Screening And Mechanism Analysis Of Natural Bacteriostatic Agents Targeting Staphylococcus Aureus Stp1

Staphylococcus aureus?S.aureus?is one of the common pathogens causing foodborne diseases,which poses a serious threat to food safety and human health.In addition,S.aureus can cause infection,skin and soft tissue damage,sepsis,pneumonia and so on.S.aureus producing multiple virulence factors is the main reason of causing foodborne diseases and infections.Eukaryotic-like Ser/Thr phosphatases?Stp1?of S.aureus can regulate the secretion and expression of multiple virulence factors and can participate in many important pathways.It is an important protein molecule for the pathogenicity of S.aureus.Therefore,using Stp1as a target of bacteriostatic agents to reduce the pathogenicity of S.aureus is a feasible strategy to solve the problem of drug resistance of S.aureus caused by antibiotic abuse..In this paper,the prokaryotic expression plasmid of PET-28a-Stp1 was constructed,and the high-purity Stp1 recombinant protein was obtained.Natural bacteriostatic agents directly interacting with Stp1 was screened The 50%inhibitory concentrations(IC₅₀)of Rosmarinic acid,Forsythoside A,Cyanidin chloride against Stp1 were determined.The interaction mechanism of Stp1 with Rosmarinic acid,Forsythoside A and Cyanidin chloride was analyzed by molecular docking,molecular dynamics simulation,calculation and decomposition of binding free energy.The interaction mechanism of natural bacteriostatic agents to Stp1 were verified by molecular dynamics simulation of mutants,calculation of binding free energy of mutants,site-directed mutagenesis assay,phosphatase activity assay and fluorescence quenching assay.The specific research results are as follows:?1?Eighteen natural bacteriostatic agents that could directly interact with Stp1were screened,among which Rosmarinic acid,Forsythoside A and Cyanidin chloride had the most obvious inhibitory effect on Stp1.?2?The IC₅₀0 of Forsythoside A against Stp1 was 2.38?g/mL.Forsythioside A was a competitive inhibitor and could bind to the active region of Stp1 mainly through hydrogen bond and hydrophobic interaction,which directly affected the activity of Stp1.The key amino acid residues of binding were Phe156,Thr157,Arg161,Tyr199,Asn229,His230,Lys232 and Asp233.Compared with wild-type Stp1,the binding region and binding mode of Forsythoside A with Stp1 mutants F156A,R161A and D233A did not change and the inhibition and binding ability decreased,indicating that Phe156,Arg161 and Asp233 were the key amino acid residues of Forsythoside A binding to Stp1,which confirmed that the inhibition mechanism of Forsythoside A to Stp1 activity was competitive inhibition.?3?The IC₅₀0 of Rosmarinic acid against Stp1 was 0.91?g/mL.Rosmarinic acid was a competitive inhibitor and could bind to the active region of Stp1 mainly through hydrogen bond and hydrophobic interaction,which directly affected the activityofStp1.Thekeyaminoacidresiduesofbindingwere Arg14,Met39,Gly40,Lys43 and Lys232.Compared with wild-type Stp1,the binding region and binding mode of Rosmarinic acid with Stp1 mutants R14A,G40A and K232A did not change and the inhibition and binding ability decreased,indicating that Arg14,Gly40 and Lys232 were the key amino acid residues of Rosmarinic acid binding to Stp1,which confirmed that the inhibition mechanism of Rosmarinic acid to Stp1 activity was competitive inhibition.?4?The IC₅₀0 of Cyanidin chloride against Stp1 was 14.59?g/mL.Cyanidin chloride was a competitive inhibitor and could bind to the active region of Stp1mainly through hydrogen bond and hydrophobic interaction,which directly affected the activity of Stp1.The key amino acid residues of binding were Asp194,Asp198,Tyr199,Ala226,Asp228 and His230.Compared with wild-type Stp1,the binding region and binding mode of Cyanidin chloride with Stp1 mutants D198A and H230A did not change and the inhibition and binding ability decreased,indicating that Asp198 and His230 were the key amino acid residues of Cyanidin chloride binding to Stp1,which confirmed that the inhibition mechanism of Cyanidin chloride to Stp1 activity was competitive inhibition.This paper not only screened 18 natural bacteriostatic agents that can directly interact with Stp1 but also analyzed the interaction mechanism of Stp1 with Rosmarinic acid,Forsythoside A and Cyanidin chloride.It would provide theoretical references for the development of natural bacteriostaticl agents targeting Stp1 and the application of Rosmarinic acid,Forsythoside A and Cyanidin chloride in food industry and pharmaceutical industry.