Therapeutic Effect Of Amentoflavone As MgrA Inhibitor Of Staphylococcus Aureus On Pneumonia In Mice

Staphylococcus aureus（S.aureus）is a common gram-positive pathogen,which hasstrong pathogenicity and can cause a variety of zoonotic diseases.From mild skin infections to fatal invasive infections of endocarditis,pneumonia,sepsis,etc,the morbidity and mortality rate of the host after infection is relatively high.In recent years,with the aggravation of drug resistance,the emergence and widespread dissemination of methicillin-resistant Staphylococcus aureus（MRSA）has cause serious impact on public health and human health,and the clinical treatment of S.aureus infections is increasingly limited.Exploring new therapeutic approaches and new drug targets has become a newoptions against complicated S.aureus infections.S.aureus can infect multiple tissues and organs of the host,and its pathogenicity depends on a vast repertoire of virulence factors.Anti-virulence strategy is a new alternative approach to clinical antibacterial therapy.Anti-virulenc drugs can inhibit the virulence of S.aureus at the same time without selective pressure on the bacteria itself.They can not only exert the efficacy of drugs but also avoid the emergence of drug resistance.MgrA（multiple gene regulator）belongs to the Mar R family and is an important global transcriptional regulator in S.aureus.It regulates the virulence of S.aureus in a variety of ways.Co-regulates the expression of more than 350 genes,including capsular polysaccharides,alpha hemolysin,proteases,adhesin,etc.MgrA plays a vital role in the pathogenesis of S.aureus and is considered to be an ideal target for the development of antibacterial drugs.Therefore,screening and developingof MgrA inhibitors can provide a new theoretical basis and excellent lead compounds for the clinical treatment of S.aureus infections.Natural compounds have many advantages of wide sources,multiple types and rich pharmacological effects.Screening inhibitors from natural compounds has broad application prospects.In this study,amentoflavone was screened as a highly effective inhibitor of MgrA from a small molecule library of natural compounds using fluorescence anisotropy analysis.Firstly,the inhibitory effect of Amentoflavone on MgrA activity and its mechanism were verified in vitro Subsequently,the in vitro combined antibacterial experiments further clarified the development potential of Amentoflavone as a clinical synergistic antibacterial infection drug;finally,the therapeutic effect of Amentoflavonealone and with combined with antibiotics on S.aureus infection was verified by establishing a mouse pneumonia model.First,the fluorescence anisotropy analysis proved that Amentoflavone significantly inhibited the activity of MgrA at a low concentration(IC₅₀=3.26±0.18μg/m L).The minimum inhibitory concentration and growth curve confirmed that ametoflavone has no effect the growth of bacteria.Gel migration assay showed that Amentoflavone inhibited the binding of MgrA to hla promoter fragments in a dose-dependent manner,which proved that Amentoflavone can inhibit the biological activity of MgrA in vitro.The effect of Amentoflavone on downstream genes regulated by MgrA was determined by fluorescence quantitative PCR.The transcription levels of RNAⅢand fibronectin binding protein A encoding gene Fnb A were significantly inhibited,which decreased by 3-fold,2.5-fold,1.5-fold,respectively.The transcription level ofα-hemolysin was most significantly inhibited in S.aureus.The transcription of genes encoding surface proteins and foreign proteins such as Ebh,Srap,and Spa were up-regulated by 2.9-fold,3.5-foldand 2.2-fold,respectively.It was further confirmed by western blot and hemolysis experiment that Amentoflavone significantly inhibited the expression and biological activity ofα-hemolysin;Fibrinogen binding experiment showed that Amentoflavone inhibited the adhesion of S.aureus USA300 to fibrinogen;Heat stable migration experiment,intracellular heat stable migration and fluorescence quenching experiments revealed that Amentoflavone inhibits its activity by directly binding to MgrA.Subsequently,molecular docking,molecular dynamics simulations and point mutation experiments confirmed that Gln-19 and Cys-12 are the key amino acid residues for the binding of Amentoflavone to MgrA.To further explore its clinical application value,the checkerboard method proved that Amentoflavone can produce a synergistic anti-MRSA effect with a variety of commonly used clinical antibiotics,of which ceftriaxone sodium had the strongest synergistic effect;The bactericidal curve further confirmed that Amentoflavone can promote S aureus USA300 Sensitivity to ceftriaxone sodium,and significantly improve its antibacterial activity.Finally,through the establishment of a mouse pneumonia model,we found that Amentoflavone alone can significantly improve the survival rate of mice,reduce the amount of bacteria in the lungs and significantly reduce the pathological damage and inflammation of the lung tissue of the mice infected by S.aureus.Combined with ceftriaxone sodium,it can significantly improve the therapeutic effect of ceftriaxone sodium and reduce its dosage.In conclusion,Amentoflavone is a highly effective inhibitor of the transcriptional regulatory factor MgrA of Saureus.Through direct interaction with MgrA toinhibit its biological activity and significantly reduces the pathogenicity of MRSA,and has a significant therapeutic effect on a mouse pneumonia.In addition,Amentoflavone combination with ceftriaxone sodium can reduce the dosageof antibiotics and enhance its antibacterial activity,thereby delaying the development of drug resistance.This study provides a new theoreticaland experimental evidence for the further development of Amentoflavone as a clinical anti-S.aureus infection drug and auxiliary drug.