Inhibition of Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin.

Angiogenesis plays an important role in the growth and metastasis of solid tumors including melanoma. Inhibiting tumor-associated angiogenesis is a tactic in treating melanoma. Dioscin restrains angiogenesis in colon tumor and has anti-melanoma effects in cell and animal models. In a previous study, we found that dioscin inhibits Src/STAT3 signaling in melanoma cells. Activation of the Src/STAT3 pathway has been shown to promote tumor angiogenesis. This study aimed to determine whether dioscin's anti-melanoma effects is related to inhibiting Src/STAT3 signaling-mediated angiogenesis. In a B16F10 allograft mouse model, we found that dioscin inhibited melanoma growth and angiogenesis. To exclude the impact of tumor growth on angiogenesis, a chicken chorioallantoic membrane (CAM) model was used to verify the anti-angiogenic effect of dioscin. Results showed that dioscin suppressed vessel formation in CAM. To determine if tumor secreted pro-angiogenic cytokines are involved in the anti-angiogenic effect of dioscin, conditioned media from dioscin-treated A375 melanoma cells were used to culture human umbilical vein endothelial cells (HUVECs), and tube formation was monitored. It was observed that the tube formation of HUVECs was inhibited. Mechanistic studies revealed that dioscin inhibited the activation of Src and STAT3, and lowered mRNA and protein levels of STAT3 transcriptionally-regulated genes, in B16F10 melanomas. ELISA assays showed that dioscin decreased the secretion of MMP-2, MMP-9 and VEGF from A375 cells. Over-activation of STAT3 lessened the effects of dioscin in decreasing the secretion of pro-angiogenic cytokines from melanoma cells, and in inhibiting tube formation of HUVECs cultured with conditioned media from melanoma cell cultures. In summary, we for the first time demonstrated that inhibiting Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin. This study provides further pharmacological groundwork for developing dioscin as an anti-melanoma agent.

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.

Discovery of the Novel Inhibitor Against New Delhi Metallo-ß-Lactamase Based on Virtual Screening and Molecular Modelling.

New Delhi metallo-ß-lactamase (NDM-1), one of the metallo-ß-lactamases (MBLs), leads to antibiotic resistance in clinical treatments due to the strong ability of hydrolysis to almost all kinds of ß-lactam antibiotics. Therefore, there is the urgent need for the research and development of the novel drug-resistant inhibitors targeting NDM-1. In this study, ZINC05683641 was screened as potential NDM-1 inhibitor by virtual screening and the inhibitor mechanism of this compound was explored based on molecular dynamics simulation. The nitrocefin assay showed that the IC50 value of ZINC05683641 was 13.59 ± 0.52 µM, indicating that the hydrolytic activity of NDM-1 can be obviously suppressed by ZINC05683641. Further, the binding mode of ZINC05683641 with NDM-1 was obtained by molecular modeling, binding free energy calculation, mutagenesis assays and fluorescence-quenching assays. As results, ILE-35, MET-67, VAL-73, TRP-93, CYS-208, ASN-220 and HIS-250 played the key roles in the binding of NDM-1 with ZINC05683641. Interestingly, these key residues were exactly located in the catalytic activity region of NDM-1, implying that the inhibitor mechanism of ZINC05683641 against NDM-1 was the competitive inhibition. These findings will provide an available approach to research and develop new drug against NDM-1 and treatment for bacterial resistance.

Quercetin protects rats from catheter-related Staphylococcus aureus infections by inhibiting coagulase activity.

Coagulase (Coa) activity is essential for the virulence of Staphylococcus aureus (S aureus), one of the most important pathogenic bacteria leading to catheter-related bloodstream infections (CRBSI). We have demonstrated that the mutation of coagulase improved outcomes in disease models of S aureus CRBSI, suggesting that targeting Coa may represent a novel antiinfective strategy for CRBSI. Here, we found that quercetin, a natural compound that does not affect S aureus viability, could inhibit Coa activity. Chemical biological analysis revealed that the direct engagement of quercetin with the active site (residues Tyr187, Leu221 and His228) of Coa inhibited its activity. Furthermore, treatment with quercetin reduced the retention of bacteria on catheter surfaces, decreased the bacterial load in the kidneys and alleviated kidney abscesses in vivo. These data suggest that antiinfective therapy targeting Coa with quercetin may represent a novel strategy and provide a new leading compound with which to combat bacterial infections.

