Daphnoretin as a Potent Sortase A Inhibitor Effectively Disarms Drug-Resistant Staphylococcus aureus Infections

Daphnoretin as a Potent Sortase A Inhibitor Effectively Disarms Drug-Resistant Staphylococcus aureus Infections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Daphnoretin as a Potent Sortase A Inhibitor Effectively Disarms Drug-Resistant Staphylococcus aureus Infections Yun Sun, Tong Zhao, Dongbin Guo, Peitong Jiang, Xinyao Liu, Zhangyu Du, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7820020/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract The rapid spread of drug-resistant Staphylococcus aureus ( S. aureus ) has severely undermined the efficacy of existing antibiotics, highlighting the urgent need to explore novel antiinfective strategies to address this global public health challenge. Sortase A (SrtA) is a critical virulence factor of S. aureus that covalently anchors multiple surface proteins to the bacterial cell wall, thereby mediating adhesion, invasion, immune evasion, and biofilm formation, playing a pivotal role in pathogenesis. In this study, potential inhibitors were screened using a fluorescence resonance energy transfer (FRET) assay, and daphnoretin was identified as a potent SrtA inhibitor with a half-maximal inhibitory concentration (IC₅₀) of 8.501 µg/mL. In vitro experiments demonstrated that daphnoretin effectively suppressed SrtA-mediated pathogenic phenotypes, including bacterial adhesion, invasion, and biofilm formation, thereby attenuating virulence. Further fluorescence quenching and molecular docking analyses revealed the binding mechanism between daphnoretin and SrtA. In vivo, daphnoretin significantly improved host survival in Galleria. mellonella infection model induced by the MRSA strain and alleviated joint damage and infection symptoms in a rat model of septic arthritis while markedly reducing the bacterial burden in infected tissues. Collectively, this study is the first to elucidate the mechanism of daphnoretin as a SrtA-targeting inhibitor and its anti-infective potential, providing important experimental evidence for the development of novel therapeutic strategies against drug-resistant S. aureus . S. aureus SrtA Daphnoretin Ativirulence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Septic arthritis (SA), also known as bacterial arthritis or pyogenic arthritis, is a disease caused by pyogenic bacteria that leads to rapid destruction of joint tissues, resulting in joint dysfunction and even disability. Among the causative pathogens, S. aureus is the most common gram-positive opportunistic pathogen associated with SA(He et al., 2023 ), accounting for 64% of cases, with methicillin-resistant S. aureus (MRSA) reaching as high as 23%(Gobao et al., 2021 ). In recent years, with the increasing number of immunocompromised patients, joint replacement surgeries, and invasive procedures, the incidence of S. aureus -associated SA has been increasing annually(Wu et al., 2024 , He et al., 2023 ). Epidemiological investigations have shown that community-acquired methicillin-resistant S. aureus (CA-MRSA) not only causes skin and soft tissue infections but also has become an important etiological agent of SA, with a particularly marked increase among adolescents and athletes(Kaplan et al., 2005 , Braun and Kahanov, 2018 ). Despite advances in antibiotics and surgical debridement, S. aureus -induced septic arthritis still has high recurrence and disability rates, indicating significant limitations of conventional therapeutic strategies(Wang and Wang, 2021 , Joo et al., 2023 , Kim et al., 2023 ). The current treatment for S. aureus -associated septic arthritis (SA) mainly relies on a combination of antibiotics and surgical intervention(Davis and Zamora, 2020 ). In clinical practice, glycopeptides (such as vancomycin), linezolid, or daptomycin are commonly used and, when necessary, are combined with β-lactams or aminoglycosides(Xu et al., 2022 ). However, S. aureus continually undermines the efficacy of antibiotics through multiple mechanisms, including the acquisition of resistance genes (such as mecA and vanA ) (Nepal et al., 2023 ), biofilm formation, and the presence of persister cells (Theis et al., 2023 ). In particular, in prosthetic joint infections, biofilms formed by bacteria can significantly resist antibiotic penetration and host immune responses, greatly increasing the difficulty of treatment (Rottier et al., 2023). Moreover, prolonged and high-dose use of antibiotics is often accompanied by toxic side effects and further dissemination of resistant strains (Mithuna et al., 2024 , Lau et al., 2018 ). Therefore, reliance solely on conventional antibiotics and surgical drainage is no longer sufficient to meet clinical needs. Antivirulence therapy has recently been regarded as an important new approach against S. aureus infections(Jonsson et al., 2003 ). The core concept is to attenuate bacterial pathogenicity by inhibiting key virulence mechanisms, such as toxins, adhesion factors, or regulatory systems, rather than directly killing bacteria. This strategy is expected to reduce selective pressure for resistance and act synergistically with antibiotics(Touati et al., 2025 , Ford et al., 2020 ). For example, the inhibition of α-hemolysin, surface adhesion proteins, and the regulatory factor SaeRS has demonstrated potential in improving host outcomes(Frank et al., 2012 , Gao et al., 2023 ). Both animal models and preclinical studies have shown that antivirulence agents can effectively reduce tissue damage, alleviate inflammatory responses, and enhance host immune clearance(Sully et al., 2014 , Le et al., 2016 , Tkaczyk et al., 2018 , Yang et al., 2016 ). Therefore, the development of potent antivirulence molecules represents a cutting-edge strategy for combating S. aureus infections. SrtA is an important transmembrane enzyme of S. aureus that is responsible for covalently anchoring surface proteins containing the LPXTG motif to the peptidoglycan layer(Ton-That et al., 2000 ). These surface proteins play central roles in host cell adhesion, tissue invasion, and immune evasion. Studies have shown that the deletion or inhibition of SrtA activity can significantly reduce the virulence and pathogenicity of S. aureus without affecting its basic growth(Zhang et al., 2014 ). Therefore, SrtA is widely considered an ideal target for antivirulence therapy, and multiple small-molecule inhibitors have been developed and validated. Targeting SrtA holds promise for blocking key steps of S. aureus infection, thereby reducing the risk of recurrence and chronic infection. Natural products have increasingly emerged in recent years as promising antivirulence candidates against S. aureus infections because of their multitarget activities and relatively high safety profiles. Unlike traditional antibiotics that inhibit bacterial growth or directly kill bacteria, natural products often act by interfering with key bacterial virulence factors. Daphnoretin, a naturally occurring dicoumarin compound, is distributed mainly in plants of the Thymelaeaceae family, such as Daphne genkwa and Wikstroemia indica. Previous studies have demonstrated that daphnoretin has significant activities in antitumor, antiviral, anti-inflammatory, and immunomodulatory contexts, with mechanisms involving the induction of apoptosis, the regulation of multiple signaling pathways, and the modulation of the cell cycle(Gu and He, 2012 , Wang et al., 2021 ). With the growing attention given to natural products in the fields of antivirulence therapy and multitarget drug development, daphnoretin has gradually gained recognition as a potential lead compound, providing important insights for the development of novel antiinfective and immunomodulatory strategies. Materials and methods Strains, culture conditions, and reagents All strains were cultured at 37°C with shaking at 220 rpm in Luria–Bertani (LB) medium or tryptic soy broth (TSB). The strains used in this study included methicillin-resistant Staphylococcus aureus ATCC BAA-1717ᵀ (USA300-HOU-MR). SrtA deletion mutant (Δ srtA ), and Escherichia coli BL21 (DE3) pET28a- srtA ΔN59, all of which were obtained from and preserved in our laboratory. Daphnoretin (purity > 98%; quality control report in Supplementary Fig. 1) was purchased from Desite Biotechnology Co., Ltd. (Chengdu, China). The fluorescent substrate peptide Abz-LPATG-Dap (Dnp)-NH₂ was synthesized and provided by LifeTein (Beijing, China). Vancomycin and kanamycin were purchased from MedChemExpress (Shanghai, China). Purification of SrtA and its mutants The recombinant plasmid pET28a-srtA ΔN59 was transformed into Escherichia coli BL21 (DE3) and cultured in LB media supplemented with kanamycin (50 µg/mL) at 37°C with shaking at 220 rpm until the optical density at 600 nm (OD₆₀₀) reached approximately 0.8. Protein expression was then induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by incubation at 16°C with shaking at 180 rpm. After induction, the bacterial cells were harvested via centrifugation and resuspended in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl₂, pH 7.4; Beyotime, China). The suspension was disrupted by ultrasonication, and the lysate was centrifuged at 12,000 rpm for 1 h at 4°C to collect the supernatant. The soluble proteins were purified via a Ni-NTA affinity chromatography system (Beyotime, China), in which the nonspecific proteins were washed away with low-concentration imidazole, and the target SrtA protein was eluted with high-concentration imidazole. Site-directed mutagenesis of SrtA (N114A, V168A, T180A, and K175A) was performed via a site-directed mutagenesis kit (Tiangen, China), with the primer sequences listed in Supplementary Table 2. The mutant plasmids were subsequently transformed into E. coli BL21 (DE3), and the expression and purification of the mutant proteins were carried out following the same protocol as that used for wild-type SrtA. SrtA inhibitor screening and the IC₅₀ assay To screen potential SrtA inhibitors, a fluorescence resonance energy transfer (FRET) assay was employed to evaluate the inhibitory effects of small-molecule compounds on the enzymatic activity of SrtA. Purified SrtA protein (final concentration of 5 µM) was dissolved in 100 µL of protein buffer and incubated with candidate small-molecule compounds (64 µg/mL) at 37°C for 1 h under light-protected conditions. The fluorescent substrate Abz-LPATG-Dap (Dnp)-NH₂ (10 µM) was subsequently added to the mixture, which was further incubated for 20 min under the same conditions. A parallel control group containing only buffer and fluorescent substrate was included to eliminate nonspecific signal interference. After the reaction, the fluorescence intensity was measured via a microplate reader (Thermo Fisher Scientific, USA) at an excitation wavelength of 309 nm and an emission wavelength of 420 nm. Compounds exhibiting ≥ 70% inhibition of SrtA activity were selected as candidate inhibitors. These compounds were then tested at a series of concentrations (0.125–128 µg/mL) via the same protocol, and concentration‒response curves were generated with GraphPad Prism 10 software to calculate the half-maximal inhibitory concentration (IC₅₀), thereby assessing their inhibitory potency. MIC determination and growth curve analysis of daphnoretin According to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) (Delgado-Valverde et al., 2017, Oh et al., 2021). The minimum inhibitory concentration (MIC) of daphnoretin against S. aureus was determined using the broth microdilution method. Briefly, resuscitated bacterial cultures were adjusted to 1 × 10⁵ CFU and distributed into 96-well plates containing serial concentrations of daphnoretin (1–256 µg/mL). To each well, 2.5 µL of resazurin (5 mg/mL) was added as a viability indicator. Positive controls (bacterial suspension only) and negative controls (medium only) were included. After incubation at 37°C for 16 h, bacterial growth was assessed on the basis of the color change of resazurin, and the MIC values were determined. In parallel, to evaluate the effects of daphnoretin on bacterial growth kinetics, cultures were incubated with daphnoretin at different concentrations (32 and 64 µg/mL), and the optical density at 600 nm (OD₆₀₀) was measured at defined time intervals to generate growth curves. MTT assay for cell viability The cytotoxicity of daphnoretin on ATDC5 cells was evaluated using the MTT assay. The ATDC5 cell line, derived from mouse teratocarcinoma, is commonly used for studies on chondrogenic differentiation and function. The cells were seeded into 96-well plates at a density of 2 × 10⁴ cells/well and cultured at 37°C with 5% CO₂ for 24 h. Subsequently, different concentrations of daphnoretin (0–64 µg/mL) were added, and the mixture was incubated for another 24 h. The medium was then removed, and MTT solution (5 mg/mL; Beyotime, China) was added to each well, followed by incubation for 4 h. The resulting formazan crystals were dissolved in 100 µL of DMSO, and the absorbance was measured at 490 nm to evaluate cell viability. Rabbit erythrocyte hemolysis assay To evaluate the hemolytic safety of the compound, fresh rabbit blood (containing anticoagulant) was collected and centrifuged at 3000 rpm to remove the plasma. The erythrocytes were washed three times with sterile PBS (pH 7.4), the supernatant was discarded, and the cells were finally resuspended in PBS to obtain a 2.5% red blood cell suspension. For the assay, 100 µL of the RBC suspension was mixed with an equal volume of solutions containing different concentrations of daphnoretin (0–64 µg/mL) and incubated at 37°C for 1 h. The negative control consisted of RBCs in PBS without drug treatment, whereas the positive control consisted of RBCs treated with 1% Triton X-100. After incubation, all the samples were centrifuged at 3000 rpm for 10 min, and the supernatants were collected. The absorbance of the supernatants was measured at 543 nm via a microplate reader. Effect of daphnoretin on S. aureus adhesion The 96–well plates were coated with bovine fibrinogen (20 µg/mL; Source Leaf, China) and incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS), the wells were blocked with 0.5% bovine serum albumin (BSA; Sigma, USA) for 2 h. S. aureus was cultured with different concentrations of daphnoretin (32–64 µg/mL) until reaching the logarithmic growth phase and then inoculated into the coated plates, followed by incubation at 37°C for 2 h. After incubation, the wells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min. Excess dye was removed by washing, and absorbance was measured at 570 nm using a microplate reader. FITC-IgG–based detection of S. aureus surface protein A S. aureus was cultured with shaking until the OD₆₀₀ reached 0.3, after which different concentrations of daphnoretin (32–64 µg/mL) were added, and incubation was continued until the OD₆₀₀ reached 1.0. The bacterial cells were collected by centrifugation (5,000 rpm, 5 min), washed twice with PBS, and blocked with 0.5% bovine serum albumin (BSA) for 20 min. The cells were then fixed with 4% paraformaldehyde for 30 min and incubated in the dark at 37°C for 2 h with a FITC-labeled goat anti-rabbit IgG antibody (Source Leaf, China) diluted 1:200. After washing with PBS, the bacteria were resuspended in PBS. One hundred microliters of resuspended bacteria was added to a black 96-well plate. The fluorescence intensity was then measured using a microplate reader (Thermo Fisher, USA) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The Δ srtA strain was used as a negative control. Daphnoretin protects ATDC5 cells After being resuscitated, ATDC5 cells were seeded at a density of 2 × 10⁴ cells per well in 24-well plates containing DMEM supplemented with 10% FBS and cultured overnight. The culture medium was then discarded, and the cells were washed with PBS. S. aureus was subsequently suspended in fresh RPMI 1640 medium containing 10% serum, and the cells were infected with 500 µL of the bacterial suspension at a multiplicity of infection (MOI) of 50.