Research On The Inhibitory Effect And Mechanism Of Hispidulin On Virulence Factor Of Staphylococcus Aureus

Staphylococcus aureus（S.aureus）can cause acute infectious diseases that threaten the safety of human life and health.The emergence of drug-resistant S.aureus has limited clinical treatment options and has led to a dramatic increase in infection and mortality rates.Currently,therapeutic strategies targeting bacterial virulence have shown great potential.The aim of this study was to investigate the inhibitory effect of natural compounds hispidulin on S.aureus virulence.Method:1.Effective inhibitors against hla were screened by hemolytic activity,and the transcript and protein expression levels of virulence factors were examined by RT-qPCR and Western blot;2.The interaction between hispidulin and hla or agrAC proteins was assessed by oligomerization assay,thermal shift assay（TSA）,cellular thermal shift assay（CETSA）,electrophoretic mobility shift assay（EMSA）,fluorescence quenching and molecular docking assay;3.The anti-infective effects of hispidulin were evaluated in vitro by double fluorescence staining and LDH assay after infection of A549 cells by S.aureus.A mouse pneumonia infection model was also constructed to detect the lethality,lung histopathological changes,lung tissue bacterial load,and the level of inflammatory factors in each group of mice to assess the therapeutic effect of hispidulin on S.aureus infection in vivo;4.The synergistic effect of hispidulin and cefoxitin was detected by the checkerboard method,and the problem of resistance of S.aureus to hispidulin was investigated by continuous passaging method.Result:1.Hispidulin effectively inhibited the hemolytic activity of hla in a variety of S.aureus.Evidenced by the transcriptional and translational levels of hla,hispidulin significantly inhibited the expression of hla,while hispidulin inhibited the transcriptional levels of agrA and RNAIII.2.Hispidulin did not affect the formation of hla heptamers.Further experiments revealed that hispidulin affected the agr quorum-sensing system by binding to the agrA protein.3.In vitro,hispidulin effectively inhibited the infection of A549 cells and the level of cellular LDH release by S.aureus.In vivo,hispidulin reduced the mortality of pneumonia infection in mice,reduced lung inflammation,decreased lung tissue bacterial load,and inhibited the levels of inflammatory factors IL-2,IFN-α,and TNF-α in alveolar lavage fluid of mice,effectively reducing the pneumonia infection caused by S.aureus in mice.4.In the short-term S.aureus USA300 did not develop resistance to hispidulin.The combination of hispidulin with antibiotic cefoxitin has a synergistic antimicrobial effect and had a stronger protective effect in pneumonia infections.Conclusion:Our study demonstrated that hispidulin acted on agrAC,a key protein in the agr quorum-sensing system of S.aureus,significantly inhibited the hemolytic activity of S.aureus.Hispidulin can inhibit the infection of A549 cells by S.aureus in vitro and attenuate the infection of mouse pneumonia caused by S.aureus in vivo.It was also found that the combination of hispidulin and cefoxitin could provide a synergistic effect,and did not develop resistance to it.In conclusion,the natural flavonoid compound hispidulin is promising to be developed as an effective anti-virulence agent against S.aureus infections.Innovation Point:In this study,the inhibitory effect of the natural compound hispidulin on S.aureus hla was investigated for the first time,and its action on agrAC,a key protein in the agr quorum-sensing system,was revealed.