Chalcone Attenuates Staphylococcus Aureus Virulence By Targeting Sortase A And Alpha-Hemolysin

Staphylococcus aureus（S.aureus）,a clinically common gram positive bacterium,is the etiologic agent of a wide range of clinical infections,including bacteremia,infective endocarditis,and osteoarticular infections,as well as skin and soft tissue infections.S.aureus resistance,considered a dilemma for the clinical treatment of this bacterial infection,is becoming increasingly intractable.Conventional antibiotics kill bacteria directly and typically lead to the development of resistance,which means a more selective pressure for bacteria and a higher risk of drug resistance.Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections.Sortase A（SrtA）,an enzyme responsible for anchoring virulence-related surface proteins.One of the shared features of these proteins that mediate bacterial adhesion and evade host immune defenses is that they all contain LPXTG（Leu-Pro-X-Thr-Gly）sorting signals,which SrtA can identify and use to catalyze the anchor further.Another important target,alpha-hemolysin（Hla）,which is encoded by the hla gene and is generally secreted late in the exponential phase of growth,is a water-soluble pore-forming cytotoxin leading to the damage and death of cells,such as erythrocytes and epithelial cells,owing to its lytic property.SrtA and Hla have aroused great scientific interest,as they have been regarded as targets for promising agents against S.aureus infection.In this study,we discovered that chalcone,a natural small compound with little anti-S.aureus activity,could significantly inhibit SrtA activity with an IC₅₀ of 53.15μM and Hla hemolysis activity with an IC₅₀ of 17.63μM using a fluorescence resonance energy transfer（FRET）assay and a hemolysis assay,respectively.In addition,chalcone was proven to reduce protein A（SpA）display in intact bacteria,binding to fibronectin,formation of biofilm and S.aureus invasion.Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene,thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury;more importantly,chalcone could also reduce mortality in infected mice.Additionally,molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA,which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168,Ile182,and Arg197.Taken together,the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S.aureus infection via targeting Srt A and Hla.These findings could be the foundation for further design of novel anti-infection agents,and thus making a contribution for opening a new horizon for the treatment of S.aureus infection.