(Chang et al., 2024) At the same time, different concentrations of daphnoretin (32 and 64 µg/mL) were added and incubated for 6 h. Cell viability was assessed using a calcein-AM/PI assay kit, and images were acquired with a cell imaging workstation to distinguish live and dead cells. Untreated cells served as the blank control, and the ratio of live to dead cells was quantitatively analyzed using ImageJ software. Assessment of S. aureus invasion into ATDC5 cells S. aureus was cultured in the presence of daphnoretin (32–64 µg/mL) at 37°C with shaking at 220 rpm until the OD₆₀₀ reached 1.0. The bacteria were then fluorescently labeled with 5(6)-FAM dye, collected by centrifugation, and resuspended in an equal volume of high-glucose DMEM. ATDC5 cells were routinely maintained in high-glucose DMEM (Servicebio, China) and seeded into 24-well plates at a density of 1 × 10⁵ cells/well. After 36 h of incubation, the cells were infected with S. aureus at a multiplicity of infection (MOI) of 5 and incubated for 1 h(Xu et al., 2025). After treatment, extracellular viable bacteria were removed by applying gentamicin (50 µg/mL, prepared in PBS), followed by two washes with PBS. The cytoskeleton and nuclei were then stained sequentially with Acti-stain 555 phalloidin and DAPI, respectively, and fluorescence images were acquired using a cell imaging workstation. Biofilm formation assay Overnight-cultured S. aureus was diluted 1:100 in BHI medium and inoculated into 96-well plates precoated with 20% freeze-dried rabbit plasma at 4°C overnight (100 µL per well). After treatment with different concentrations of daphnoretin (0–128 µg/mL) and incubation at 37°C for 24 h, the wells were washed with sterile PBS and stained with 50 µL of crystal violet solution for 15 min. Excess dye was discarded, and the wells were thoroughly rinsed, after which images of the biofilms were captured, and the minimum biofilm inhibitory concentration (MBIC) was determined on the basis of visual inspection. The bound dye was then eluted with 33% glacial acetic acid, and 100 µL of the eluate was transferred to a new 96-well plate. The absorbance was measured at 570 nm to quantify the biofilm biomass. To further evaluate the effects of daphnoretin on different stages of biofilm development, 96-well plates precoated with rabbit plasma were inoculated with S. aureus , and daphnoretin was added at different time points (0–24 h) to a final concentration of 64 µg/mL. After 24 h of coincubation, the biofilms were stained with crystal violet, and the absorbance was measured at OD 570 nm. Biofilm extracellular matrix component analysis To systematically evaluate the impact of daphnoretin on the extracellular matrix composition of S. aureus biofilms, three major components—extracellular polysaccharides, proteins, and extracellular DNA (eDNA)—were analyzed. Briefly, biofilms were cultured in BHI medium supplemented with various concentrations of daphnoretin for 24 h. Extracellular polysaccharides were quantified via the phenol–sulfuric acid method, and the absorbance was measured at 490 nm via a microplate reader. For protein analysis, biofilms treated under the same conditions were harvested, washed with PBS, and subjected to isotonic buffer extraction followed by cold acetone precipitation; protein concentrations were subsequently determined via a BCA assay, with the absorbance measured at 595 nm. For eDNA quantification, biofilms were cultured as described above, digested with proteinase K, precipitated with ethanol, and analyzed via a Nanodrop spectrophotometer, with validation performed via agarose gel electrophoresis. Collectively, these assays provide a comprehensive assessment of the modulatory effects of daphnoretin on the biofilm extracellular matrix. Confocal laser scanning microscopy (CLSM) analysis of biofilms In accordance with previous studies with modifications (Walker and Horswill, 2012), overnight-resuscitated S. aureus was inoculated into BHI media at a 1:100 dilution. Glass coverslips were placed into six-well plates and precoated with freeze-dried rabbit plasma at 4°C overnight. A 500 µL bacterial suspension containing different concentrations of daphnoretin was then added to each well and incubated at 37°C for 24 h to allow mature biofilm formation. After incubation, the supernatant was removed, and the biofilms on the coverslips were washed with PBS (pH 7.4). The samples were stained with SYTO-9 (Foryou Biotech, China) for 30 min at room temperature, and three-dimensional fluorescence images were subsequently captured via a confocal laser scanning microscope (CLSM; Leica TCS DMI8, Germany). Scanning electron microscopy (SEM) analysis of biofilms The pretreatment of the coverslips for SEM observation was the same as that for CLSM. After coincubation of the coverslips with S. aureus and daphnoretin, the samples were gently washed with sterile PBS to remove nonadherent cells. The samples were then fixed in 4% glutaraldehyde solution at 4°C overnight. After fixation, the samples were washed three times with PBS and dehydrated sequentially with graded ethanol solutions (35%, 50%, and 70%). Following dehydration, the samples were dried via a liquid CO₂ critical point dryer. Finally, the samples were sputter-coated with platinum and imaged via a scanning electron microscope (HITACHI Regulus 8100, Japan) to observe the biofilm structures(Kong et al., 2018). Bacterial sliding motility assay To evaluate the sliding motility of S. aureus , bacteria were inoculated onto plates containing tryptic soy broth (TSB) supplemented with 1% agar to assess their spreading ability. The experimental procedure was performed according to previously reported methods with minor modifications(Shen et al., 2020). Briefly, a standardized suspension of S. aureus (1 × 10⁸ CFU/mL, 15 µL) was spot inoculated onto the center of the plates, with treatment groups containing daphnoretin (16–128 µg/mL) or without drug as controls. After inoculation, the plates were air-dried at room temperature for 20 min and then incubated at 37°C for 24 h. The spreading zones were observed to evaluate sliding motility. Western blot analysis After resuspension, S. aureus was cultured to an OD₆₀₀ of 0.3, and different concentrations of daphnoretin were added. The cultures were then incubated at 37°C with shaking until they reached an OD₆₀₀ of 2.5 (late logarithmic phase). The bacterial cells were harvested by centrifugation and lysed with lysozyme and lysostaphin by incubation on ice for 30 min. The total protein concentration was determined 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 at 80 rpm/min for 2 h, followed by overnight incubation at 4°C with polyclonal anti-SrtA serum (1:200, prepared in our laboratory). After being washed with TBS-T, the membranes were incubated with an HRP-conjugated goat anti-rabbit secondary antibody (1:10,000; Beyotime, China) for 2 h at room temperature. The protein bands were visualized via an enhanced chemiluminescence (ECL) Plus substrate (Beyotime, China). The Δ srtA strain was used as a negative control. Molecular docking Molecular docking of daphnoretin with SrtA was performed via AutoDock Vina 1.1.2. The three-dimensional structure of SrtA (PDB ID: 1T2P) was obtained from the Protein Data Bank (www.rcsb.org). The 3D structure of daphnoretin was retrieved from the PubChem database (PubChem CID: 5281406) and further generated via ChemBioDraw Ultra 14.0. The preparation of the ligand and receptor files was carried out via AutoDockTools 1.5.6. During docking, nonpolar hydrogen atoms were merged, and rotatable bonds of the ligand were defined. The docking grid was centered on the catalytic cysteine with coordinates center_x = − 30.329, center_y = − 19.713, center_z = 1.688, dimensions size_x = 43.5, size_y = 40.5, and size_z = 46.5. All other parameters were set to their defaults. The best docking conformation obtained was used to generate the ligand–protein complex to predict the binding mode of daphnoretin with SrtA. Fluorescence quenching Fluorescence quenching experiments were conducted to determine the binding constant ( K A ) of daphnoretin with SrtA and its mutant proteins. Purified SrtA (4 µM) was mixed with different concentrations of daphnoretin in PBS. Fluorescence emission spectra were recorded in the range of 280–400 nm with an excitation wavelength of 260 nm via a microplate reader (Thermo Fisher Scientific, China), and the K A values were calculated via the Stern–Volmer equation. (Papadopoulou et al., 2005). G. mellonella infection model To assess the efficacy of daphnoretin in the G. mellonella infection model, larvae (220–260 mg) were divided into five groups: the MRSA-infected group, the untreated control group, the daphnoretin-treated groups (25 and 50 mg/kg), and the vancomycin-treated group (50 mg/kg), with 10 larvae in each group. A 10 µL suspension of MRSA (5 × 10⁶ CFU/mL) was injected into the last left proleg of the larvae, and treatment was initiated 1 h post infection. The control group received PBS containing 0.1% dimethyl sulfoxide (DMSO). Larvae were maintained at a constant temperature of 37°C, and survival rates and health scores were monitored every 12 h for a total of 120 h(Guo et al., 2024). For bacterial burden assessment, a method similar to that used in the survival assay was employed. At 48 h post infection, the larvae were collected, surface sterilized, homogenized, and plated on TSB agar plates, followed by incubation at 37°C for 24 h to evaluate the bacterial loads within the larvae. Rat septic arthritis model To evaluate the therapeutic effect of daphnoretin in a septic arthritis model, thirty 6–8-week-old Sprague–Dawley (SD) rats were randomly divided into five groups (n = 6 per group). The animals were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg), and the skin around the knee joint was disinfected with povidone-iodine. S. aureus (4 × 10⁶ CFU/10 µL) was then injected into the joint cavity beneath the patella using a Hamilton syringe. The treatment groups received daily subcutaneous injections of daphnoretin (25 or 50 mg/kg) or vancomycin (50 mg/kg) at the nape of the neck for six consecutive days, whereas the control group was administered an equal volume of PBS containing 0.1% DMSO. On day 7, the animals were sacrificed, and relevant parameters were evaluated. The severity of arthritis was monitored using multiple approaches. First, on day 7, knee joint diameters were measured with a digital caliper to assess swelling. Second, to track the infection process in real time, bioluminescent S. aureus Xen29 was injected using the same method described above, and bioluminescent signals were recorded daily for 7 consecutive days. At the end of the experiment, knee joint tissues were fixed and subjected to micro-CT scanning of the distal femur and proximal tibia, followed by three-dimensional reconstruction to analyze cartilage surface damage and changes in subchondral bone density. The bone volume fraction (BV/TV) and bone mineral density (BMD) of the femoral and tibial condyles were further calculated using Cruiser CT software to quantify bone destruction. All the rats were housed in an SPF-grade animal facility under controlled conditions (22 ± 3°C, relative humidity 50–70%, 12 h light/dark cycle) with free access to sterilized food and water. Results Daphnoretin inhibits SrtA activity Screening for SrtA inhibitors typically relies on an in vitro fluorescence resonance energy transfer (FRET)-based assay. This method employs a fluorescent substrate peptide containing the LPXTG motif, with donor and acceptor fluorophores labeled at both termini. When the substrate is cleaved by SrtA, the fluorophores are separated, resulting in an enhanced fluorescence signal that enables real-time monitoring of enzymatic activity (Fig. 1 A). Using this system, several candidate compounds preserved in our laboratory were screened (Supplementary Table 1), and daphnoretin exhibited the most pronounced inhibitory effect on SrtA, with an inhibition rate as high as 78.34% at a concentration of 64 µg/mL; therefore, it was selected for further investigation. (Fig. 1 B). Subsequent concentration‒gradient assays revealed that daphnoretin significantly inhibited SrtA activity in a dose-dependent manner, with enzymatic activity progressively decreasing as the drug concentration increased. Nonlinear regression analysis revealed that the half-maximal inhibitory concentration (IC₅₀) was 8.501 µg/mL (Fig. 1 C), indicating that daphnoretin is a promising SrtA inhibitor. Notably, although daphnoretin inhibited the enzymatic activity of SrtA, it had no significant effect on the growth of S. aureus (Fig. 1 D), and the minimum inhibitory concentration (MIC) against this strain was > 256 µg/mL (Fig. 1 E), far exceeding the therapeutic concentration. These findings suggest that daphnoretin does not inhibit bacterial growth and thus does not impose selective pressure on the strain, thereby preventing the emergence of resistant mutants. This further underscores its potential advantage as an antivirulence candidate compound. Safety assessment of daphnoretin To evaluate the safety of daphnoretin at therapeutically relevant doses, we first performed an MTT assay on ATDC5 cells. Compared with the control treatment, treatment with 64 µg/mL daphnoretin did not significantly affect cell viability (Fig. 1 F). A rabbit red blood cell hemolysis assay was subsequently conducted to further examine the potential in vivo toxicity of daphnoretin, and no hemolytic response was observed even at concentrations higher than the IC 50 (Fig. 1 G), suggesting that daphnoretin has extremely low cytotoxicity. In addition, an in vivo toxicity evaluation was performed using the G. mellonella larval model, in which daphnoretin (25 and 50 mg/kg) was injected into the penultimate left proleg, and larval survival and melanization were monitored for five consecutive days post-treatment. The results demonstrated that neither dose induced melanization nor mortality (Fig. 1 H). Collectively, these findings indicate that daphnoretin is safe at both the cellular level and in the G. mellonella model, providing important evidence for its pharmacological safety assessment and subsequent application studies. Daphnoretin reduces S. aureus adhesion The strong adhesive capacity of S. aureus is a critical step for colonization, host invasion, and subsequent pathogenesis(Berry et al., 2022 ). In this process, SrtA catalyzes the anchoring of cell wall–associated proteins, thereby mediating bacterial attachment to host tissues, and is considered an indispensable enzyme in pathogenesis. To evaluate the effect of daphnoretin on bacterial adhesion, a fibrinogen-binding assay was performed. The results revealed that the adhesion capacity decreased significantly in the daphnoretin-treated group in a dose-dependent manner, with the adhesion rate of the Δ SrtA strain reducing to only 38.87 ± 6.09% of that of the wild-type (WT) strain (Fig. 2 A). These findings suggest that daphnoretin effectively inhibits the binding of S. aureus to fibrinogen, thereby attenuating its ability to invade the host. Daphnoretin inhibits SpA-mediated immune evasion SrtA plays a critical role in the immune evasion of S. aureus by mediating the anchoring of surface protein A (SpA) to the cell wall. SpA, a typical substrate of SrtA, binds to the Fc fragment of host IgG, thereby blocking antibody-dependent phagocytosis, helping bacteria evade immune clearance, and enabling persistent survival within the host(Falugi et al., 2013 ). Thus, the anchoring of SpA is considered one of the key mechanisms by which S. aureus achieves immune evasion and maintains infection. In this study, we employed a microplate reader to measure fluorescence intensity to assess the effect of daphnoretin on SpA anchoring. The results revealed that with increasing concentrations of daphnoretin, the FITC-labeled goat anti-rabbit IgG fluorescence signal progressively decreased, and the Δ SrtA group accounted for only 33.11 ± 3.29% of the WT group. (Fig. 2 B). These findings indicate that daphnoretin reduces the anchoring of SpA on the bacterial cell wall by inhibiting the catalytic activity of SrtA, thereby weakening SpA-dependent immune evasion. This observation further supports the potential role of daphnoretin as a SrtA inhibitor in blocking the immune evasion mechanisms of S. aureus . Daphnoretin suppresses S. aureus internalization and improves ATDC5 cell viability To evaluate the protective effect of daphnoretin against S. aureus infection, calcein-AM/PI double staining was performed on ATDC5 cells. Fluorescence microscopy revealed that with increasing concentrations of daphnoretin, the