proteins was determined via fluorescence quenching. Purified SaeR (4 µM) was mixed with increasing concentrations of BI (0–174.92 µmol/L). Fluorescence measurements were taken at an excitation wavelength of 280 nm, with the emission spectra recorded between 280 and 400 nm via a microplate reader (Thermo Fisher Scientific, China). The fluorescence quenching data were plotted as the relative fluorescence intensity (RFI = F/F0×100) against the BI concentration. A Stern‒Volmer plot was used to calculate the KA value through linear regression.SPR analysisSPR analysis was performed via a Biacore 1 K system (Cytiva, Sweden). A CM5 sensor chip was installed according to standard operating procedures, and PBS (pH 7.4) was used as the running buffer at a flow rate of 150 µL/min. SaeR protein was diluted in sodium acetate and immobilized on the chip surface. After functionalization with NHS, EDC, and ethylenediamine, different concentrations of BI (0.19–100 μmol/L) were injected at a flow rate of 30 µL/min. The association and dissociation times were set at 120 s and 180 s, respectively. The data were analyzed via a one-to-one binding model.Molecular dockingMolecular docking of BI (PubChem CID: 5488822) with SaeR was performed via AutoDock Vina 1.1.2. The three-dimensional structure of SaeR (PDB ID: 4QWQ) was downloaded from the Protein Data Bank (www.rcsb.org), and the 3D structure of BI was drawn via ChemBioDraw Ultra 14.0. The input files for docking were prepared via AutoDockTools 1.5.6, and the ligand structure was prepared by merging nonpolar hydrogen atoms and defining rotatable bonds. The docking grid for SaeR was defined at center_x: −18.047, center_y: −4.024, and center_z: 11.259, with dimensions of size_x: 45.11, size_y: 50.75, and size_z: 50.75. Exhaustiveness was set to 20 to improve accuracy. Default parameters were used unless otherwise stated.Site-directed mutagenesisOn the basis of the binding sites predicted via molecular docking, site-directed mutagenesis of key amino acid residues in SaeR was performed via a site-directed mutagenesis kit, with pET28a-saeR serving as the template. The mutations converted specific amino acids to alanine (Ala) (the primer sequences are listed in Supplementary Table 5). The mutated proteins were expressed and purified following standard induction protocols. Fluorescence quenching experiments were conducted to assess the specific binding of BI to the SaeR protein.