Theaflavin-3,3´-digallate increases the antibacterial activity of ß-lactam antibiotics by inhibiting metallo-ß-lactamase activity.

Metallo-ß-lactamases (MBLs) are some of the best known ß-lactamases produced by common Gram-positive and Gram-negative pathogens and are crucial factors in the rise of bacterial resistance against ß-lactam antibiotics. Although many types of ß-lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time-kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand-residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin-3,3´-digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time-kill assays, we observed a synergistic effect of TFDG with ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with ß-lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of ß-lactam antibiotics against pathogens in vitro and in vivo.

Correction to: Phloretin reduces cell injury and inflammation mediated by Staphylococcus aureus via targeting sortase B and the molecular mechanism.

The images of cells under microscope in Figure 2 and Figure S2 were misused from Wang G et al. Front Cell Infect Microbiol. 2018 Nov 30;8:418. These images were generated in the same set of assays.

Amentoflavone Ameliorates Streptococcus suis-Induced Infection In Vitro and In Vivo.

Streptococcus suis, an important zoonotic pathogen, has caused considerable economic losses in the swine industry and severe public health issues worldwide. The development of a novel effective strategy for the prevention and therapy of S. suis is urgently needed. Here, amentoflavone, a natural biflavonoid compound isolated from Chinese herbs that has negligible anti-S. suis activity, was identified as a potent antagonist of suilysin (SLY)-mediated hemolysis without interfering with the expression of SLY. Amentoflavone effectively inhibited SLY oligomerization, which is critical for its pore-forming activity. The treatment with amentoflavone reduced S. suis-induced cytotoxicity in macrophages (J774 cells). Furthermore, S. suis-infected mice that received amentoflavone exhibited lower mortality and bacterial burden. Additionally, amentoflavone significantly decreased the production of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in an S. suis-infected cell model. Analyses of signaling pathways demonstrated that amentoflavone reduced S. suis-induced inflammation in S. suis serotype 2 (SS2)-infected cells by regulating the p38, Jun N-terminal protein kinase 1 and 2 (JNK1/2), and NF-κB pathways. The antivirulence and anti-inflammatory properties of amentoflavone against S. suis infection provide the possibility for future pharmaceutical application of amentoflavone in the treatment of S. suis infection.IMPORTANCE The widespread use of antibiotics in therapy and in the prevention of Streptococcus suis infection in the swine industry raises concerns for the emergence of a resistant strain. The use of antivirulence agents has potential benefits, mainly because of the reduced selective pressure for the development of bacterial resistance. In this study, we found that amentoflavone is an effective agent against S. suis serotype 2 (SS2) infection both in vitro and in vivo Our results demonstrated that amentoflavone is a promising anti-infective therapeutic for S. suis infections, due to its antivirulence and anti-inflammatory effects without antibacterial activity, with fewer side effects than conventional antibacterial agents.

Phloretin reduces cell injury and inflammation mediated by Staphylococcus aureus via targeting sortase B and the molecular mechanism.

Sortase B (SrtB) is a vital virulence factor that plays a critical role in Staphylococcus aureus (S. aureus) infections, indicating that it could be a latent target for S. aureus infections. In this study, phloretin, a natural compound that primarily exists in the pericarp and velamen of apples and pears, shows little anti-S. aureus activity, but significantly inhibited SrtB activity in vitro. The results of lactate dehydrogenase release and live/dead cell assays suggested that phloretin reduced human alveolar epithelial cell damage caused by S. aureus. Additionally, an adhesion assay confirmed that phloretin lowered the colony count of S. aureus in human alveolar cells. Phloretin treatment significantly attenuated the inflammatory response in macrophage cells (J774) co-cultured with S. aureus as determined by an enzyme-linked immune-sorbent assay. Furthermore, the results of molecular dynamics simulation, site-directed mutagenesis, and fluorescence spectroscopy quenching indicated that phloretin was directly located in the active pocket of SrtB and blocked substrate binding, leading to the loss of SrtB activity. These results indicate that phloretin is a possible candidate for treatment of S. aureus infections.

Acetylcholinesterase Biosensor Based On Mesoporous Hollow Carbon Spheres/Core-Shell Magnetic Nanoparticles-Modified Electrode for the Detection of Organophosphorus Pesticides.