green fluorescence signals (viable cells) progressively increased in a dose-dependent manner, whereas the red fluorescence signals (dead cells) markedly decreased, indicating effective attenuation of S. aureus -induced cytotoxicity. Among the groups, the Δ SrtA strain presented the highest cell survival rate, reaching 91.77 ± 4.69% (Fig. 2 C–D). Further invasion assays demonstrated that daphnoretin significantly reduced the intracellular bacterial burden. Consistently, fluorescence imaging revealed a progressive decrease in the number of intracellular red fluorescence signals with increasing drug concentration, whereas the overall cell morphology remained intact (Fig. 2 E–F). Collectively, these findings indicate that daphnoretin protects host cells by alleviating cytotoxic damage and markedly inhibits MRSA invasion and colonization, thereby supporting its potential as a promising antivirulence candidate. Daphnoretin inhibits S. aureus biofilm formation Biofilm formation and development are progressive and highly dynamic processes. Under favorable environmental conditions, S. aureus initially establishes colonization through surface adhesion, followed by aggregation into early biofilms that continuously expand and mature with the support of extracellular matrix components(Peng et al., 2022 ). As the biofilm structure becomes more complete, bacterial resistance to antibiotics and host immunity are markedly enhanced; eventually, some bacteria detach from the mature biofilm and disseminate to new sites, leading to persistent infection and recurrence (Fig. 3 A). To evaluate the effect of daphnoretin on this process, biofilm formation was quantified via crystal violet staining. The results revealed a dose-dependent reduction in biofilm biomass upon treatment, and the minimum biofilm inhibitory concentration (MBIC) was 16 µg/mL (Fig. 3 B), suggesting that daphnoretin significantly inhibits biofilm development. Stage-specific intervention further revealed that the administration of 64 µg/mL daphnoretin had a major inhibitory effect during the early stage of biofilm formation (Fig. 3 C), indicating interference with initial adhesion and early matrix establishment. To further elucidate the mechanism involved, the major biofilm matrix components were examined. Daphnoretin treatment resulted in a significant decrease in the levels of extracellular DNA (eDNA), polysaccharide intercellular adhesin (PIA), and extracellular proteins. Notably, PIAs and extracellular proteins were significantly reduced at 32 µg/mL, whereas eDNA was markedly decreased at 16 µg/mL. (Fig. 3 D–G). eDNA functions as a structural scaffold facilitating intercellular adhesion and genetic exchange; PIAs play a key role in cell aggregation and biofilm maturation, while extracellular proteins not only support the matrix structure but also include adhesion- and immune-regulation–related functional proteins. By simultaneously reducing these three critical components, daphnoretin impaired the structural stability and cell aggregation capacity of biofilms, thereby weakening adhesion and immune evasion and ultimately attenuating the pathogenic potential of S. aureus . Morphological analyses further supported these findings. Confocal laser scanning microscopy (CLSM) revealed marked alterations in biofilm architecture following daphnoretin treatment: as the drug concentration increased, the biofilms shifted from dense and compact to sparse and porous (Fig. 3 H), indicating disruption of spatial organization and stability. Scanning electron microscopy (SEM) at the ultrastructural level confirmed this trend: while the control biofilms presented compact, homogeneous structures characteristic of mature biofilms, the treated groups formed only loose, irregular thin layers with markedly reduced intercellular cohesion (Fig. 3 I). Together, these data demonstrate that daphnoretin not only inhibits biofilm formation but also disrupts the three-dimensional structural integrity and stability of biofilms, thereby compromising their protective barrier function. In addition, sliding motility assays were performed to assess the surface spreading ability of S. aureus . Sliding motility, an important means of expansion for nonflagellated bacteria, is closely associated with early adhesion, population dispersal, and subsequent biofilm establishment. The results revealed that with increasing concentrations of daphnoretin, the spreading area of bacterial colonies on agar plates gradually decreased. At a concentration of 128 µg/mL, the spreading area was only 42.88 ± 1.09% that of the WT group (Fig. 3 J–K), indicating significant inhibition of bacterial motility. These findings suggest that daphnoretin interferes with bacterial sliding motility, thereby reducing early-stage colonization and dissemination and ultimately blocking key steps of biofilm establishment. This observation, which is consistent with the results of the crystal violet staining and morphological analyses, further highlights the potential of daphnoretin as an antivirulence candidate drug. Daphnoretin interacts with SrtA To elucidate the interaction mechanism between daphnoretin and SrtA, a combination of Western blot, fluorescence quenching, and molecular docking analyses was employed (Fig. 4 A). Compared with no treatment, treatment with daphnoretin at concentrations of 0–64 µg/mL did not alter the band intensity of SrtA (Fig. 4 B), suggesting that daphnoretin does not regulate SrtA expression but more likely modulates its catalytic function through direct binding. Fluorescence quenching experiments were subsequently conducted to characterize the direct drug–protein interactions. Fluorescence spectra were recorded with an excitation wavelength set at 260 nm, and emission was monitored within the range of 280–400 nm. A concentration-dependent decrease in fluorescence intensity was observed with increasing daphnoretin concentration. Stern–Volmer analysis of F₀/F versus ligand concentration yielded a binding constant of K A = 1.6383 × 10⁴ L·mol⁻¹ (Fig. 4 C), indicating a specific interaction between the two molecules. Molecular docking further revealed that daphnoretin primarily localized to a recognition pocket adjacent to the catalytic groove of SrtA, where it formed key interactions with Asn114 (N114), Val168 (V168), Lys175 (K175), and Thr180 (T180). The calculated binding free energy of the complex was ΔG_bind = − 8.5 kcal·mol⁻¹ (Fig. 4 D-E), which is consistent with a moderate-to-strong affinity typical of small molecule–protein interactions. To validate the predicted binding sites, site-directed mutants of SrtA (N114A, V168A, T180A, and K175A) were generated, and their affinities for daphnoretin were compared with those of the wild type (WT) using fluorescence quenching. The results demonstrated that the binding constants ( K A ) of all the mutants were significantly lower than those of the WT (Fig. 4 F–I), indicating that these residues are crucial for ligand recognition and anchoring. Collectively, these findings consistently support that daphnoretin inhibits SrtA activity by directly binding to key residues adjacent to the catalytic groove rather than through the regulation of protein expression, thereby elucidating the molecular basis of its inhibitory mechanism. Therapeutic efficacy of daphnoretin in the G. mellonella model The immune system of G. mellonella shows a high degree of similarity to the innate immune response of vertebrates at both the cellular and molecular levels and is therefore widely used as a reliable model for evaluating anti-infective therapies(Serrano et al., 2023 ). On the basis of this rationale, a G. mellonella infection model was established to evaluate the therapeutic effect of daphnoretin on S. aureus infection (Fig. 5 A). During the experimental period, larval activity, cocoon formation, degree of melanization, and survival were continuously monitored to assess health status comprehensively. The results revealed that the health scores of the daphnoretin-treated larvae improved progressively with increasing drug concentration. By day 5, the health conditions of the high-dose group were second only to those of the vancomycin-treated group and were significantly better than those of the untreated control group (Fig. 5 B–C), indicating that daphnoretin effectively alleviated infection-associated symptoms. Survival analysis further confirmed this finding, with survival rates in the high-dose daphnoretin group reaching 80% on day 5 (Fig. 5 D–E). In addition, sterile homogenization and colony enumeration performed at 48 h post infection revealed that the bacterial burden in larvae treated with daphnoretin was significantly lower than that in the WT group. With increasing drug concentrations, the bacterial load in the treatment groups decreased correspondingly (Fig. 5 F). Taken together, the results from health scoring, survival rate, and bacterial burden consistently demonstrate that daphnoretin exhibited significant anti-infective efficacy in the G. mellonella model. Daphnoretin alleviates septic arthritis in rats To evaluate the anti-infective and joint-protective effects of daphnoretin in vivo, a rat model of septic arthritis was established via S. aureus inoculation, and therapeutic efficacy was assessed via randomized grouping (Fig. 6 A). Bioluminescence imaging revealed that during the early phase of infection (days 1–3), the luminescence intensity increased over time and peaked on day 3. From day 4 onward, the luminescence intensity began to decrease, and by day 7, the intensities in the daphnoretin- and vancomycin-treated groups were significantly lower than those in the untreated wild-type infection group, indicating that the bacterial burden and inflammatory activity at the lesion sites were suppressed. (Fig. 6 B–C). Similarly, the knee joint diameter of the rats in the treatment groups was significantly greater than that of the WT group, with the high-dose daphnoretin group showing a diameter of 13.09 ± 0.09 mm, indicating that acute joint swelling and inflammatory signs were alleviated (Fig. 6 D). At the structural level, micro-CT was employed to perform three-dimensional reconstruction and quantitative analysis of the affected joints. In the untreated WT group, irregular cartilage surfaces and discontinuities in cortical and trabecular bone were observed. In contrast, compared with infection, daphnoretin treatment markedly improved bone parameters, with higher BV/TV and BMD values Fig. 6 E–G). Both imaging (BLI, micro-CT) and clinical endpoints (joint diameter) exhibited consistent improvement trends, collectively supporting the in vivo anti-infective and tissue-protective activities of daphnoretin. Overall, daphnoretin reduced bioluminescence intensity, mitigated joint swelling and inflammatory signs, and improved BV/TV and BMD in a rat model of septic arthritis, highlighting its strong potential as an antivirulence candidate drug for treating joint infections. Discussion To address the problem of antibiotic resistance, this study employed a strategy distinct from traditional antibiotics that act through direct bacteriostasis, namely, by attenuating the virulence of S. aureus through the inhibition of the key virulence factor SrtA. Previous studies have demonstrated that the deletion or functional inhibition of SrtA significantly reduces bacterial pathogenicity without affecting basic growth(Mazmanian et al., 1999 , Jonsson et al., 2003 ). In this study, through in vitro screening and multidimensional validation, we identified and confirmed that daphnoretin is a potent inhibitor of SrtA. Notably, daphnoretin markedly attenuated the adhesion, invasion, immune evasion, and biofilm formation of S. aureus at concentrations far below its antibacterial level without imposing significant pressure on bacterial growth. More importantly, in S. aureus infection models, daphnoretin exhibited pronounced antivirulence activity. These findings not only further validate the feasibility of targeting SrtA as an antivirulence therapeutic strategy(Cegelski et al., 2008 ) but also provide new insights into the application of natural products in the management of complex infections. Our findings further demonstrate that daphnoretin significantly attenuates critical pathogenic steps of S. aureus within the host by inhibiting the SrtA-mediated anchoring of surface proteins. We observed that daphnoretin treatment markedly reduced bacterial binding to fibrinogen and significantly diminished its ability to invade chondrocytes. Clumping factors (ClfA/ClfB) play important roles in the early adhesion and colonization of bacteria(D et al., 1998 ), whereas protein A (SpA) enhances immune evasion by binding to the Fc fragment of IgG, thereby blocking complement activation and phagocytosis(Falugi et al., 2013 ). By inhibiting the anchoring of these surface proteins to the cell wall, daphnoretin effectively reduces the adhesive and immune evasion capabilities of bacteria. Our experimental results, for the first time at the level of natural products, validated the feasibility of this strategy and provided solid evidence for the further development of antivirulence therapeutics. Intervention in biofilm formation plays a critical role in combating S. aureus infections. Our results demonstrated that biofilms formed after daphnoretin treatment presented a sparse and porous structure, with significantly reduced levels of extracellular matrix components, including polysaccharides, extracellular DNA, and proteins. These findings suggest that daphnoretin effectively blocks the early stages of biofilm formation, thereby inhibiting bacterial colonization and community stabilization. Prosthetic joint infections and chronic osteoarthritis are often closely associated with persistent or recurrent infections linked to biofilms(Otto, 2008 , Archer et al., 2011 ). This mechanism has important clinical value. Blocking biofilm formation not only reduces the long-term survival of bacteria in the joint environment but also may enhance host immune defenses and antibiotic penetration, thereby lowering the risk of refractory infections. At the molecular level, the molecular docking results indicated that daphnoretin binds to the active pocket of SrtA and forms stable interactions with key residues, including Lys175, Thr180, Val168, and Asn114. More importantly, this study confirmed the protective effect of daphnoretin in both G. mellonella and rat in vivo models. In the G. mellonella infection model, drug treatment significantly improved host survival without causing melanization or behavioral abnormalities, indicating favorable biosafety. This finding is consistent with previous studies in which G. mellonella has been widely used to evaluate the in vivo toxicity and efficacy of antibacterial candidates(Desbois and Coote, 2012 , Tsai et al., 2016 ). In a rat model of septic arthritis, animals treated with daphnoretin presented reduced joint swelling, decreased bacterial burden in tissues, and markedly improved cartilage integrity, which is consistent with previous reports that antivirulence agents can mitigate the pathogenic processes of joint infection. These in vivo findings are highly consistent with our in vitro mechanistic results, indicating that the antivirulence activity of daphnoretin also occurs in complex host environments. Notably, whether antivirulence agents can retain their activity in vivo has long been considered one of the major obstacles to clinical translation(Clatworthy et al., 2007 ), and our findings provide positive evidence addressing this issue. This study also systematically evaluated the safety profile of daphnoretin. The results showed that the compound did not induce hemolysis of rabbit erythrocytes at therapeutically relevant concentrations and did not significantly impair the viability of ATDC5 chondrocytes; moreover, no obvious signs of toxicity were observed in the G. mellonella model. These findings are consistent with previous reports that natural bicoumarin compounds generally possess high tolerability and low toxicity. As drug safety is one of the core considerations for the clinical translation of novel anti-infective agents (DiMasi et al., 2016 ), the favorable safety profile of daphnoretin not only reinforces its feasibility as a lead compound but also provides important assurance for its further preclinical