G. mellonella infection modelG. mellonella larvae (220–260 mg) were used to assess the in vivo toxicity and therapeutic potential of BI. The larvae were randomly divided into four groups (n = 5): a control group treated with PBS and two groups treated with BI (20 or 50 mg/kg). The control group received PBS (containing 0.1% DMSO) or BI via a 10 μL Hamilton syringe into the last right proleg of each larva. The survival rate and degree of melanization were monitored and recorded daily for 5 days.To evaluate the therapeutic potential of BI against systemic S. aureus infection, G. mellonella were divided into infection groups (WT), kd-saeR strain infection groups (kd-saeR), and groups treated with BI (20 or 50 mg/kg) or vancomycin (50 mg/kg). Each group contained 10 larvae (n = 10). The infection groups received 5 × 10⁸ CFU/mL S. aureus USA300 or the kd-saeR strain in 10 μL, which was injected into the last right proleg of each larva. One hour post infection, treatment interventions began. The larvae were kept at a constant temperature of 37 °C throughout the experiment. Survival rates were monitored every 12 h over a 120-hour period. For bacterial load assessment, larvae were harvested at 48 h post infection. The larvae were surface sterilized, homogenized in sterile PBS, serially diluted, and plated on BHI agar. The plates were incubated at 37 °C for 24 h, and CFUs were counted. The sensitivity threshold for detection in this assay was 100 CFU/mL of homogenized larva.MRSA-induced rat model of septic arthritisAll animal experiments were conducted in accordance with protocols approved by the Animal Care & Welfare Committee of Changchun University of Chinese Medicine (Approval No: 2024635). Male Sprague–Dawley (SD) rats (6–8 weeks old) (Liaoning Changsheng Biotechnology Co., Ltd., China) were anesthetized via an intraperitoneal injection of 1% pentobarbital. The fur around the knee joints was shaved, and the samples were gently washed with warm water. The skin around the joints was disinfected via povidone-iodine and alcohol swabs.After the joints were disinfected, a 10 μL Hamilton syringe was used to inject 4 × 106 CFU/10 μL of MRSA or kd-saeR strain into the joint cavity beneath the patella. Vancomycin (50 mg/kg) or BI (50 mg/kg) was administered subcutaneously once daily for 6 days. The control animals received the same volume of PBS. The rats were kept on a warming pad and monitored until they could move freely. All the animals were housed in ventilated cages with a 12-hour light‒dark cycle at 22 ± 3 °C and were given free access to food and water.The severity of arthritis was assessed via clinical scoring via macroscopic inspection of the knee joints, assigning a score of 0–4 for each limb (0 = normal, 1 = periarticular erythema, 2 = articular erythema, and edema, 3 = functional impairment with difficulty in locomotion and joint extension, 4 = purulent process with abscess formation). After 7 days of observation, the rats were euthanized via CO2 in a chamber filled at a low rate (30% of the chamber volume per minute). Knee joint diameters were measured, and representative images of the knees were taken.For real-time monitoring of knee joint infection, bioluminescence imaging was performed using the luminescent strain S. aureus Xen29 or SaeR-knockdown of Xen29 (knockdown). A 10 μL Hamilton syringe was used to inject 4 × 106 CFU/10 μL S. aureus Xen29 into the joint cavity beneath the patella. Bioluminescence was measured daily for 7 days to track infection progression.After 7 days, the rats were euthanized, and the knee joints were collected. The joints were fixed in 4% paraformaldehyde, decalcified with 12% EDTA, embedded in paraffin, and sectioned at a thickness of 4 μm. H&E staining was performed to assess joint tissue integrity. Images of the stained sections were captured under a light microscope.To evaluate the bacterial load in the synovial fluid, the knee joints were flushed with 10 μL of PBS via a Hamilton syringe, and the fluid was transferred into sterile microcentrifuge tubes. The mixture was plated on BHI agar containing chloramphenicol (10 μg/mL) and incubated for 24–48 h, followed by colony counting.Micro-CT analysisKnee joint tissues were fixed in 10% buffered formalin for 5 days and then transferred to 70% ethanol for storage at 4 °C. Micro-CT scans of the distal femur and proximal tibia were performed via the NEMO Micro-CT system (NMC-200, Pingseng Scientific, China) with a voltage of 80 kV, a current of 0.06 mA, and a scan time of 200 s. The acquired images were reconstructed into three-dimensional models via Recon software. Coronal and sagittal views were analyzed for the smoothness of the cartilage surface and the degree of subchondral bone sclerosis. The BV/TV and TMD of the femoral and tibial condyles were calculated via Cruiser CT software.Statistical analysisAll the statistical analyses were conducted via GraphPad Prism 8.0 (GraphPad Software). Two-tailed Student’s t tests were used to assess significant differences between two groups, whereas one-way or two-way analysis of variance was used for comparisons among multiple groups. Log-rank tests were performed for survival analyses. Differences were considered statistically significant at P < 0.05. The error bars represent the standard error of the mean. The experiments were repeated in triplicate when necessary.

Data availability

All data generated during this study are available upon request from the corresponding authors.

ReferencesBerendonk, T. U. et al. Tackling antibiotic resistance: the environmental framework. Nat. Rev. Microbiol. 13, 310–317 (2015).CAS 

PubMed 

Google Scholar 

CDC. Antibiotic Resistance Threats in the United States, https://www.cdc.gov/antimicrobial-resistance/data-research/threats/index.html (2019).Shallcross, L. J., Howard, S. J., Fowler, T. & Davies, S. C. Tackling the threat of antimicrobial resistance: from policy to sustainable action. Philos. Trans. R. Soc. B: Biol. Sci. 370, 20140082 (2015).

