The Target Confirmation And Molecular Interaction Mechanism Of Natural Inhibitor Against Staphylococcus Aureus Ser/Thr Phosphatase

Staphylococcus aureus(S.aureus)is a common food-borne pathogen that can contaminate food through multiple channels and cause bacterial infections in consumers.In addition to causing infections,Staphylococcus aureus also causes food poisoning by producing toxins,leading to vomiting,diarrhea and abdominal cramps.In China,about 20%-25%of food-borne bacterial outbreaks are caused by Staphylococcus aureus,second only to Salmonella and Vibrio parahaemolyticus.Food poisoning incidents caused by Staphylococcus aureus occur frequently,affecting food safety and human life and health,and putting tremendous pressure on global public health.At present,antibiotics are still the main drugs used clinically to treat bacterial infections.However,the abuse of antibiotics in animal husbandry and medical treatment has led to increasing resistance to bacteria and even the emergence of"super bacteria".Food contamination caused by drug-resistant strains poses a potential threat to consumers'health.As drug-resistant strains have been discovered from animal-source foods around the world,there is an urgent need to find new strategies for the treatment of bacterial infections.In recent years,scientists have thoroughly studied the pathogenic mechanism of bacteria,and the specific targeting virulence regulators has gradually become a research hotspot in anti-bacterial infections.This strategy weakens the pathogenicity of bacteria by reducing toxicity,so as to achieve the purpose of treatment.Given that most regulatory factors do not affect on the survival of bacteria,targeted inhibition of virulence regulators reduces the selection pressure on bacteria and reduces the generation of drug-resistant strains.In Staphylococcus aureus,protein phosphorylation/dephosphorylation is a common phenomenon in the process of signal transmission,which can coordinate the expression of virulence factors.Serine/threonine phosphatase Stp1 regulates substrate proteins through dephosphorylation in Staphylococcus aureus,and plays an important role as a regulatory factor.Besides,Stp1 is involved in regulating the expression of multiple virulence proteins including hemolysin,antibiotic resistance,and many important bacterial pathways.Mutant strains lacking Stp1 can significantly reduce the infection effect of Staphylococcus aureus in mouse animal models,which indicates that Stp1 is expected to become a new target for the treatment of bacterial infections.In this study,Stp1 was used as the target and binding affinity was used as an indicator to screen effective inhibitors of Stp1 through a virtual screening method.After verification of phosphatase activity,corilagin,cichoric acid,verbascoside and2-methylvaleric acid were selected as potential inhibitors.The inhibitory effects from high to low were:corilagin(IC₅₀=4.05?g/m L)>cichoric acid(IC₅₀=5.15?g/m L)>verbascoside(IC₅₀=20.02?g/m L)>2-methylvaleric acid(IC₅₀=59.60?g/m L),which was consistant with the binding affinity ranking.Based on the results of the screening,corilagin exhibited the dual inhibitory mechanism of competition and allosteric using enzymatic reaction kinetics experiments and molecular dynamics simulations.For the competitive binding mechanism,corilagin and the catalytic active center of Stp1(near Mn²⁺)are combined through hydrogen bonding and hydrophobic interaction.Residues Asn162,Ile164,Tyr199 and Lys232 played an important role in the binding of Stp1 and corilagin.The two amino acids with the strongest energy contribution were Asn162 and Tyr199,which could form stable hydrogen bonds with the carbonyl group in the 12-membered ring of corilagin.For the noncompetitive/allosteric binding mechanism,corilagin is tightly bound to the inactive region flap subdomain of Stp1 through hydrophobic interaction.Asn142,Val145,Leu146,Pro152 and Phe179 were important amino acid residues in this binding mode.These amino acids were mainly combined with the hydroxyl group on the six-membered ring of corilagin.For cichoric acid,this study used molecular docking,molecular dynamics,phosphatase activity experiments and binding energy calculations to reveal the complex binding mechanism to explore the groups that small molecules interact with Stp1 at the atomic level.The results of the study showed that cichoric acid not only exhibited the characteristics of the competitive inhibitor,but also had the characteristics of the noncompetitive inhibitor.In the competitive binding mode,cichoric acid binds neared the Mn²⁺of Stp1 and reduced the activity of Stp1 by competing with the substrate.Among them,Met39,Gly41,Asn162,Ile163 and Ile164residues were important sites for the complex binding.There was a stable hydrogen bond between the-NH₂group of Asn162 and cichoric acid.In the allosteric binding mode,cichoric acid interacted with the Ser136,Val145,Leu146,Pro152 and Ala155residues of Stp1 through hydrophobic interaction.Verbascoside plays a dual role,competition and non-competition mechanism,in inhibiting Stp1.Molecular docking and dynamic simulation showed that the Met39,Gly41,His42,Arg161 and Asn162 residues of Stp1 participated in the competitive binding of the complex through hydrogen bonding and hydrophobic interaction,while the Arg122,Ser136,Asp137,Asn142 and Val145 residues were related to the allosteric binding of verbascoside.Based on the result of molecular dynamics simulation and enzymatic kinetic reaction experiments,2-methylvaleric acid was a competitive inhibitor of Stp1.Binding free energy calculations showed that Met39,Thr102,Ile164,Val167 and Thr170 bound to 2-methylvaleric acid through hydrogen bonding and hydrophobic interaction.At the same time,after 2-methylvaleric acid bound to the active center of Stp1,the protein flap subdomain gradually deviated from the horizontal position,the active pocket was closed,and the enzyme activity was reduced.Due to the single binding mechanism of 2-methylvaleric acid and Stp1,the binding free energy of complex was lower than that of the other three inhibitors,so 2-methylvaleric acid had the lowest inhibitory activity on the enzyme.According to the molecular mechanism proposed by theoretical calculations,point mutations,fluorescence quenching and binding free energy calculation experimental methods were used to verify.It was found that after the mutation of the amino acid at the binding site,the weakened binding free energy between the inhibitor and the protein,the reduced interaction force,and the lower binding force,lead to a decrease in the inhibitory effect.At the cellular level,cichoric acid,verbascoside and 2-methylvaleric acid reduced the cytotoxicity of Staphylococcus aureus and protected the damage of alveolar epithelial cells A549,indicating their potential therapeutic effect on Staphylococcus aureus pneumonia.These three small molecules had no inhibitory effect on Staphylococcus aureus and helped to reduce bacterial resistance.In summary,the inhibitory mechanism of small molecules on Stp1 was clarified using theoretical calculations and experiments,which provided a theoretical basis for the development of new inhibitors.Point mutation and fluorescence quenching experiments verified the above-mentioned mechanism and improved the credibility and accuracy of theoretical calculations.In addition,small molecules protect A549cell damage induced by Staphylococcus aureus toxin,laying the foundation for the development of inhibitors against Staphylococcus aureus infection.