development. From a clinical perspective, daphnoretin has multiple advantages as a candidate antivirulence agent. First, it does not directly inhibit bacterial growth, thereby markedly reducing the risk of resistance development. Second, it targets multiple steps of pathogenesis, including adhesion, immune evasion, and biofilm formation, resulting in an overall attenuation of virulence. Third, as a natural product, it is readily accessible and amenable to structural modification, offering potential for further optimization. These features suggest that daphnoretin could not only be used prophylactically in high-risk populations, such as patients undergoing joint replacement but also serve as an adjuvant to antibiotics, improving treatment success rates and reducing recurrence. Previous studies have indicated that the combination of antivirulence agents with conventional antibiotics can increase efficacy and reduce resistance pressure(Rasko and Sperandio, 2010 , Cegelski et al., 2008 ). Therefore, future research on daphnoretin may also focus on its synergistic effects with existing antimicrobial agents. Although this study systematically revealed the antivirulence activity of daphnoretin as an SrtA inhibitor, several limitations remain. First, molecular docking only provides theoretical support, and structural evidence, such as crystallography or cryo-electron microscopy, is still lacking to precisely define the binding sites and conformational stability (Zong et al., 2004 , Suree et al., 2009 ). Second, the in vivo investigations were limited mainly to G. mellonella and rat models, which, despite demonstrating favorable protective effects and safety, did not address pharmacokinetics, tissue distribution, or long-term toxicity in a systematic manner. In addition, as a natural product, daphnoretin may suffer from limited affinity and bioavailability, necessitating structural modification and medicinal chemistry optimization to enhance its efficacy and pharmacokinetic properties(Newman and Cragg, 2012 ). Therefore, future studies should integrate structural analyses, drug optimization, and validation in higher-order animal models to advance its clinical translation. In summary, this study systematically evaluated the antivirulence activity of daphnoretin as an SrtA inhibitor and demonstrated its ability to inhibit adhesion, immune evasion, and biofilm formation in vitro, as well as its protective efficacy and safety in G. mellonella and rat septic arthritis models. Molecular docking analysis further elucidated its binding mechanism, providing a theoretical basis for subsequent drug optimization. These findings provide important evidence supporting the development of natural products as antivirulence agents and offer new directions for combating infections caused by drug-resistant S. aureus . With further progress in structural validation, pharmacokinetic studies, and preclinical evaluations, daphnoretin and its derivatives hold promise as important components for the future treatment of septic arthritis and related infections. Statistical analysis All statistical analyses were performed using GraphPad Prism 10 software. Experimental data are presented as the mean ± standard deviation (SD), with each experiment conducted in at least three independent biological replicates. Differences between two groups were analyzed using two-tailed Student’s t -tests, and multiple group comparisons were performed using one-way or two-way analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant. Declarations Acknowledgements This work was supported by the Jilin Provincial Science and Technology Development Plan (Grant No. YDZJ202501ZYTS177). Author contributions Yun Sun and Tong Zhao: Writing and original draft preparation;Dongbin Guo, Peitong Jiang,Xinyao Liu, Zhangyu Du, and Luanbiao Sun: Data curation and analysis;Yuan Gao and Han Gao: Participation in in vivo and in vitro experiments;Li Wang, Bingmei Wang, and Ming Yan: Study conception and design.All authors have read and approved the final manuscript. Ethics statement All animal experiments in this study were conducted in accordance with national guidelines for the care and use of laboratory animals, and the experimental protocol was reviewed and approved by the Experimental Animal Ethics Committee of Changchun University of Chinese Medicine. Availability of data and materials All data generated or analyzed during this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no conflict of interest. References ARCHER, N. K., MAZAITIS, M. J., COSTERTON, J. W., LEID, J. G., POWERS, M. 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Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice. PLoS One , 11 , e0149460.https://doi.org/10.1371/journal.pone.0149460 ZHANG, J., LIU, H., ZHU, K., GONG, S., DRAMSI, S., WANG, Y. T., LI, J., CHEN, F., ZHANG, R., ZHOU, L., LAN, L., JIANG, H., SCHNEEWIND, O., LUO, C. & YANG, C. G. (2014). Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase. Proc Natl Acad Sci U S A , 111 , 13517-13522.https://doi.org/10.1073/pnas.1408601111 ZONG, Y., BICE, T. W., TON-THAT, H., SCHNEEWIND, O. & NARAYANA, S. V. (2004). Crystal structures of Staphylococcus aureus sortase A and its substrate complex. J Biol Chem , 279 , 31383-31389.https://doi.org/10.1074/jbc.M401374200 Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Graphicalabstract.tif Graphical Abstract Mechanism by whichdaphnoretin alleviates S. aureus infection via SrtA inhibition Daphnoretin, a major active component derived from traditional Chinese medicine, targets the key virulence factor SrtA of S. aureus . SrtA recognizes and cleaves the threonine (T) and glycine (G) residues within the LPXTG motif, thereby covalently anchoring surface proteins to the bacterial cell wall. This process promotes bacterial adhesion and invasion of host cells and plays a central role in immune evasion as well as biofilm formation and maturation. By inhibiting SrtA activity, daphnoretin blocks the anchoring of surface proteins, thereby reducing the ability of S. aureus to adhere, invade, evade immune responses, and form biofilms, ultimately attenuating its overall pathogenicity. Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 09 Jan, 2026 Reviews received at journal 28 Dec, 2025 Reviewers agreed at journal 15 Dec, 2025 Reviews received at journal 18 Nov, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviewers invited by journal 22 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Submission checks completed at journal 10 Oct, 2025 First submitted to journal 09 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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19:15:13","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183495,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/22e728a4b5495eeb9bc53e21.html"},{"id":95053641,"identity":"47aa7a1e-6b58-4283-b324-4571f4553dfc","added_by":"auto","created_at":"2025-11-03 19:15:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2917468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaphnoretin inhibits SrtA activity and shows favorable safety in vitro and in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the inhibitor screening principle: SrtA catalyzes the specific cleavage of the substrate Abz-LPATG-Dap(Dnp)-NH₂, resulting in changes in the FRET (fluorescence resonance energy transfer) signal for enzymatic activity detection. (B) Chemical structure of daphnoretin (CAS: 2034-69-7). (C) FRET-based inhibition curve showing the IC₅₀ value of daphnoretin against SrtA (8.501 μg/mL). (D) Growth curves of \u003cem\u003eS. aureus\u003c/em\u003e and the Δ\u003cem\u003esrtA\u003c/em\u003e mutant treated with different concentrations of daphnoretin. (E) Determination of the minimum inhibitory concentration (MIC) of daphnoretin against MRSA. (F) MTT assay showing the viability of ATDC5 cells after treatment with various concentrations of daphnoretin. (G) Evaluating the hemolytic effect of daphnoretin on erythrocytes. (H, I) In vivo toxicity assessment in \u003cem\u003eG. mellonella\u003c/em\u003elarvae. All experiments were independently performed in triplicate. The data are presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/76a227c1732eeccf8704946d.png"},{"id":95053643,"identity":"d388bb0f-3d65-4dce-ba6f-2590add6b924","added_by":"auto","created_at":"2025-11-03 19:15:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7035822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaphnoretin reduces \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e virulence via SrtA inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effects of different concentrations of daphnoretin (0–64 μg/mL) on the adhesion ability of \u003cem\u003eS. aureus\u003c/em\u003e. (B) FITC-labeled goat anti-rabbit IgG was used to assess the effect of daphnoretin on the expression level of the surface protein SpA in \u003cem\u003eS. aureus\u003c/em\u003e, with Δ\u003cem\u003esrtA\u003c/em\u003e as a control. (C–D) Calcein-AM/PI double-staining assay was used to evaluate the live/dead status of ATDC5 cells infected with \u003cem\u003eS. aureus\u003c/em\u003e and treated with various concentrations of daphnoretin; fluorescence microscopy revealed viable cells in green and dead cells in red. (E–F) Invasion assay of \u003cem\u003eS. aureus\u003c/em\u003e into ATDC5 cells following treatment with different concentrations of daphnoretin; infected cells were visualized viafluorescence imaging after staining with FITC-labeled bacteria, TRITC–phalloidin, and DAPI. All experiments were independently repeated three times. The data are presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/56ea21c57a7c57f8c06e94fa.png"},{"id":95222746,"identity":"1b16b8c9-0c25-4862-81b4-1575d88ce156","added_by":"auto","created_at":"2025-11-05 16:21:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4627830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of daphnoretin on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram illustrating the four typical stages of biofilm development: adhesion, formation, maturation, and dispersal. (B) Crystal violet staining was used to quantify the inhibitory effects of different concentrations of daphnoretin on biofilm formation. (C) Intervention assay assessing the effectsof the addition of daphnoretin (64 μg/mL) at different stages of biofilm development (0–24 h), followed by quantification at 24 h viacrystal violet staining. Effects of daphnoretin on biofilm extracellular matrix components, including extracellular proteins (D), polysaccharides (E), and extracellular DNA (F). (G) CLSM 3D imaging showing structural changes in biofilms treated with different concentrations of daphnoretin. (H) SEM image of the ultrastructural alterations in biofilms following daphnoretin treatment. (I-K) Sliding motility assay demonstrating the inhibitory effect of daphnoretin on bacterial motility. All experiments were independently repeated three times. The data are presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/4618f6c969f0873d3bfb7d65.png"},{"id":95053645,"identity":"ee640447-48de-4731-bace-400c33299198","added_by":"auto","created_at":"2025-11-03 19:15:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaphnoretin directly interacts with SrtA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration showing the validation of the daphnoretin–SrtA interaction via Western blotting, fluorescence quenching, and molecular docking. (B) Western blot analysis of the SrtA protein expression level in \u003cem\u003eS. aureus\u003c/em\u003e treated with different concentrations of daphnoretin (0–64 μg/mL), with quantification of the band intensity. (C) Fluorescence quenching assay characterizing the binding properties of daphnoretin with SrtA and calculation of the binding constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) via the Stern–Volmer relationship. (D-E) The molecular visualization tool ABEE was used to analyze the 3D electrostatic potential map of daphnoretin, and a molecular docking simulation was used to predict the binding mode of daphnoretin with SrtA and identify key amino acid residues. (F–I) Fluorescence quenching analysis comparing the binding affinities of daphnoretin with those of wild-type SrtA and individual site-directed mutants to validate the predicted key binding residues. All experiments were independently repeated three times. The data are presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/e357af35f5e509f5e3ec87c9.png"},{"id":95312387,"identity":"1be6bd4d-92c6-4751-8943-2ccd9f5cd16e","added_by":"auto","created_at":"2025-11-06 15:49:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6516530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaphnoretin protects \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG. mellonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the \u003cem\u003eG. mellonella\u003c/em\u003e infection model. (B–C) Health scores of larvae (n = 10 per group) at120 h post infection in the control, infection, daphnoretin (25 and 50 mg/kg), and vancomycin (50 mg/kg) groups. (D–E) Survival analysis of the above groups within 120 h postinfection. (F) Colony counts showing the bacterial burden in larvae from each treatment group. All experiments were independently repeated at least three times. The dataare presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/ce62d9c2589bd05288d0d298.png"},{"id":95222462,"identity":"617c1d07-ebfe-45b9-ad53-847033fd75b8","added_by":"auto","created_at":"2025-11-05 16:20:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5792354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaphnoretin in a rat model of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-induced septic arthritis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the experimental design for the rat model of knee joint infection induced by \u003cem\u003eS. aureus\u003c/em\u003e. (B–C) In vivo bioluminescence imaging to dynamically monitor infection progression in different treatment groups, including the untreated infection, daphnoretin treatment (25 and 50 mg/kg, subcutaneous injection), and vancomycin treatment (50 mg/kg) groups. Representative images and corresponding quantitative analysis of fluorescence intensity were recorded on day 7 postinfection. (D) Knee joint swelling was assessed by direct measurement of the joint diameter on day 7 post infection. (E–G) Microcomputedtomography (micro-CT) was used for three-dimensional reconstruction of knee joint structures, and quantitative analysis of the bone volume fraction (BV/TV) and bone mineral density (BMD) of the femoral and tibial condyles was performed. All experiments were independently repeated at least three times. The data are presented as the means ± standard deviations (means ± SD). Statistical significance: *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/dd38614bb9b98f624db4f1eb.png"},{"id":106343267,"identity":"46c97ef4-a9ea-41d4-8490-2944845cfabb","added_by":"auto","created_at":"2026-04-07 16:00:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27534457,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/0029580d-e1c3-4b08-9574-952a0a0eda1b.pdf"},{"id":95053642,"identity":"b1b0c08c-8192-45f8-a5d3-28736d09cc6e","added_by":"auto","created_at":"2025-11-03 19:15:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":116686,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/35ae06cd781a4874d73ee4b6.docx"},{"id":95222496,"identity":"12dfc0f7-4858-4889-8fae-8bcac2738d67","added_by":"auto","created_at":"2025-11-05 16:20:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1809764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanism by whichdaphnoretin alleviates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection via SrtA inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaphnoretin, a major active component derived from traditional Chinese medicine, targets the key virulence factor SrtA of \u003cem\u003eS. aureus\u003c/em\u003e. SrtA recognizes and cleaves the threonine (T) and glycine (G) residues within the LPXTG motif, thereby covalently anchoring surface proteins to the bacterial cell wall. This process promotes bacterial adhesion and invasion of host cells and plays a central role in immune evasion as well as biofilm formation and maturation. By inhibiting SrtA activity, daphnoretin blocks the anchoring of surface proteins, thereby reducing the ability of \u003cem\u003eS. aureus\u003c/em\u003e to adhere, invade, evade immune responses, and form biofilms, ultimately attenuating its overall pathogenicity.