The present study investigated the synthesis of mesoporous hollow carbon spheres (MHCS) and magnetic mesoporous hollow carbon spheres with core-shell structures (Fe3O4@MHCS). Two acetylcholinesterase sensors (acetylcholinesterase/mesoporous hollow carbon spheres/glassy carbon electrode (AChE/MHCS/GCE) and acetylcholinesterase/core-shell magnetic mesoporous hollow carbon spheres/glassy carbon electrode (AChE/Fe3O4@MHCS/GCE) based on mesoporous carbon materials were prepared. Under the optimum conditions, using Malathion as the model compound, the developed biosensors showed a wide detection range, low detection limit, good reproducibility, and high stability. The AChE/MHCS/GCE electrochemical sensor response exhibited two good linear ranges at the incubation time of 10 min at the Malathion concentration ranges of 0.01 to 100 ppb and 100 to 600 ppb, with a detection limit of 0.0148 ppb (S/N = 3). The AChE/Fe3O4@MHCS/GCE electrochemical sensor that was operated with an incubation time of 12 min at the malathion concentration ranges between 0.01⁻50 ppb and 50⁻600 ppb had a detection limit of 0.0182 ppb (S/N = 3). Moreover, the AChE/MHCS/GCE and AChE/Fe3O4@MHCS/GCE biosensors were effective for the detection of real samples, and were demonstrated to be suitable for the field-testing of organophosphorus pesticide (OP) residues.

Novel Inhibitor Discovery of Staphylococcus aureus Sortase B and the Mechanism Confirmation via Molecular Modeling.

SortaseB (SrtB) plays a critical role in Staphylococcus aureus (S. aureus) infections. According to the reports in the literature, SrtB can anchor the IsdC to the cell wall to capture iron from the host to achieve a successful invasion. On the other hand, SrtB could also affect the adhesion of S. aureus to host cells based on previous studies. Here, we report about a novel SrtB inhibitor, coptisine, a natural compound that does not exhibit antibacterial activity but can inhibit the SrtB activity in vitro. A cytotoxicity test indicated that coptisine protects human lung epithelial cells from S. aureus. In addition, coptisine can reduce the adhesion of S. aureus to human lung epithelial cells based on the result of plate colony counting assay. Molecular dynamics simulation revealed that coptisine can bind to the active pocket of SrtB, leading to its activity loss. Through the calculation of binding free energy between ligand and protein, site-directed mutagenesis and fluorescence spectroscopy quenching methods, it was confirmed that residues of Arg115, Asn116, and Ile182 played a vital role in the interaction of SrtB with coptisine. These data provide the theoretical basis for the therapy option to the infections caused by S. aureus.

Epigallocatechin gallate inhibits Streptococcus pneumoniae virulence by simultaneously targeting pneumolysin and sortase A.

Streptococcus pneumoniae (pneumococcus), the causative agent of several human diseases, possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. Pneumolysin (PLY), an important virulence factor, is a member of the cholesterol-dependent cytolysin family and has cytolytic activity. Sortase A (SrtA), another crucial pneumococcal virulence determinate, contributes greatly to the anchoring of many virulence-associated surface proteins to the cell wall. In this study, epigallocatechin gallate (EGCG), a natural compound with little known antipneumococcal activity, was shown to directly inhibit PLY-mediated haemolysis and cytolysis by blocking the oligomerization of PLY and simultaneously reduce the peptidase activity of SrtA. The biofilm formation, production of neuraminidase A (NanA, the pneumococcal surface protein anchored by SrtA), and bacterial adhesion to human epithelial cells (Hep2) were inhibited effectively when S. pneumoniae D39 was cocultured with EGCG. The results from molecular dynamics simulations and mutational analysis confirmed the interaction of EGCG with PLY and SrtA, and EGCG binds to Glu277, Tyr358, and Arg359 in PLY and Thr169, Lys171, and Phe239 in SrtA. In vivo studies further demonstrated that EGCG protected mice against S. pneumoniae pneumonia. Our results imply that EGCG is an effective inhibitor of both PLY and SrtA and that an antivirulence strategy that directly targets PLY and SrtA using EGCG is a promising therapeutic option for S. pneumoniae pneumonia.

Verbascoside Alleviates Pneumococcal Pneumonia by Reducing Pneumolysin Oligomers.