Google Scholar 

Barber, M. Methicillin-resistant staphylococci. J. Clin. Pathol. 14, 385 (1961).CAS 

PubMed 

PubMed Central 

Google Scholar 

David, M. Z. et al. What is community-associated methicillin-resistant Staphylococcus aureus? J. Infect. Dis. 197, 1235–1243 (2008).PubMed 

Google Scholar 

Nicholson, T. L., Shore, S. M., Smith, T. C. & Fraena, T. S. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of swine origin form robust biofilms. PLoS One 8, e73376 (2013).CAS 

PubMed 

PubMed Central 

Google Scholar 

Melo-Cristino, J., Resina, C., Manuel, V., Lito, L. & Ramirez, M. First case of infection with vancomycin-resistant Staphylococcus aureus in Europe. Lancet 382, 205 (2013).PubMed 

Google Scholar 

Erkilinc, M., Gilmore, A., Weber, M. & Mistovich, R. J. Current concepts in pediatric septic arthritis. JAAOS J. Am. Acad. Orthop. Surg. 29, 196–206 (2021).PubMed 

Google Scholar 

Bragginton, E. C. & Piddock, L. J. UK and European Union public and charitable funding from 2008 to 2013 for bacteriology and antibiotic research in the UK: an observational study. Lancet Infect. Dis. 14, 857–868 (2014).PubMed 

Google Scholar 

Vuong, C., Yeh, A. J., Cheung, G. Y. & Otto, M. Investigational drugs to treat methicillin-resistant Staphylococcus aureus. Expert Opin. Investig. drugs 25, 73–93 (2016).CAS 

PubMed 

Google Scholar 

Smeltzer, M. S. Staphylococcus aureus pathogenesis: the importance of reduced cytotoxicity. Trends Microbiol. 24, 681–682 (2016).CAS 

PubMed 

Google Scholar 

Otto, M. Staphylococcus aureus toxins. Curr. Opin. Microbiol. 17, 32–37 (2014).CAS 

PubMed 

Google Scholar 

Wang, J. & Wang, L. Novel therapeutic interventions towards improved management of septic arthritis. BMC Musculoskelet. Disord. 22, 530 (2021).PubMed 

PubMed Central 

Google Scholar 

Thammavongsa, V., Kim, H. K., Missiakas, D. & Schneewind, O. Staphylococcal manipulation of host immune responses. Nat. Rev. Microbiol. 13, 529–543 (2015).CAS 

PubMed 

PubMed Central 

Google Scholar 

Flemming, H.-C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).CAS 

PubMed 

Google Scholar 

Akbarian, M. et al. A review on biofilms and the currently available antibiofilm approaches: matrix-destabilizing hydrolases and anti-bacterial peptides as promising candidates for the food industries. Int. J. Biol. Macromol.219, 1163–1179 (2022).CAS 

PubMed 

Google Scholar 

Mashruwala, A. A., Gries, C. M., Scherr, T. D., Kielian, T. & Boyd, J. M. SaeRS is responsive to cellular respiratory status and regulates fermentative biofilm formation in Staphylococcus aureus. Infect. Immun. 85, 00157–00117 (2017).

Google Scholar 

Novick, R. P. & Jiang, D. The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing. Microbiology 149, 2709–2717 (2003).CAS 

PubMed 

Google Scholar 

Schurig-Briccio, L. A. et al. Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence. EMBO Rep. 21, e45832 (2020).CAS 

PubMed 

PubMed Central 

Google Scholar 

Giraudo, A. T., Calzolari, A., Cataldi, A. A., Bogni, C. & Nagel, R. The sae locus of Staphylococcus aureus encodes a two-component regulatory system. FEMS Microbiol. Lett. 177, 15–22 (1999).CAS 

PubMed 

Google Scholar 

Liu, Q., Yeo, W.-S. & Bae, T. The SaeRS two-component system of Staphylococcus aureus. Genes 7, 81 (2016).PubMed 

PubMed Central 

Google Scholar 

Kuroda, H., Kuroda, M., Cui, L. & Hiramatsu, K. Subinhibitory concentrations of β-lactam induce haemolytic activity in Staphylococcus aureus through the SaeRS two-component system. FEMS Microbiol. Lett. 268, 98–105 (2007).CAS 

PubMed 

Google Scholar 

Liang, X. et al. Inactivation of a two-component signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus. Infect. Immun. 74, 4655–4665 (2006).CAS 

PubMed 

PubMed Central 

Google Scholar 

Ahmad, I., Husain, F. M., Maheshwari, M. & Zahin, M. Medicinal plants and phytocompounds: a potential source of novel antibiofilm agents. Antibiofilm Agents 8, 205–232 (2014).

Google Scholar 

Duan, J. et al. Subinhibitory concentrations of resveratrol reduce alpha-hemolysin production in Staphylococcus aureus isolates by downregulating saeRS. Emerg. microbes Infect. 7, 1–10 (2018).