\u003c/p\u003e","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7820020/v1/bd9f0d76e5877b8bb4b44021.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Daphnoretin as a Potent Sortase A Inhibitor Effectively Disarms Drug-Resistant Staphylococcus aureus Infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSeptic arthritis (SA), also known as bacterial arthritis or pyogenic arthritis, is a disease caused by pyogenic bacteria that leads to rapid destruction of joint tissues, resulting in joint dysfunction and even disability. Among the causative pathogens, \u003cem\u003eS. aureus\u003c/em\u003e is the most common gram-positive opportunistic pathogen associated with SA(He et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), accounting for 64% of cases, with methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) reaching as high as 23%(Gobao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In recent years, with the increasing number of immunocompromised patients, joint replacement surgeries, and invasive procedures, the incidence of \u003cem\u003eS. aureus\u003c/em\u003e-associated SA has been increasing annually(Wu et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, He et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Epidemiological investigations have shown that community-acquired methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (CA-MRSA) not only causes skin and soft tissue infections but also has become an important etiological agent of SA, with a particularly marked increase among adolescents and athletes(Kaplan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Braun and Kahanov, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite advances in antibiotics and surgical debridement, \u003cem\u003eS. aureus\u003c/em\u003e-induced septic arthritis still has high recurrence and disability rates, indicating significant limitations of conventional therapeutic strategies(Wang and Wang, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Joo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Kim et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe current treatment for \u003cem\u003eS. aureus\u003c/em\u003e-associated septic arthritis (SA) mainly relies on a combination of antibiotics and surgical intervention(Davis and Zamora, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In clinical practice, glycopeptides (such as vancomycin), linezolid, or daptomycin are commonly used and, when necessary, are combined with β-lactams or aminoglycosides(Xu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, \u003cem\u003eS. aureus\u003c/em\u003e continually undermines the efficacy of antibiotics through multiple mechanisms, including the acquisition of resistance genes (such as \u003cem\u003emecA\u003c/em\u003e and \u003cem\u003evanA\u003c/em\u003e) (Nepal et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), biofilm formation, and the presence of persister cells (Theis et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In particular, in prosthetic joint infections, biofilms formed by bacteria can significantly resist antibiotic penetration and host immune responses, greatly increasing the difficulty of treatment (Rottier et al., 2023). Moreover, prolonged and high-dose use of antibiotics is often accompanied by toxic side effects and further dissemination of resistant strains (Mithuna et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Lau et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, reliance solely on conventional antibiotics and surgical drainage is no longer sufficient to meet clinical needs.\u003c/p\u003e\u003cp\u003eAntivirulence therapy has recently been regarded as an important new approach against \u003cem\u003eS. aureus\u003c/em\u003e infections(Jonsson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The core concept is to attenuate bacterial pathogenicity by inhibiting key virulence mechanisms, such as toxins, adhesion factors, or regulatory systems, rather than directly killing bacteria. This strategy is expected to reduce selective pressure for resistance and act synergistically with antibiotics(Touati et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Ford et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, the inhibition of α-hemolysin, surface adhesion proteins, and the regulatory factor SaeRS has demonstrated potential in improving host outcomes(Frank et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Gao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Both animal models and preclinical studies have shown that antivirulence agents can effectively reduce tissue damage, alleviate inflammatory responses, and enhance host immune clearance(Sully et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Le et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Tkaczyk et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Yang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the development of potent antivirulence molecules represents a cutting-edge strategy for combating \u003cem\u003eS. aureus\u003c/em\u003e infections.\u003c/p\u003e\u003cp\u003eSrtA is an important transmembrane enzyme of \u003cem\u003eS. aureus\u003c/em\u003e that is responsible for covalently anchoring surface proteins containing the LPXTG motif to the peptidoglycan layer(Ton-That et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). These surface proteins play central roles in host cell adhesion, tissue invasion, and immune evasion. Studies have shown that the deletion or inhibition of SrtA activity can significantly reduce the virulence and pathogenicity of \u003cem\u003eS. aureus\u003c/em\u003e without affecting its basic growth(Zhang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, SrtA is widely considered an ideal target for antivirulence therapy, and multiple small-molecule inhibitors have been developed and validated. Targeting SrtA holds promise for blocking key steps of \u003cem\u003eS. aureus\u003c/em\u003e infection, thereby reducing the risk of recurrence and chronic infection.\u003c/p\u003e\u003cp\u003eNatural products have increasingly emerged in recent years as promising antivirulence candidates against \u003cem\u003eS. aureus\u003c/em\u003e infections because of their multitarget activities and relatively high safety profiles. Unlike traditional antibiotics that inhibit bacterial growth or directly kill bacteria, natural products often act by interfering with key bacterial virulence factors. Daphnoretin, a naturally occurring dicoumarin compound, is distributed mainly in plants of the Thymelaeaceae family, such as Daphne genkwa and Wikstroemia indica. Previous studies have demonstrated that daphnoretin has significant activities in antitumor, antiviral, anti-inflammatory, and immunomodulatory contexts, with mechanisms involving the induction of apoptosis, the regulation of multiple signaling pathways, and the modulation of the cell cycle(Gu and He, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). With the growing attention given to natural products in the fields of antivirulence therapy and multitarget drug development, daphnoretin has gradually gained recognition as a potential lead compound, providing important insights for the development of novel antiinfective and immunomodulatory strategies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eStrains, culture conditions, and reagents\u003c/h2\u003e\n \u003cp\u003eAll strains were cultured at 37\u0026deg;C with shaking at 220 rpm in Luria\u0026ndash;Bertani (LB) medium or tryptic soy broth (TSB). The strains used in this study included methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC BAA-1717ᵀ (USA300-HOU-MR). SrtA deletion mutant (\u0026Delta;\u003cem\u003esrtA\u003c/em\u003e), and \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) pET28a-\u003cem\u003esrtA\u003c/em\u003e \u0026Delta;N59, all of which were obtained from and preserved in our laboratory. Daphnoretin (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%; quality control report in Supplementary Fig.\u0026nbsp;1) was purchased from Desite Biotechnology Co., Ltd. (Chengdu, China). The fluorescent substrate peptide Abz-LPATG-Dap (Dnp)-NH₂ was synthesized and provided by LifeTein (Beijing, China). Vancomycin and kanamycin were purchased from MedChemExpress (Shanghai, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePurification of SrtA and its mutants\u003c/h3\u003e\n\u003cp\u003eThe recombinant plasmid pET28a-srtA \u0026Delta;N59 was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) and cultured in LB media supplemented with kanamycin (50 \u0026micro;g/mL) at 37\u0026deg;C with shaking at 220 rpm until the optical density at 600 nm (OD₆₀₀) reached approximately 0.8. Protein expression was then induced by the addition of isopropyl-\u0026beta;-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by incubation at 16\u0026deg;C with shaking at 180 rpm. After induction, the bacterial cells were harvested via centrifugation and resuspended in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl₂, pH 7.4; Beyotime, China). The suspension was disrupted by ultrasonication, and the lysate was centrifuged at 12,000 rpm for 1 h at 4\u0026deg;C to collect the supernatant. The soluble proteins were purified via a Ni-NTA affinity chromatography system (Beyotime, China), in which the nonspecific proteins were washed away with low-concentration imidazole, and the target SrtA protein was eluted with high-concentration imidazole.\u003c/p\u003e\n\u003cp\u003eSite-directed mutagenesis of SrtA (N114A, V168A, T180A, and K175A) was performed via a site-directed mutagenesis kit (Tiangen, China), with the primer sequences listed in Supplementary Table\u0026nbsp;2. The mutant plasmids were subsequently transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), and the expression and purification of the mutant proteins were carried out following the same protocol as that used for wild-type SrtA.\u003c/p\u003e\n\u003ch3\u003eSrtA inhibitor screening and the IC₅₀ assay\u003c/h3\u003e\n\u003cp\u003eTo screen potential SrtA inhibitors, a fluorescence resonance energy transfer (FRET) assay was employed to evaluate the inhibitory effects of small-molecule compounds on the enzymatic activity of SrtA. Purified SrtA protein (final concentration of 5 \u0026micro;M) was dissolved in 100 \u0026micro;L of protein buffer and incubated with candidate small-molecule compounds (64 \u0026micro;g/mL) at 37\u0026deg;C for 1 h under light-protected conditions. The fluorescent substrate Abz-LPATG-Dap (Dnp)-NH₂ (10 \u0026micro;M) was subsequently added to the mixture, which was further incubated for 20 min under the same conditions. A parallel control group containing only buffer and fluorescent substrate was included to eliminate nonspecific signal interference.\u003c/p\u003e\n\u003cp\u003eAfter the reaction, the fluorescence intensity was measured via a microplate reader (Thermo Fisher Scientific, USA) at an excitation wavelength of 309 nm and an emission wavelength of 420 nm. Compounds exhibiting\u0026thinsp;\u0026ge;\u0026thinsp;70% inhibition of SrtA activity were selected as candidate inhibitors. These compounds were then tested at a series of concentrations (0.125\u0026ndash;128 \u0026micro;g/mL) via the same protocol, and concentration‒response curves were generated with GraphPad Prism 10 software to calculate the half-maximal inhibitory concentration (IC₅₀), thereby assessing their inhibitory potency.\u003c/p\u003e\n\u003ch3\u003eMIC determination and growth curve analysis of daphnoretin\u003c/h3\u003e\n\u003cp\u003eAccording to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) (Delgado-Valverde et al., 2017, Oh et al., 2021). The minimum inhibitory concentration (MIC) of daphnoretin against \u003cem\u003eS. aureus\u003c/em\u003e was determined using the broth microdilution method. Briefly, resuscitated bacterial cultures were adjusted to 1 \u0026times; 10⁵ CFU and distributed into 96-well plates containing serial concentrations of daphnoretin (1\u0026ndash;256 \u0026micro;g/mL). To each well, 2.5 \u0026micro;L of resazurin (5 mg/mL) was added as a viability indicator. Positive controls (bacterial suspension only) and negative controls (medium only) were included. After incubation at 37\u0026deg;C for 16 h, bacterial growth was assessed on the basis of the color change of resazurin, and the MIC values were determined. In parallel, to evaluate the effects of daphnoretin on bacterial growth kinetics, cultures were incubated with daphnoretin at different concentrations (32 and 64 \u0026micro;g/mL), and the optical density at 600 nm (OD₆₀₀) was measured at defined time intervals to generate growth curves.\u003c/p\u003e\n\u003ch3\u003eMTT assay for cell viability\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity of daphnoretin on ATDC5 cells was evaluated using the MTT assay. The ATDC5 cell line, derived from mouse teratocarcinoma, is commonly used for studies on chondrogenic differentiation and function. The cells were seeded into 96-well plates at a density of 2 \u0026times; 10⁴ cells/well and cultured at 37\u0026deg;C with 5% CO₂ for 24 h. Subsequently, different concentrations of daphnoretin (0\u0026ndash;64 \u0026micro;g/mL) were added, and the mixture was incubated for another 24 h. The medium was then removed, and MTT solution (5 mg/mL; Beyotime, China) was added to each well, followed by incubation for 4 h. The resulting formazan crystals were dissolved in 100 \u0026micro;L of DMSO, and the absorbance was measured at 490 nm to evaluate cell viability.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eRabbit erythrocyte hemolysis assay\u003c/h2\u003e\n \u003cp\u003eTo evaluate the hemolytic safety of the compound, fresh rabbit blood (containing anticoagulant) was collected and centrifuged at 3000 rpm to remove the plasma. The erythrocytes were washed three times with sterile PBS (pH 7.4), the supernatant was discarded, and the cells were finally resuspended in PBS to obtain a 2.5% red blood cell suspension. For the assay, 100 \u0026micro;L of the RBC suspension was mixed with an equal volume of solutions containing different concentrations of daphnoretin (0\u0026ndash;64 \u0026micro;g/mL) and incubated at 37\u0026deg;C for 1 h. The negative control consisted of RBCs in PBS without drug treatment, whereas the positive control consisted of RBCs treated with 1% Triton X-100. After incubation, all the samples were centrifuged at 3000 rpm for 10 min, and the supernatants were collected. The absorbance of the supernatants was measured at 543 nm via a microplate reader.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of daphnoretin on\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003eadhesion\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe 96\u0026ndash;well plates were coated with bovine fibrinogen (20 \u0026micro;g/mL; Source Leaf, China) and incubated overnight at 4\u0026deg;C. After washing with phosphate-buffered saline (PBS), the wells were blocked with 0.5% bovine serum albumin (BSA; Sigma, USA) for 2 h. \u003cem\u003eS. aureus\u003c/em\u003e was cultured with different concentrations of daphnoretin (32\u0026ndash;64 \u0026micro;g/mL) until reaching the logarithmic growth phase and then inoculated into the coated plates, followed by incubation at 37\u0026deg;C for 2 h. After incubation, the wells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min. Excess dye was removed by washing, and absorbance was measured at 570 nm using a microplate reader.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFITC-IgG\u0026ndash;based detection of\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003esurface protein A\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e was cultured with shaking until the OD₆₀₀ reached 0.3, after which different concentrations of daphnoretin (32\u0026ndash;64 \u0026micro;g/mL) were added, and incubation was continued until the OD₆₀₀ reached 1.0. The bacterial cells were collected by centrifugation (5,000 rpm, 5 min), washed twice with PBS, and blocked with 0.5% bovine serum albumin (BSA) for 20 min. The cells were then fixed with 4% paraformaldehyde for 30 min and incubated in the dark at 37\u0026deg;C for 2 h with a FITC-labeled goat anti-rabbit IgG antibody (Source Leaf, China) diluted 1:200. After washing with PBS, the bacteria were resuspended in PBS. One hundred microliters of resuspended bacteria was added to a black 96-well plate. The fluorescence intensity was then measured using a microplate reader (Thermo Fisher, USA) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The \u0026Delta;\u003cem\u003esrtA\u003c/em\u003e strain was used as a negative control.