Pneumolysin (PLY), an essential virulence factor of Streptococcus pneumoniae (pneumococcus), can penetrate the physical defenses of the host and possesses inflammatory properties. The vital role PLY plays in pneumococcus pathogenesis makes this virulence factor one of the most promising targets for the treatment of pneumococcal infection. Verbascoside (VBS) is an agent that does not exhibit bacteriostatic activity but has been shown to inhibit PLY-mediated cytotoxicity. The results from molecular dynamics simulations and mutational analysis indicated that VBS binds to the cleft between domains 3 and 4 of PLY, thereby blocking PLY's oligomerization and counteracting its hemolytic activity. Moreover, VBS can effectively alleviate PLY-mediated human alveolar epithelial (A549) cell injury, and treatment with VBS provides significant protection against lung damage and reduces mortality in a pneumococcal pneumonia murine model. Our results demonstrate that VBS is a strong candidate as a novel therapeutic in the treatment of Streptococcus pneumoniae infection.

Baicalin inhibits the lethality of ricin in mice by inducing protein oligomerization.

Toxic ribosome-inactivating proteins abolish cell viability by inhibiting protein synthesis. Ricin, a member of these lethal proteins, is a potential bioterrorism agent. Despite the grave challenge posed by these toxins to public health, post-exposure treatment for intoxication caused by these agents currently is unavailable. In this study, we report the identification of baicalin extracted from Chinese herbal medicine as a compound capable of inhibiting the activity of ricin. More importantly, post-exposure treatment with baicalin significantly increased the survival of mice poisoned by ricin. We determined the mechanism of action of baicalin by solving the crystal structure of its complex with the A chain of ricin (RTA) at 2.2 Å resolution, which revealed that baicalin interacts with two RTA molecules at a novel binding site by hydrogen bond networks and electrostatic force interactions, suggesting its role as molecular glue of the RTA. Further biochemical and biophysical analyses validated the amino acids directly involved in binding the inhibitor, which is consistent with the hypothesis that baicalin exerts its inhibitory effects by inducing RTA to form oligomers in solution, a mechanism that is distinctly different from previously reported inhibitors. This work offers promising leads for the development of therapeutics against ricin and probably other ribosome-inactivating proteins.

The use of chlorogenic acid and its analogues as inhibitors: an investigation of the inhibition of sortase A of Staphylococcus aureus using molecular docking and dynamic simulation.

OBJECTIVES: To use molecular docking and dynamic simulation to investigate the inhibitory action of chlorogenic acid (CHA) and its analogues against sortase A of Staphylococcus aureus. RESULTS: Five novel, natural inhibitors with different activities were discovered for sortase A (SrtA). The inhibition mechanism of the novel inhibitors was consistent with the mechanism of CHA, which was reported previously by Wang et al. (Front Microbiol 6:1031, 2015). Based on structure-activity relationship analysis, the hydroxyl moiety (C1) of the inhibitors is critical in the catalytic region of SrtA, which could be confirmed by the calculation of the binding free energy between SrtA and the inhibitors. CONCLUSIONS: The mechanism obtained by molecular dynamics simulation is thus useful for the development of novel, selective SrtA inhibitors.

Fisetin inhibits Listeria monocytogenes virulence by interfering with the oligomerization of listeriolysin O.

Listeriolysin O (LLO), an essential virulence determinant of Listeria monocytogenes, is a pore-forming toxin whose primary function is to facilitate cytosolic bacterial replication by breaching the phagosomal membranes, which is critical for the pathogen to evade host immune recognition. The critical role of LLO in the virulence of L. monocytogenes renders it an ideal target for designing novel antivirulence therapeutics. We found that fisetin, a natural flavonoid without antimicrobial activity, is a potent antagonist of LLO-mediated hemolysis. Fisetin effectively inhibits L. monocytogenes infection in both tissue culture and animal infection models. Molecular modeling and mutational analysis revealed that fisetin directly engages loop 2 and loop 3 of LLO, leading to the blockage of cholesterol binding and the reduction of its oligomerization, thus inhibiting its hemolytic activity. Our results establish fisetin as an effective antitoxin agent for LLO, which can be further developed into novel therapeutics against infections caused by L. monocytogenes.

Baicalin inhibits the lethality of Shiga-like toxin 2 in mice.

