The Mechanism Of Edible Rosmarinic Acid Complex Hydrogel Against Staphylococcus Aureus Infection

Staphylococcus aureus（S.aureus）is a common Gram-positive pathogen that can cause various diseases including food poisoning,skin infections,and tissue abscesses.Antibiotics,while treating,have given bacteria strong selective pressure,leading to the widespread spread of drug-resistant S.aureus,and novel approaches are urgently needed to address S.aureus infection.With the deepening understanding of the pathogenesis of S.aureus,the treatment strategy for S.aureus infection has shifted towards interfering with its key virulence factors.Serine/threonine phosphatase（Stp1）is an important virulence regulator in S.aureus,involved in important pathways such as bacterial cell wall metabolism and antibiotic sensitivity.Literature reports indicate that the deletion of the stp1 gene significantly reduces the pathogenicity of S.aureus,and Stp1 is an important target for resistance to S.aureus infection.In previous studies,we found that the food additive rosmarinic acid（RA）is an effective Stp1 inhibitor through virtual screening methods.RA has antioxidant,anti-tumor,anti diabetes and other biological activities,and is widely used in the food and pharmaceutical industry.However,the defects of physicochemical properties of RA have affected its use,seriously limiting its further promotion and application.The hydrogels synthesized from food raw materials are characterized by their hydrophilicity,flexibility and biocompatibility.They have the advantages of safety,low price and industrial production,and are good drug delivery carriers.Therefore,we prepared the hydrogel CS9P1-RA with RA and food derived bacteriostat chitosan（CS）as raw materials,and this hydrogel showed significant bacteriostatic and anti-virulent activities.The potential mechanism of CS9P1-RA hydrogel against S.aureus infection was elucidated by computational biology and molecular biology experiments.The anti-S.aureus effect of CS9P1-RA hydrogel was verified by animal infection model.The main research contents and results are as follows:（1）The preparation and structural characterization of CS9P1-RA hydrogel.The effective inhibitor RA was obtained through virtual screening targeting S.aureus Stp1and validated by phosphatase activity tests,and CS9P1-RA hydrogel was prepared by loading RA in a cross-linked polymer network with CS as the skeleton.The scanning electron microscopy,X-ray diffraction,Fourier transform infrared spectroscopy and thermal stability analysis revealed the porous structure and crystallization characteristics of CS9P1-RA hydrogel,and confirmed the successful synthesis of CS9P1-RA hydrogel.In addition,in vitro drug release tests showed that RA could be slowly released at different pH in CS9P1-RA hydrogel.（2）CS9P1-RA hydrogel has dual functions of bacteriostasis and anti-virulence.The results of phosphatase activity tests and enzymatic reaction kinetics tests showed that CS9P1-RA hydrogel could inhibit Stp1 activity in a dose-dependent manner(IC₅₀=7.424μM).By downregulating the gene expression levels of virulence factors（α-hemolysin,δ-hemolysin,Panton-Valentine leukocidin,and phenol soluble modulin）in S.aureus,inhibiting the secretion ofα-hemolysin protein and weakening the hemolytic activity of S.aureus,CS9P1-RA hydrogel effectively reduced the virulence of S.aureus.On the other hand,CS9P1-RA hydrogel showed significant antibacterial activity（MIC=1 mg/m L）based on the synergistic effect of natural bacteriostat CS.In addition,CS9P1-RA protected A549 cells from the invasion of S.aureus,demonstrating the ability to resist S.aureus infection.（3）The study on the bacteriostatic and anti-virulent mechanism of CS9P1-RA hydrogel.The binding affinity and interactions between CS,the bacteriostatic component in CS9P1-RA hydrogel,and teichoic acid,the main component of cell wall and membrane of S.aureus,were studied based on molecular docking.By constructing the complex models using CS repeat units with ribol phosphate（Rbo-P）and glycerol phosphate（Gbo-P）repeat units that make up teichoic acid,it was found that CS had strong binding affinity with Rbo-P and Gbo-P（-8.3 and-7.9 kcal/mol,respectively）,which came from the hydrogen bond interactions between them.In addition,the competitive inhibitory mechanism and noncompetitive inhibitory mechanism of the anti-virulent component RA on Stp1 was studied based on molecular dynamics simulations and binding free energy calculations.In the competitive binding mode,RA stably bound to the catalytic active region of Stp1,resulting in hindrance of substrate binding to Stp1,thereby inhibiting the activity of Stp1.Residues MET-39,GLN-160,ILE-164,and VAL-167 were the main energy contributors to their binding.In the noncompetitive binding mode,RA stably bound to the flap subdomain of Stp1,causing the conformation of flap subdomain to change from horizontal to vertical,and the catalytic active region to change from open to closed,resulting in a decrease in Stp1activity.Residues ARG-122,SER-139,VAL-141,and ARG-161 were the key residues in the noncompetitive binding sites of RA and Stp1.The subsequent amino acid site-specific mutation tests,mutant protein phosphatase activity tests,fluorescence quenching tests,and protein in vitro thermal shift tests further confirmed the above inhibitory mechanism.（4）The study on the application of CS9P1-RA hydrogel against S.aureus infection.Through swelling tests,water retention tests and water vapor permeability tests,it was found that CS9P1-RA hydrogel had good water absorption and water vapor permeability,which could absorb wound exudate and provide a moist environment for the wound.The tensile test results showed that CS9P1-RA hydrogel had a certain mechanical strength,which was conducive to maintaining its structural integrity during use,and confirmed the potential of CS9P1-RA hydrogel as a wound dressing.CS9P1-RA hydrogel could effectively eliminate DPPH free radicals and ABTS free radicals,and had safety and significant antioxidant property,which help to reduce oxidative stress at the wound.In addition,by constructing the mice skin wound model infected with S.aureus,and tracking the wound healing rate,the number of S.aureus and the content of hydroxyproline in the wound treated with CS9P1-RA hydrogel,it was found that CS9P1-RA hydrogel could effectively combat S.aureus infection and promote wound healing.In conclusion,this study combined the natural food additive RA with the food derived bacteriostat CS to construct a multifunctional hydrogel CS9P1-RA for S.aureus infection.CS9P1-RA hydrogel showed significant efficacy in reducing the virulence of S.aureus and inhibiting S.aureus.Based on computational biology and molecular biology experiments,the bacteriostatic and anti-virulent mechanisms of CS9P1-RA hydrogel were clarified.The synergistic effect of CS and RA in CS9P1-RA hydrogel enables it to effectively treat S.aureus infection,which not only expands the application range of food raw materials,provides a new research direction for the development of food hydrogels in the future,but also provides a promising solution for the treatment of skin damage caused by S.aureus infection.