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDaphnoretin protects ATDC5 cells\u003c/h3\u003e\n\u003cp\u003eAfter being resuscitated, ATDC5 cells were seeded at a density of 2 \u0026times; 10⁴ cells per well in 24-well plates containing DMEM supplemented with 10% FBS and cultured overnight. The culture medium was then discarded, and the cells were washed with PBS. \u003cem\u003eS. aureus\u003c/em\u003e was subsequently suspended in fresh RPMI 1640 medium containing 10% serum, and the cells were infected with 500 \u0026micro;L of the bacterial suspension at a multiplicity of infection (MOI) of 50.(Chang et al., 2024) At the same time, different concentrations of daphnoretin (32 and 64 \u0026micro;g/mL) were added and incubated for 6 h. Cell viability was assessed using a calcein-AM/PI assay kit, and images were acquired with a cell imaging workstation to distinguish live and dead cells. Untreated cells served as the blank control, and the ratio of live to dead cells was quantitatively analyzed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003einvasion into ATDC5 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e was cultured in the presence of daphnoretin (32\u0026ndash;64 \u0026micro;g/mL) at 37\u0026deg;C with shaking at 220 rpm until the OD₆₀₀ reached 1.0. The bacteria were then fluorescently labeled with 5(6)-FAM dye, collected by centrifugation, and resuspended in an equal volume of high-glucose DMEM. ATDC5 cells were routinely maintained in high-glucose DMEM (Servicebio, China) and seeded into 24-well plates at a density of 1 \u0026times; 10⁵ cells/well. After 36 h of incubation, the cells were infected with \u003cem\u003eS. aureus\u003c/em\u003e at a multiplicity of infection (MOI) of 5 and incubated for 1 h(Xu et al., 2025). After treatment, extracellular viable bacteria were removed by applying gentamicin (50 \u0026micro;g/mL, prepared in PBS), followed by two washes with PBS. The cytoskeleton and nuclei were then stained sequentially with Acti-stain 555 phalloidin and DAPI, respectively, and fluorescence images were acquired using a cell imaging workstation.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation assay\u003c/h3\u003e\n\u003cp\u003eOvernight-cultured \u003cem\u003eS. aureus\u003c/em\u003e was diluted 1:100 in BHI medium and inoculated into 96-well plates precoated with 20% freeze-dried rabbit plasma at 4\u0026deg;C overnight (100 \u0026micro;L per well). After treatment with different concentrations of daphnoretin (0\u0026ndash;128 \u0026micro;g/mL) and incubation at 37\u0026deg;C for 24 h, the wells were washed with sterile PBS and stained with 50 \u0026micro;L of crystal violet solution for 15 min. Excess dye was discarded, and the wells were thoroughly rinsed, after which images of the biofilms were captured, and the minimum biofilm inhibitory concentration (MBIC) was determined on the basis of visual inspection. The bound dye was then eluted with 33% glacial acetic acid, and 100 \u0026micro;L of the eluate was transferred to a new 96-well plate. The absorbance was measured at 570 nm to quantify the biofilm biomass.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the effects of daphnoretin on different stages of biofilm development, 96-well plates precoated with rabbit plasma were inoculated with \u003cem\u003eS. aureus\u003c/em\u003e, and daphnoretin was added at different time points (0\u0026ndash;24 h) to a final concentration of 64 \u0026micro;g/mL. After 24 h of coincubation, the biofilms were stained with crystal violet, and the absorbance was measured at OD\u003csub\u003e570\u003c/sub\u003e nm.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBiofilm extracellular matrix component analysis\u003c/h2\u003e\n \u003cp\u003eTo systematically evaluate the impact of daphnoretin on the extracellular matrix composition of \u003cem\u003eS. aureus\u003c/em\u003e biofilms, three major components\u0026mdash;extracellular polysaccharides, proteins, and extracellular DNA (eDNA)\u0026mdash;were analyzed. Briefly, biofilms were cultured in BHI medium supplemented with various concentrations of daphnoretin for 24 h. Extracellular polysaccharides were quantified via the phenol\u0026ndash;sulfuric acid method, and the absorbance was measured at 490 nm via a microplate reader. For protein analysis, biofilms treated under the same conditions were harvested, washed with PBS, and subjected to isotonic buffer extraction followed by cold acetone precipitation; protein concentrations were subsequently determined via a BCA assay, with the absorbance measured at 595 nm. For eDNA quantification, biofilms were cultured as described above, digested with proteinase K, precipitated with ethanol, and analyzed via a Nanodrop spectrophotometer, with validation performed via agarose gel electrophoresis. Collectively, these assays provide a comprehensive assessment of the modulatory effects of daphnoretin on the biofilm extracellular matrix.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eConfocal laser scanning microscopy (CLSM) analysis of biofilms\u003c/h2\u003e\n \u003cp\u003eIn accordance with previous studies with modifications (Walker and Horswill, 2012), overnight-resuscitated \u003cem\u003eS. aureus\u003c/em\u003e was inoculated into BHI media at a 1:100 dilution. Glass coverslips were placed into six-well plates and precoated with freeze-dried rabbit plasma at 4\u0026deg;C overnight. A 500 \u0026micro;L bacterial suspension containing different concentrations of daphnoretin was then added to each well and incubated at 37\u0026deg;C for 24 h to allow mature biofilm formation. After incubation, the supernatant was removed, and the biofilms on the coverslips were washed with PBS (pH 7.4). The samples were stained with SYTO-9 (Foryou Biotech, China) for 30 min at room temperature, and three-dimensional fluorescence images were subsequently captured via a confocal laser scanning microscope (CLSM; Leica TCS DMI8, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eScanning electron microscopy (SEM) analysis of biofilms\u003c/h2\u003e\n \u003cp\u003eThe pretreatment of the coverslips for SEM observation was the same as that for CLSM. After coincubation of the coverslips with \u003cem\u003eS. aureus\u003c/em\u003e and daphnoretin, the samples were gently washed with sterile PBS to remove nonadherent cells. The samples were then fixed in 4% glutaraldehyde solution at 4\u0026deg;C overnight. After fixation, the samples were washed three times with PBS and dehydrated sequentially with graded ethanol solutions (35%, 50%, and 70%). Following dehydration, the samples were dried via a liquid CO₂ critical point dryer. Finally, the samples were sputter-coated with platinum and imaged via a scanning electron microscope (HITACHI Regulus 8100, Japan) to observe the biofilm structures(Kong et al., 2018).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eBacterial sliding motility assay\u003c/h2\u003e\n \u003cp\u003eTo evaluate the sliding motility of \u003cem\u003eS. aureus\u003c/em\u003e, bacteria were inoculated onto plates containing tryptic soy broth (TSB) supplemented with 1% agar to assess their spreading ability. The experimental procedure was performed according to previously reported methods with minor modifications(Shen et al., 2020). Briefly, a standardized suspension of \u003cem\u003eS. aureus\u003c/em\u003e (1 \u0026times; 10⁸ CFU/mL, 15 \u0026micro;L) was spot inoculated onto the center of the plates, with treatment groups containing daphnoretin (16\u0026ndash;128 \u0026micro;g/mL) or without drug as controls. After inoculation, the plates were air-dried at room temperature for 20 min and then incubated at 37\u0026deg;C for 24 h. The spreading zones were observed to evaluate sliding motility.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eWestern blot analysis\u003c/h2\u003e\n \u003cp\u003eAfter resuspension, \u003cem\u003eS. aureus\u003c/em\u003e was cultured to an OD₆₀₀ of 0.3, and different concentrations of daphnoretin were added. The cultures were then incubated at 37\u0026deg;C with shaking until they reached an OD₆₀₀ of 2.5 (late logarithmic phase). The bacterial cells were harvested by centrifugation and lysed with lysozyme and lysostaphin by incubation on ice for 30 min. The total protein concentration was determined via a BCA protein assay kit (Beyotime, China). Equal amounts of protein (20 \u0026micro;g) were separated by SDS‒PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk at 80 rpm/min for 2 h, followed by overnight incubation at 4\u0026deg;C with polyclonal anti-SrtA serum (1:200, prepared in our laboratory). After being washed with TBS-T, the membranes were incubated with an HRP-conjugated goat anti-rabbit secondary antibody (1:10,000; Beyotime, China) for 2 h at room temperature. The protein bands were visualized via an enhanced chemiluminescence (ECL) Plus substrate (Beyotime, China). The \u0026Delta;\u003cem\u003esrtA\u003c/em\u003e strain was used as a negative control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eMolecular docking\u003c/h2\u003e\n \u003cp\u003eMolecular docking of daphnoretin with SrtA was performed via AutoDock Vina 1.1.2. The three-dimensional structure of SrtA (PDB ID: 1T2P) was obtained from the Protein Data Bank (www.rcsb.org). The 3D structure of daphnoretin was retrieved from the PubChem database (PubChem CID: 5281406) and further generated via ChemBioDraw Ultra 14.0. The preparation of the ligand and receptor files was carried out via AutoDockTools 1.5.6. During docking, nonpolar hydrogen atoms were merged, and rotatable bonds of the ligand were defined. The docking grid was centered on the catalytic cysteine with coordinates center_x = \u0026minus;\u0026thinsp;30.329, center_y = \u0026minus;\u0026thinsp;19.713, center_z\u0026thinsp;=\u0026thinsp;1.688, dimensions size_x\u0026thinsp;=\u0026thinsp;43.5, size_y\u0026thinsp;=\u0026thinsp;40.5, and size_z\u0026thinsp;=\u0026thinsp;46.5. All other parameters were set to their defaults. The best docking conformation obtained was used to generate the ligand\u0026ndash;protein complex to predict the binding mode of daphnoretin with SrtA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eFluorescence quenching\u003c/h2\u003e\n \u003cp\u003eFluorescence quenching experiments were conducted to determine the binding constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) of daphnoretin with SrtA and its mutant proteins. Purified SrtA (4 \u0026micro;M) was mixed with different concentrations of daphnoretin in PBS. Fluorescence emission spectra were recorded in the range of 280\u0026ndash;400 nm with an excitation wavelength of 260 nm via a microplate reader (Thermo Fisher Scientific, China), and the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e values were calculated via the Stern\u0026ndash;Volmer equation. (Papadopoulou et al., 2005).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eG. mellonella\u003c/strong\u003e \u003cstrong\u003einfection model\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo assess the efficacy of daphnoretin in the \u003cem\u003eG. mellonella\u003c/em\u003e infection model, larvae (220\u0026ndash;260 mg) were divided into five groups: the MRSA-infected group, the untreated control group, the daphnoretin-treated groups (25 and 50 mg/kg), and the vancomycin-treated group (50 mg/kg), with 10 larvae in each group. A 10 \u0026micro;L suspension of MRSA (5 \u0026times; 10⁶ CFU/mL) was injected into the last left proleg of the larvae, and treatment was initiated 1 h post infection. The control group received PBS containing 0.1% dimethyl sulfoxide (DMSO). Larvae were maintained at a constant temperature of 37\u0026deg;C, and survival rates and health scores were monitored every 12 h for a total of 120 h(Guo et al., 2024). For bacterial burden assessment, a method similar to that used in the survival assay was employed. At 48 h post infection, the larvae were collected, surface sterilized, homogenized, and plated on TSB agar plates, followed by incubation at 37\u0026deg;C for 24 h to evaluate the bacterial loads within the larvae.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eRat septic arthritis model\u003c/h2\u003e\n \u003cp\u003eTo evaluate the therapeutic effect of daphnoretin in a septic arthritis model, thirty 6\u0026ndash;8-week-old Sprague\u0026ndash;Dawley (SD) rats were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;6 per group). The animals were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg), and the skin around the knee joint was disinfected with povidone-iodine. \u003cem\u003eS. aureus\u003c/em\u003e (4 \u0026times; 10⁶ CFU/10 \u0026micro;L) was then injected into the joint cavity beneath the patella using a Hamilton syringe. The treatment groups received daily subcutaneous injections of daphnoretin (25 or 50 mg/kg) or vancomycin (50 mg/kg) at the nape of the neck for six consecutive days, whereas the control group was administered an equal volume of PBS containing 0.1% DMSO. On day 7, the animals were sacrificed, and relevant parameters were evaluated.\u003c/p\u003e\n \u003cp\u003eThe severity of arthritis was monitored using multiple approaches. First, on day 7, knee joint diameters were measured with a digital caliper to assess swelling. Second, to track the infection process in real time, bioluminescent \u003cem\u003eS. aureus\u003c/em\u003e Xen29 was injected using the same method described above, and bioluminescent signals were recorded daily for 7 consecutive days. At the end of the experiment, knee joint tissues were fixed and subjected to micro-CT scanning of the distal femur and proximal tibia, followed by three-dimensional reconstruction to analyze cartilage surface damage and changes in subchondral bone density. The bone volume fraction (BV/TV) and bone mineral density (BMD) of the femoral and tibial condyles were further calculated using Cruiser CT software to quantify bone destruction. All the rats were housed in an SPF-grade animal facility under controlled conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, relative humidity 50\u0026ndash;70%, 12 h light/dark cycle) with free access to sterilized food and water.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eDaphnoretin inhibits SrtA activity\u003c/h2\u003e\u003cp\u003eScreening for SrtA inhibitors typically relies on an in vitro fluorescence resonance energy transfer (FRET)-based assay. This method employs a fluorescent substrate peptide containing the LPXTG motif, with donor and acceptor fluorophores labeled at both termini. When the substrate is cleaved by SrtA, the fluorophores are separated, resulting in an enhanced fluorescence signal that enables real-time monitoring of enzymatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using this system, several candidate compounds preserved in our laboratory were screened (Supplementary Table\u0026nbsp;1), and daphnoretin exhibited the most pronounced inhibitory effect on SrtA, with an inhibition rate as high as 78.34% at a concentration of 64 \u0026micro;g/mL; therefore, it was selected for further investigation. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequent concentration‒gradient assays revealed that daphnoretin significantly inhibited SrtA activity in a dose-dependent manner, with enzymatic activity progressively decreasing as the drug concentration increased. Nonlinear regression analysis revealed that the half-maximal inhibitory concentration (IC₅₀) was 8.501 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that daphnoretin is a promising SrtA inhibitor. Notably, although daphnoretin inhibited the enzymatic activity of SrtA, it had no significant effect on the growth of \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and the minimum inhibitory concentration (MIC) against this strain was \u0026gt;\u0026thinsp;256 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), far exceeding the therapeutic concentration. These findings suggest that daphnoretin does not inhibit bacterial growth and thus does not impose selective pressure on the strain, thereby preventing the emergence of resistant mutants. This further underscores its potential advantage as an antivirulence candidate compound.