Shiga-like toxins (Stxs), produced by pathogenic Escherichia coli, are a major virulence factor involved in severe diseases in human and animals. These toxins are ribosome-inactivating proteins, and treatment for diseases caused by them is not available. Therefore, there is an urgent need for agents capable of effectively targeting this lethal toxin. In this study, we identified baicalin, a flavonoid compound used in Chinese traditional medicine, as a compound against Shiga-like toxin 2 (Stx2). We found that baicalin significantly improves renal function and reduces Stx2-induced lethality in mice. Further experiments revealed that baicalin induces the formation of oligomers by the toxin by direct binding. We also identified the residues important for such interactions and analyzed their roles in binding baicalin by biophysical and biochemical analyses. Our results establish baicalin as a candidate compound for the development of therapeutics against diseases caused by Stxs.

Quercitrin, an inhibitor of Sortase A, interferes with the adhesion of Staphylococcal aureus.

Sortase A (SrtA) is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN), a natural compound that does not affect Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg) cell-clumping and immobilized fibronectin (Fn) adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

Molecular dynamics simulation of the processive endocellulase Cel48F from Clostridium cellulolyticum: a novel "water-control mechanism" in enzymatic hydrolysis of cellulose.

Glycoside hydrolase of Cel48F from Clostridium cellulolyticum is an important processive cellulose, which can hydrolyze cellulose into cellobiose. Molecular dynamics simulations were used to investigate the hydrolysis mechanism of cellulose. The two conformations of the Cel48F-cellotetrose complex in which the cellotetroses are bound at different sites (known as the sliding conformation and the hydrolyzing conformation) were simulated. By comparing these two conformations, a water-control mechanism is proposed, in which the hydrolysis proceeds by providing a water molecule for every other glucosidic linkage. The roles of certain key residues are determined: Glu55 and Asp230 are the most probable candidates for acid and base, respectively, in the mechanism of inverting anomeric carbon. Met414 and Trp417 constitute the water-control system. Glu44 might keep the substrate at a certain location within the active site or help the substrate chain to move from the sliding conformation to the hydrolyzing conformation. The other hydrophobic residues around the substrate can decrease the sliding energy barrier or provide a hydrophobic environment to resist entry of the surrounding water molecules into the active site, except for those coming from a specific water channel.

Oroxylin A inhibits hemolysis via hindering the self-assembly of α-hemolysin heptameric transmembrane pore.

Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical "Loop" region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus.

A natural inhibitor of diapophytoene desaturase attenuates methicillin-resistant Staphylococcus aureus (MRSA) pathogenicity and overcomes drug-resistance.

BACKGROUND AND PURPOSE: At present, the inhibition of staphyloxanthin biosynthesis has emerged as a prominent strategy in combating methicillin-resistant Staphylococcus aureus (MRSA) infection. Nonetheless, there remains a limited understanding regarding the bio-structural characteristics of staphyloxanthin biosynthetic enzymes, as well as the molecular mechanisms underlying the interaction between inhibitors and proteins. Furthermore, the functional scope of these inhibitors is relatively narrow. EXPERIMENTAL APPROACH: In this study, we address these limitations by harnessing the power of deep learning techniques to construct the 3D structure of diapophytoene desaturase (CrtN). We perform efficient virtual screening and unveil alnustone as a potent inhibitor of CrtN. Further investigations employing molecular modelling, site-directed mutagenesis and biolayer interferometry (BLI) confirmed that alnustone binds to the catalytic active site of CrtN. Transcriptomic analysis reveals that alnustone significantly down-regulates genes associated with staphyloxanthin, histidine and peptidoglycan biosynthesis. KEY RESULTS: Under the effects of alnustone, MRSA strains exhibit enhanced sensitivity to various antibiotics and the host immune system, accompanied by increased cell membrane permeability. In a mouse model of systemic MRSA infection, the combination of alnustone and antibiotics exhibited a significant therapeutic effect, leading to reduced bacterial colony counts and attenuated pathological damage. CONCLUSION AND IMPLICATIONS: Alnustone, as a natural inhibitor targeting CrtN, exhibits outstanding antibacterial properties that are single-targeted yet multifunctional. This finding provides a novel strategy and theoretical basis for the development of drugs targeting staphyloxanthin producing bacteria.