Google Scholar 

Coenye, T. et al. Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds. Phytomedicine 19, 409–412 (2012).CAS 

PubMed 

Google Scholar 

He, X. et al. Global downregulation of penicillin resistance and biofilm formation by MRSA is associated with the interaction between kaempferol rhamnosides and quercetin. Microbiol. Spectr. 10, e02782–02722 (2022).PubMed 

PubMed Central 

Google Scholar 

Qian, H.-q et al. A systematic review of traditional uses, phytochemistry, pharmacology and toxicity of Epimedium koreanum Nakai. J. Ethnopharmacol. 318, 116957 (2024).CAS 

PubMed 

Google Scholar 

Zhang, L.-B. et al. Epimedii Herba: An ancient Chinese herbal medicine in the prevention and treatment of rheumatoid arthritis. Front. Chem. 10, 1023779 (2022).CAS 

PubMed 

PubMed Central 

Google Scholar 

Sun, X. et al. Biofilm formation in erythromycin-resistant Staphylococcus aureus and the relationship with antimicrobial susceptibility and molecular characteristics. Microb. Pathog. 124, 47–53 (2018).CAS 

PubMed 

Google Scholar 

Zhao, Y. et al. Effect of drying processes on prenylflavonoid content and antioxidant activity of Epimedium koreanum Nakai. J. Food Drug Anal. 26, 796–806 (2018).CAS 

PubMed 

Google Scholar 

Krishna, S. N. et al. A fluorescence-based thermal shift assay identifies inhibitors of mitogen activated protein kinase kinase 4. PloS One 8, e81504 (2013).PubMed 

PubMed Central 

Google Scholar 

Thänert, R., Goldmann, O., Beineke, A. & Medina, E. Host-inherent variability influences the transcriptional response of Staphylococcus aureus during in vivo infection. Nat. Commun. 8, 14268 (2017).PubMed 

PubMed Central 

Google Scholar 

Payne, J. A. et al. Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus. Nat. Commun. 12, 6157 (2021).CAS 

PubMed 

PubMed Central 

Google Scholar 

Butler, M. T., Wang, Q. & Harshey, R. M. Cell density and mobility protect swarming bacteria against antibiotics. Proc. Natl. Acad. Sci. 107, 3776–3781 (2010).CAS 

PubMed 

PubMed Central 

Google Scholar 

Gordon, C. P., Williams, P. & Chan, W. C. Attenuating Staphylococcus aureus virulence gene regulation: a medicinal chemistry perspective. J. Med. Chem. 56, 1389–1404 (2013).CAS 

PubMed 

PubMed Central 

Google Scholar 

Crossley, K. B., Jefferson, K. K., Archer, G. L. & Fowler, V. G. Jr Staphylococci in human disease. http://onlinelibrary.wiley.com/doi/book/10.1002/9781444308464 (2009).Löffler, B. et al. Staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils. PLoS Pathog. 6, e1000715 (2010).PubMed 

PubMed Central 

Google Scholar 

Sun, F. et al. In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS. J. Bacteriol. 192, 2111–2127 (2010).CAS 

PubMed 

PubMed Central 

Google Scholar 

Jiang, F. et al. Repurposed Fenoprofen targeting SaeR attenuates Staphylococcus aureus virulence in implant-associated infections. ACS Cent. Sci. 9, 1354–1373 (2023).CAS 

PubMed 

PubMed Central 

Google Scholar 

Mainiero, M. et al. Differential target gene activation by the Staphylococcus aureus two-component system saeRS. J. Bacteriol. 192, 613–623 (2010).CAS 

PubMed 

Google Scholar 

Hunter, J. G., Gross, J. M., Dahl, J. D., Amsdell, S. L. & Gorczyca, J. T. Risk factors for failure of a single surgical debridement in adults with acute septic arthritis. JBJS 97, 558–564 (2015).

Google Scholar 

Moldovan, A. & Fraunholz, M. J. In or out: phagosomal escape of Staphylococcus aureus. Cell. Microbiol. 21, e12997 (2019).PubMed 

Google Scholar 

Yu, K. et al. Recalcitrant methicillin-resistant Staphylococcus aureus infection of bone cells: intracellular penetration and control strategies. Bone Jt. Res. 9, 49–59 (2020).