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eSafety assessment of daphnoretin\u003c/h2\u003e\u003cp\u003eTo evaluate the safety of daphnoretin at therapeutically relevant doses, we first performed an MTT assay on ATDC5 cells. Compared with the control treatment, treatment with 64 \u0026micro;g/mL daphnoretin did not significantly affect cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). A rabbit red blood cell hemolysis assay was subsequently conducted to further examine the potential in vivo toxicity of daphnoretin, and no hemolytic response was observed even at concentrations higher than the IC\u003csub\u003e50\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting that daphnoretin has extremely low cytotoxicity. In addition, an in vivo toxicity evaluation was performed using the \u003cem\u003eG. mellonella\u003c/em\u003e larval model, in which daphnoretin (25 and 50 mg/kg) was injected into the penultimate left proleg, and larval survival and melanization were monitored for five consecutive days post-treatment. The results demonstrated that neither dose induced melanization nor mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Collectively, these findings indicate that daphnoretin is safe at both the cellular level and in the \u003cem\u003eG. mellonella\u003c/em\u003e model, providing important evidence for its pharmacological safety assessment and subsequent application studies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDaphnoretin reduces\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003eadhesion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe strong adhesive capacity of \u003cem\u003eS. aureus\u003c/em\u003e is a critical step for colonization, host invasion, and subsequent pathogenesis(Berry et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this process, SrtA catalyzes the anchoring of cell wall\u0026ndash;associated proteins, thereby mediating bacterial attachment to host tissues, and is considered an indispensable enzyme in pathogenesis. To evaluate the effect of daphnoretin on bacterial adhesion, a fibrinogen-binding assay was performed. The results revealed that the adhesion capacity decreased significantly in the daphnoretin-treated group in a dose-dependent manner, with the adhesion rate of the Δ\u003cem\u003eSrtA\u003c/em\u003e strain reducing to only 38.87\u0026thinsp;\u0026plusmn;\u0026thinsp;6.09% of that of the wild-type (WT) strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These findings suggest that daphnoretin effectively inhibits the binding of \u003cem\u003eS. aureus\u003c/em\u003e to fibrinogen, thereby attenuating its ability to invade the host.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eDaphnoretin inhibits SpA-mediated immune evasion\u003c/h2\u003e\u003cp\u003eSrtA plays a critical role in the immune evasion of \u003cem\u003eS. aureus\u003c/em\u003e by mediating the anchoring of surface protein A (SpA) to the cell wall. SpA, a typical substrate of SrtA, binds to the Fc fragment of host IgG, thereby blocking antibody-dependent phagocytosis, helping bacteria evade immune clearance, and enabling persistent survival within the host(Falugi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, the anchoring of SpA is considered one of the key mechanisms by which \u003cem\u003eS. aureus\u003c/em\u003e achieves immune evasion and maintains infection. In this study, we employed a microplate reader to measure fluorescence intensity to assess the effect of daphnoretin on SpA anchoring. The results revealed that with increasing concentrations of daphnoretin, the FITC-labeled goat anti-rabbit IgG fluorescence signal progressively decreased, and the Δ\u003cem\u003eSrtA\u003c/em\u003e group accounted for only 33.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29% of the WT group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings indicate that daphnoretin reduces the anchoring of SpA on the bacterial cell wall by inhibiting the catalytic activity of SrtA, thereby weakening SpA-dependent immune evasion. This observation further supports the potential role of daphnoretin as a SrtA inhibitor in blocking the immune evasion mechanisms of \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDaphnoretin suppresses\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003einternalization and improves ATDC5 cell viability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the protective effect of daphnoretin against \u003cem\u003eS. aureus\u003c/em\u003e infection, calcein-AM/PI double staining was performed on ATDC5 cells. Fluorescence microscopy revealed that with increasing concentrations of daphnoretin, the green fluorescence signals (viable cells) progressively increased in a dose-dependent manner, whereas the red fluorescence signals (dead cells) markedly decreased, indicating effective attenuation of \u003cem\u003eS. aureus\u003c/em\u003e-induced cytotoxicity. Among the groups, the Δ\u003cem\u003eSrtA\u003c/em\u003e strain presented the highest cell survival rate, reaching 91.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;D). Further invasion assays demonstrated that daphnoretin significantly reduced the intracellular bacterial burden. Consistently, fluorescence imaging revealed a progressive decrease in the number of intracellular red fluorescence signals with increasing drug concentration, whereas the overall cell morphology remained intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;F). Collectively, these findings indicate that daphnoretin protects host cells by alleviating cytotoxic damage and markedly inhibits MRSA invasion and colonization, thereby supporting its potential as a promising antivirulence candidate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDaphnoretin inhibits\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003ebiofilm formation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBiofilm formation and development are progressive and highly dynamic processes. Under favorable environmental conditions, \u003cem\u003eS. aureus\u003c/em\u003e initially establishes colonization through surface adhesion, followed by aggregation into early biofilms that continuously expand and mature with the support of extracellular matrix components(Peng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As the biofilm structure becomes more complete, bacterial resistance to antibiotics and host immunity are markedly enhanced; eventually, some bacteria detach from the mature biofilm and disseminate to new sites, leading to persistent infection and recurrence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To evaluate the effect of daphnoretin on this process, biofilm formation was quantified via crystal violet staining. The results revealed a dose-dependent reduction in biofilm biomass upon treatment, and the minimum biofilm inhibitory concentration (MBIC) was 16 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that daphnoretin significantly inhibits biofilm development. Stage-specific intervention further revealed that the administration of 64 \u0026micro;g/mL daphnoretin had a major inhibitory effect during the early stage of biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating interference with initial adhesion and early matrix establishment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the mechanism involved, the major biofilm matrix components were examined. Daphnoretin treatment resulted in a significant decrease in the levels of extracellular DNA (eDNA), polysaccharide intercellular adhesin (PIA), and extracellular proteins. Notably, PIAs and extracellular proteins were significantly reduced at 32 \u0026micro;g/mL, whereas eDNA was markedly decreased at 16 \u0026micro;g/mL. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;G). eDNA functions as a structural scaffold facilitating intercellular adhesion and genetic exchange; PIAs play a key role in cell aggregation and biofilm maturation, while extracellular proteins not only support the matrix structure but also include adhesion- and immune-regulation\u0026ndash;related functional proteins. By simultaneously reducing these three critical components, daphnoretin impaired the structural stability and cell aggregation capacity of biofilms, thereby weakening adhesion and immune evasion and ultimately attenuating the pathogenic potential of \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eMorphological analyses further supported these findings. Confocal laser scanning microscopy (CLSM) revealed marked alterations in biofilm architecture following daphnoretin treatment: as the drug concentration increased, the biofilms shifted from dense and compact to sparse and porous (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), indicating disruption of spatial organization and stability. Scanning electron microscopy (SEM) at the ultrastructural level confirmed this trend: while the control biofilms presented compact, homogeneous structures characteristic of mature biofilms, the treated groups formed only loose, irregular thin layers with markedly reduced intercellular cohesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Together, these data demonstrate that daphnoretin not only inhibits biofilm formation but also disrupts the three-dimensional structural integrity and stability of biofilms, thereby compromising their protective barrier function.\u003c/p\u003e\u003cp\u003eIn addition, sliding motility assays were performed to assess the surface spreading ability of \u003cem\u003eS. aureus\u003c/em\u003e. Sliding motility, an important means of expansion for nonflagellated bacteria, is closely associated with early adhesion, population dispersal, and subsequent biofilm establishment. The results revealed that with increasing concentrations of daphnoretin, the spreading area of bacterial colonies on agar plates gradually decreased. At a concentration of 128 \u0026micro;g/mL, the spreading area was only 42.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09% that of the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u0026ndash;K), indicating significant inhibition of bacterial motility. These findings suggest that daphnoretin interferes with bacterial sliding motility, thereby reducing early-stage colonization and dissemination and ultimately blocking key steps of biofilm establishment. This observation, which is consistent with the results of the crystal violet staining and morphological analyses, further highlights the potential of daphnoretin as an antivirulence candidate drug.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eDaphnoretin interacts with SrtA\u003c/h2\u003e\u003cp\u003eTo elucidate the interaction mechanism between daphnoretin and SrtA, a combination of Western blot, fluorescence quenching, and molecular docking analyses was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared with no treatment, treatment with daphnoretin at concentrations of 0\u0026ndash;64 \u0026micro;g/mL did not alter the band intensity of SrtA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that daphnoretin does not regulate SrtA expression but more likely modulates its catalytic function through direct binding. Fluorescence quenching experiments were subsequently conducted to characterize the direct drug\u0026ndash;protein interactions. Fluorescence spectra were recorded with an excitation wavelength set at 260 nm, and emission was monitored within the range of 280\u0026ndash;400 nm. A concentration-dependent decrease in fluorescence intensity was observed with increasing daphnoretin concentration. Stern\u0026ndash;Volmer analysis of F₀/F versus ligand concentration yielded a binding constant of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e = 1.6383 \u0026times; 10⁴ L\u0026middot;mol⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating a specific interaction between the two molecules.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMolecular docking further revealed that daphnoretin primarily localized to a recognition pocket adjacent to the catalytic groove of SrtA, where it formed key interactions with Asn114 (N114), Val168 (V168), Lys175 (K175), and Thr180 (T180). The calculated binding free energy of the complex was ΔG_bind\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;8.5 kcal\u0026middot;mol⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), which is consistent with a moderate-to-strong affinity typical of small molecule\u0026ndash;protein interactions. To validate the predicted binding sites, site-directed mutants of SrtA (N114A, V168A, T180A, and K175A) were generated, and their affinities for daphnoretin were compared with those of the wild type (WT) using fluorescence quenching. The results demonstrated that the binding constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) of all the mutants were significantly lower than those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;I), indicating that these residues are crucial for ligand recognition and anchoring.\u003c/p\u003e\u003cp\u003eCollectively, these findings consistently support that daphnoretin inhibits SrtA activity by directly binding to key residues adjacent to the catalytic groove rather than through the regulation of protein expression, thereby elucidating the molecular basis of its inhibitory mechanism.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTherapeutic efficacy of daphnoretin in the\u003c/b\u003e \u003cb\u003eG. mellonella\u003c/b\u003e \u003cb\u003emodel\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe immune system of \u003cem\u003eG. mellonella\u003c/em\u003e shows a high degree of similarity to the innate immune response of vertebrates at both the cellular and molecular levels and is therefore widely used as a reliable model for evaluating anti-infective therapies(Serrano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the basis of this rationale, a \u003cem\u003eG. mellonella\u003c/em\u003e infection model was established to evaluate the therapeutic effect of daphnoretin on \u003cem\u003eS. aureus\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). During the experimental period, larval activity, cocoon formation, degree of melanization, and survival were continuously monitored to assess health status comprehensively. The results revealed that the health scores of the daphnoretin-treated larvae improved progressively with increasing drug concentration. By day 5, the health conditions of the high-dose group were second only to those of the vancomycin-treated group and were significantly better than those of the untreated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;C), indicating that daphnoretin effectively alleviated infection-associated symptoms. Survival analysis further confirmed this finding, with survival rates in the high-dose daphnoretin group reaching 80% on day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E). In addition, sterile homogenization and colony enumeration performed at 48 h post infection revealed that the bacterial burden in larvae treated with daphnoretin was significantly lower than that in the WT group. With increasing drug concentrations, the bacterial load in the treatment groups decreased correspondingly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, the results from health scoring, survival rate, and bacterial burden consistently demonstrate that daphnoretin exhibited significant anti-infective efficacy in the \u003cem\u003eG. mellonella\u003c/em\u003e model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDaphnoretin alleviates septic arthritis in rats\u003c/b\u003eTo evaluate the anti-infective and joint-protective effects of daphnoretin in vivo, a rat model of septic arthritis was established via \u003cem\u003eS. aureus\u003c/em\u003e inoculation, and therapeutic efficacy was assessed via randomized grouping (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Bioluminescence imaging revealed that during the early phase of infection (days 1\u0026ndash;3), the luminescence intensity increased over time and peaked on day 3. From day 4 onward, the luminescence intensity began to decrease, and by day 7, the intensities in the daphnoretin- and vancomycin-treated groups were significantly lower than those in the untreated wild-type infection group, indicating that the bacterial burden and inflammatory activity at the lesion sites were suppressed. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;C). Similarly, the knee joint diameter of the rats in the treatment groups was significantly greater than that of the WT group, with the high-dose daphnoretin group showing a diameter of 13.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mm, indicating that acute joint swelling and inflammatory signs were alleviated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the structural level, micro-CT was employed to perform three-dimensional reconstruction and quantitative analysis of the affected joints. In the untreated WT group, irregular cartilage surfaces and discontinuities in cortical and trabecular bone were observed. In contrast, compared with infection, daphnoretin treatment markedly improved bone parameters, with higher BV/TV and BMD values Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;G). Both imaging (BLI, micro-CT) and clinical endpoints (joint diameter) exhibited consistent improvement trends, collectively supporting the in vivo anti-infective and tissue-protective activities of daphnoretin.\u003c/p\u003e\u003cp\u003eOverall, daphnoretin reduced bioluminescence intensity, mitigated joint swelling and inflammatory signs, and improved BV/TV and BMD in a rat model of septic arthritis, highlighting its strong potential as an antivirulence candidate drug for treating joint infections.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo address the problem of antibiotic resistance, this study employed a strategy distinct from traditional antibiotics that act through direct bacteriostasis, namely, by attenuating the virulence of \u003cem\u003eS. aureus\u003c/em\u003e through the inhibition of the key virulence factor SrtA. Previous studies have demonstrated that the deletion or functional inhibition of SrtA significantly reduces bacterial pathogenicity without affecting basic growth(Mazmanian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Jonsson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In this study, through in vitro screening and multidimensional validation, we identified and confirmed that daphnoretin is a potent inhibitor of SrtA. Notably, daphnoretin markedly attenuated the adhesion, invasion, immune evasion, and biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e at concentrations far below its antibacterial level without imposing significant pressure on bacterial growth. More importantly, in \u003cem\u003eS. aureus\u003c/em\u003e infection models, daphnoretin exhibited pronounced antivirulence activity. These findings not only further validate the feasibility of targeting SrtA as an antivirulence therapeutic strategy(Cegelski et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) but also provide new insights into the application of natural products in the management of complex infections.\u003c/p\u003e\u003cp\u003eOur findings further demonstrate that daphnoretin significantly attenuates critical pathogenic steps of \u003cem\u003eS. aureus\u003c/em\u003e within the host by inhibiting the SrtA-mediated anchoring of surface proteins. We observed that daphnoretin treatment markedly reduced bacterial binding to fibrinogen and significantly diminished its ability to invade chondrocytes. Clumping factors (ClfA/ClfB) play important roles in the early adhesion and colonization of bacteria(D et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), whereas protein A (SpA) enhances immune evasion by binding to the Fc fragment of IgG, thereby blocking complement activation and phagocytosis(Falugi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). By inhibiting the anchoring of these surface proteins to the cell wall, daphnoretin effectively reduces the adhesive and immune evasion capabilities of bacteria. Our experimental results, for the first time at the level of natural products, validated the feasibility of this strategy and provided solid evidence for the further development of antivirulence therapeutics.\u003c/p\u003e\u003cp\u003eIntervention in biofilm formation plays a critical role in combating \u003cem\u003eS. aureus\u003c/em\u003e infections. Our results demonstrated that biofilms formed after daphnoretin treatment presented a sparse and porous structure, with significantly reduced levels of extracellular matrix components, including polysaccharides, extracellular DNA, and proteins. These findings suggest that daphnoretin effectively blocks the early stages of biofilm formation, thereby inhibiting bacterial colonization and community stabilization. Prosthetic joint infections and chronic osteoarthritis are often closely associated with persistent or recurrent infections linked to biofilms(Otto, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Archer et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This mechanism has important clinical value. Blocking biofilm formation not only reduces the long-term survival of bacteria in the joint environment but also may enhance host immune defenses and antibiotic penetration, thereby lowering the risk of refractory infections. At the molecular level, the molecular docking results indicated that daphnoretin binds to the active pocket of SrtA and forms stable interactions with key residues, including Lys175, Thr180, Val168, and Asn114.\u003c/p\u003e\u003cp\u003eMore importantly, this study confirmed the protective effect of daphnoretin in both \u003cem\u003eG. mellonella\u003c/em\u003e and rat in vivo models. In the \u003cem\u003eG. mellonella\u003c/em\u003e infection model, drug treatment significantly improved host survival without causing melanization or behavioral abnormalities, indicating favorable biosafety. This finding is consistent with previous studies in which \u003cem\u003eG. mellonella\u003c/em\u003e has been widely used to evaluate the in vivo toxicity and efficacy of antibacterial candidates(Desbois and Coote, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Tsai et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In a rat model of septic arthritis, animals treated with daphnoretin presented reduced joint swelling, decreased bacterial burden in tissues, and markedly improved cartilage integrity, which is consistent with previous reports that antivirulence agents can mitigate the pathogenic processes of joint infection. These \u003cem\u003ein vivo\u003c/em\u003e findings are highly consistent with our in vitro mechanistic results, indicating that the antivirulence activity of daphnoretin also occurs in complex host environments. Notably, whether antivirulence agents can retain their activity in vivo has long been considered one of the major obstacles to clinical translation(Clatworthy et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and our findings provide positive evidence addressing this issue. This study also systematically evaluated the safety profile of daphnoretin. The results showed that the compound did not induce hemolysis of rabbit erythrocytes at therapeutically relevant concentrations and did not significantly impair the viability of ATDC5 chondrocytes; moreover, no obvious signs of toxicity were observed in the \u003cem\u003eG. mellonella\u003c/em\u003e model. These findings are consistent with previous reports that natural bicoumarin compounds generally possess high tolerability and low toxicity. As drug safety is one of the core considerations for the clinical translation of novel anti-infective agents (DiMasi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the favorable safety profile of daphnoretin not only reinforces its feasibility as a lead compound but also provides important assurance for its further preclinical development.\u003c/p\u003e\u003cp\u003eFrom a clinical perspective, daphnoretin has multiple advantages as a candidate antivirulence agent. First, it does not directly inhibit bacterial growth, thereby markedly reducing the risk of resistance development. Second, it targets multiple steps of pathogenesis, including adhesion, immune evasion, and biofilm formation, resulting in an overall attenuation of virulence. Third, as a natural product, it is readily accessible and amenable to structural modification, offering potential for further optimization. These features suggest that daphnoretin could not only be used prophylactically in high-risk populations, such as patients undergoing joint replacement but also serve as an adjuvant to antibiotics, improving treatment success rates and reducing recurrence. Previous studies have indicated that the combination of antivirulence agents with conventional antibiotics can increase efficacy and reduce resistance pressure(Rasko and Sperandio, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Cegelski et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, future research on daphnoretin may also focus on its synergistic effects with existing antimicrobial agents.\u003c/p\u003e\u003cp\u003eAlthough this study systematically revealed the antivirulence activity of daphnoretin as an SrtA inhibitor, several limitations remain. First, molecular docking only provides theoretical support, and structural evidence, such as crystallography or cryo-electron microscopy, is still lacking to precisely define the binding sites and conformational stability (Zong et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Suree et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Second, the in vivo investigations were limited mainly to \u003cem\u003eG. mellonella\u003c/em\u003e and rat models, which, despite demonstrating favorable protective effects and safety, did not address pharmacokinetics, tissue distribution, or long-term toxicity in a systematic manner. In addition, as a natural product, daphnoretin may suffer from limited affinity and bioavailability, necessitating structural modification and medicinal chemistry optimization to enhance its efficacy and pharmacokinetic properties(Newman and Cragg, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, future studies should integrate structural analyses, drug optimization, and validation in higher-order animal models to advance its clinical translation.\u003c/p\u003e\u003cp\u003eIn summary, this study systematically evaluated the antivirulence activity of daphnoretin as an SrtA inhibitor and demonstrated its ability to inhibit adhesion, immune evasion, and biofilm formation in vitro, as well as its protective efficacy and safety in \u003cem\u003eG. mellonella\u003c/em\u003e and rat septic arthritis models. Molecular docking analysis further elucidated its binding mechanism, providing a theoretical basis for subsequent drug optimization. These findings provide important evidence supporting the development of natural products as antivirulence agents and offer new directions for combating infections caused by drug-resistant \u003cem\u003eS. aureus\u003c/em\u003e. With further progress in structural validation, pharmacokinetic studies, and preclinical evaluations, daphnoretin and its derivatives hold promise as important components for the future treatment of septic arthritis and related infections.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 10 software. Experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), with each experiment conducted in at least three independent biological replicates. Differences between two groups were analyzed using two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests, and multiple group comparisons were performed using one-way or two-way analysis of variance (ANOVA). A \u003cem\u003eP\u003c/em\u003e value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Jilin Provincial Science and Technology Development Plan (Grant No. YDZJ202501ZYTS177).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYun Sun and Tong Zhao: Writing and original draft preparation;Dongbin Guo, Peitong Jiang,Xinyao Liu, Zhangyu Du, and Luanbiao Sun: Data curation and analysis;Yuan Gao and Han Gao: Participation in in vivo and in vitro experiments;Li Wang, Bingmei Wang, and Ming Yan: Study conception and design.All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in this study were conducted in accordance with national guidelines for the care and use of laboratory animals, and the experimental protocol was reviewed and approved by the Experimental Animal Ethics Committee of Changchun University of Chinese Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eARCHER, N. 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J Biol Chem\u003cem\u003e,\u003c/em\u003e 279\u003cstrong\u003e,\u003c/strong\u003e 31383-31389.https://doi.org/10.1074/jbc.M401374200\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"S. aureus, SrtA, Daphnoretin, Ativirulence","lastPublishedDoi":"10.21203/rs.3.rs-7820020/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7820020/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rapid spread of drug-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) has severely undermined the efficacy of existing antibiotics, highlighting the urgent need to explore novel antiinfective strategies to address this global public health challenge. Sortase A (SrtA) is a critical virulence factor of \u003cem\u003eS. aureus\u003c/em\u003e that covalently anchors multiple surface proteins to the bacterial cell wall, thereby mediating adhesion, invasion, immune evasion, and biofilm formation, playing a pivotal role in pathogenesis. In this study, potential inhibitors were screened using a fluorescence resonance energy transfer (FRET) assay, and daphnoretin was identified as a potent SrtA inhibitor with a half-maximal inhibitory concentration (IC₅₀) of 8.501 \u0026micro;g/mL. In vitro experiments demonstrated that daphnoretin effectively suppressed SrtA-mediated pathogenic phenotypes, including bacterial adhesion, invasion, and biofilm formation, thereby attenuating virulence. Further fluorescence quenching and molecular docking analyses revealed the binding mechanism between daphnoretin and SrtA. In vivo, daphnoretin significantly improved host survival in \u003cem\u003eGalleria. mellonella\u003c/em\u003e infection model induced by the MRSA strain and alleviated joint damage and infection symptoms in a rat model of septic arthritis while markedly reducing the bacterial burden in infected tissues. Collectively, this study is the first to elucidate the mechanism of daphnoretin as a SrtA-targeting inhibitor and its anti-infective potential, providing important experimental evidence for the development of novel therapeutic strategies against drug-resistant \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Daphnoretin as a Potent Sortase A Inhibitor Effectively Disarms Drug-Resistant Staphylococcus aureus Infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 19:15:07","doi":"10.21203/rs.3.rs-7820020/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-09T09:27:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-28T21:57:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174267574512851860059217430916931645079","date":"2025-12-15T16:15:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T17:18:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309998819764811038370042316911337544150","date":"2025-10-22T17:59:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T09:52:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T18:09:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T07:00:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-10-09T17:13:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3dfc2ccd-0295-4188-86eb-54bfa1359509","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:00:19+00:00","versionOfRecord":{"articleIdentity":"rs-7820020","link":"https://doi.org/10.1007/s11274-026-04901-5","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2026-03-30 15:57:22","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2025-11-03 19:15:07","video":"","vorDoi":"10.1007/s11274-026-04901-5","vorDoiUrl":"https://doi.org/10.1007/s11274-026-04901-5","workflowStages":[]},"version":"v1","identity":"rs-7820020","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7820020","identity":"rs-7820020","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}