Google Scholar 

Masters, E. A. et al. Evolving concepts in bone infection: redefining “biofilm”,“acute vs. chronic osteomyelitis”,“the immune proteome” and “local antibiotic therapy. Bone Res. 7, 20 (2019).PubMed 

PubMed Central 

Google Scholar 

Yang, D. et al. Novel insights into Staphylococcus aureus deep bone infections: the involvement of osteocytes. MBio 9, 00415–00418 (2018).

Google Scholar 

Birmingham, P., Helm, J. M., Manner, P. A. & Tuan, R. S. Simulated joint infection assessment by rapid detection of live bacteria with real-time reverse transcription polymerase chain reaction. JBJS 90, 602–608 (2008).

Google Scholar 

Moormeier, D. E. & Bayles, K. W. Staphylococcus aureus biofilm: a complex developmental organism. Mol. Microbiol. 104, 365–376 (2017).CAS 

PubMed 

PubMed Central 

Google Scholar 

Lister, J. L. & Horswill, A. R. Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Front. Cell. Infect. Microbiol. 4, 178 (2014).PubMed 

PubMed Central 

Google Scholar 

Balcázar, J. L., Subirats, J. & Borrego, C. M. The role of biofilms as environmental reservoirs of antibiotic resistance. Front. Microbiol. 6, 1216 (2015).PubMed 

PubMed Central 

Google Scholar 

Ricciardi, B. F. et al. Staphylococcus aureus evasion of host immunity in the setting of prosthetic joint infection: biofilm and beyond. Curr. Rev. Musculoskelet. Med. 11, 389–400 (2018).PubMed 

PubMed Central 

Google Scholar 

Anderson, M. J. et al. Alpha-toxin contributes to biofilm formation among Staphylococcus aureus wound isolates. Toxins 10, 157 (2018).PubMed 

PubMed Central 

Google Scholar 

Wang, T., Zhang, P., Lv, H., Deng, X. & Wang, J. A natural dietary flavone myricetin as an α-hemolysin inhibitor for controlling Staphylococcus aureus infection. Front. Cell. Infect. Microbiol. 10, 330 (2020).CAS 

PubMed 

PubMed Central 

Google Scholar 

Zheng, X. et al. Antibacterial and anti-biofilm efficacy of chinese dragon’s blood against staphylococcus aureus isolated from infected wounds. Front. Microbiol. 12, 672943 (2021).PubMed 

PubMed Central 

Google Scholar 

Malachowa, N. & DeLeo, F. R. Staphylococcus aureus survival in human blood. Virulence 2, 567–569 (2011).PubMed 

PubMed Central 

Google Scholar 

Pietrocola, G., Nobile, G., Rindi, S. & Speziale, P. Staphylococcus aureus manipulates innate immunity through own and host-expressed proteases. Front. Cell. Infect. Microbiol. 7, 166 (2017).PubMed 

PubMed Central 

Google Scholar 

van Wamel, W. J., Rooijakkers, S. H., Ruyken, M., van Kessel, K. P. & van Strijp, J. A. The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on β-hemolysin-converting bacteriophages. J. Bacteriol. 188, 1310–1315 (2006).PubMed 

PubMed Central 

Google Scholar 

Chen, W., Zhang, Y., Yeo, W.-S., Bae, T. & Ji, Q. Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system. J. Am. Chem. Soc. 139, 3790–3795 (2017).CAS 

PubMed 

Google Scholar 

Kadurugamuwa, J. L. et al. Direct continuous method for monitoring biofilm infection in a mouse model. Infect. Immun. 71, 882–890 (2003).CAS 

PubMed 

PubMed Central 

Google Scholar 

Sulimov, A. V., Kutov, D. C., Katkova, E. V., Ilin, I. S. & Sulimov, V. B. New generation of docking programs: Supercomputer validation of force fields and quantum-chemical methods for docking. J. Mol. Graph. Model. 78, 139–147 (2017).CAS 

PubMed 

Google Scholar 

Bhardwaj, P., Biswas, G. P. & Bhunia, B. Docking-based inverse virtual screening strategy for identification of novel protein targets for triclosan. Chemosphere 235, 976–984 (2019).CAS 

PubMed 

Google Scholar 

Delgado-Valverde, M. et al. MIC of amoxicillin/clavulanate according to CLSI and EUCAST: discrepancies and clinical impact in patients with bloodstream infections due to Enterobacteriaceae. J. Antimicrob. Chemother. 72, 1478–1487 (2017).CAS 

PubMed 

Google Scholar 

Opperman, T. J. et al. Aryl rhodanines specifically inhibit staphylococcal and enterococcal biofilm formation. Antimicrob. Agents Chemother. 53, 4357–4367 (2009).CAS 

PubMed 

PubMed Central 

Google Scholar 

Gao, P. et al. Suppression of Staphylococcus aureus virulence by a small-molecule compound. Proc. Natl. Acad. Sci. USA 115, 8003–8008 (2018).CAS 

PubMed 

PubMed Central 

Google Scholar 

Download referencesAcknowledgementsThis work was supported by the Jilin Provincial Scientific and Technological Development Program (Grant No. 20240207010CX to Yicheng Zhao), the National Natural Science Foundation of China (Grant No. U22A20340 to Yicheng Zhao), the Jilin Provincial Science and Technology Development Plan (Grant No. YDZJ202401113ZYTS to Li Wang), the Jilin Provincial Traditional Chinese Medicine Science and Technology Program (Grant No. 2024069 to Li Wang).Author informationAuthor notesThese authors contributed equally: Yueshan Xu, Li Wang.Authors and AffiliationsIntegrated Chinese and Western Medicine, Changchun University of Chinese Medicine, Changchun, ChinaYueshan Xu, Li Wang, Dongbin Guo, Yueying Wang, Xinyao Liu, Yun Sun, Rong Wang, Bingmei Wang & Ming YanDepartment of Orthopedics, The Third Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, ChinaYueshan Xu, Yueying Wang & Ming YanClinical Medical College, Changchun University of Chinese Medicine, Changchun, ChinaLi Wang & Bingmei WangChina-Japan Union Hospital of Jilin University, Changchun, ChinaLuanbiao SunCollege of Pharmacy, Changchun University of Chinese Medicine, Changchun, ChinaPeitong JiangState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Jilin University, Changchun, ChinaQuan Liu & Yicheng ZhaoChinese Medicine Guangdong Laboratory, Guangdong, Hengqin, ChinaYicheng ZhaoAuthorsYueshan XuView author publicationsYou can also search for this author inPubMed Google ScholarLi WangView author publicationsYou can also search for this author inPubMed Google ScholarDongbin GuoView author publicationsYou can also search for this author inPubMed Google ScholarYueying WangView author publicationsYou can also search for this author inPubMed Google ScholarXinyao LiuView author publicationsYou can also search for this author inPubMed Google ScholarYun SunView author publicationsYou can also search for this author inPubMed Google ScholarRong WangView author publicationsYou can also search for this author inPubMed Google ScholarLuanbiao SunView author publicationsYou can also search for this author inPubMed Google ScholarPeitong JiangView author publicationsYou can also search for this author inPubMed Google ScholarQuan LiuView author publicationsYou can also search for this author inPubMed Google ScholarBingmei WangView author publicationsYou can also search for this author inPubMed Google ScholarMing YanView author publicationsYou can also search for this author inPubMed Google ScholarYicheng ZhaoView author publicationsYou can also search for this author inPubMed Google ScholarContributionsB.W., M.Y., and Y.Z. designed the study. Y.X. and X.L. participated in the expression and purification of the SaeR protein. Y.W., Y.S., and L.W. participated in the SaeR inhibitor screening and SaeR-related assay. D.G., R.W., and L.S. participated in the in vitro assays. Y.X., Y.W., and L.W. participated in the animal experiments and molecular docking. Q.L. and P.J. conducted the analysis. Y.X. and L.W. drafted the manuscript. All the authors have read and approved the final manuscript.Corresponding authorsCorrespondence to

Bingmei Wang, Ming Yan or Yicheng Zhao.Ethics declarations

Competing interests

The authors declare no competing interests.

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationSupplementary informationRights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissionsAbout this articleCite this articleXu, Y., Wang, L., Guo, D. et al. Baohuoside I targets SaeR as an antivirulence strategy to disrupt MRSA biofilm formation and pathogenicity.

npj Biofilms Microbiomes 11, 45 (2025). https://doi.org/10.1038/s41522-025-00681-2Download citationReceived: 29 September 2024Accepted: 08 March 2025Published: 19 March 2025DOI: https://doi.org/10.1038/s41522-025-00681-2Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard

Provided by the Springer Nature SharedIt content-sharing initiative

Read full news in source page