Piceatannol Inhibits The Activity Of Cholesterol-dependent Cytolytic And The Mechanism

Cholesterol-dependent cytolysins(CDCs)are mainly secreted by varieties of gram-positive pathogens during infection,which are known for the characters of forming fore on targeting cells that possess them another name of pore-forming toxins.Gram-positive pathogens secreting CDCs can cause a series of infectious diseases in humans and animals,such as meningitis,bacteremia,arthritis,pneumonia,gastroenteritis and necrotic enteritis,which have seriously threatened human health,animal husbandry economy benefits and food safety.More than 40 gram-positive pathogens can secrete CDCs in the process of infection,Listeriolysin O(LLO),Suilysin(SLY)and Pneumolysin(PLY)that produced by L.monocytogenes,S.suis and S.pneumonia,are critical members of CDCs.LLO,SLY and PLY have been revealed promote their host pathgoens infections through many aspects,such as lyze various cells,causing the leakage of cellular content;helping their host pass through blood brain barrier and causing meningitis;promoting bacteria to escape the elimination from the host immune system;inducing cell apoptosis and leading to inflammation,which make CDCs important target for screening inhibitor to combat their pathogens.In addition,the high homology of the primary structure and the similarity of the spatial structure make CDCs members possess similar pore-forming mechanism and biological functions,which providing theoretical basis for screening broad-spectrum anti-virulence inhibitors.In this study,we take LLO?SLY and PLY as targets and harvest a natural food-borne compound piceatannol(PN)that possess wide-spectrum anti-CDCs properties.Antimicrobial assay indicates that PN did not affect the growth of pathogens that produce CDCs under effective concentrations.We detected the formation of oligomers by using Wstern-blot methods and found that PN inhibited the oligomerization of CDCs members directly.At the cellular level,PN reduced the cytotoxicity caused by pathogens that produced CDCs,and alleviated the inflammatory effect mediated by pathogens that produced CDCs.In addition,PN showed systematic protective effect on infected mice by significantly reducing inflammation,bacterial burden and the mortality.We explored the mechanism between PN and CDCs based on computational biology and biochemical methods.The stable complex system of PN and CDCs were obtained based on molecular docking and molecular dynamics simulation.We analyzed the binding model,the binding free energy contribution of residues,the change of residues flexibilities before and after binding with PN and the formation of hydrogen bond,and found that PN located in the junctional zone between domain 2 and domain 4,causing the spatial conformation of CDCs changed,especially the increasing of angle between domain 2 and domain 4 resulted in the binding of domain 4 and the cholesterol receptor on the targeting cell membranes was not sufficient,blocking the formation of oligomers and finally leading to the reduction of pore-forming.Asparaginate contribute free energy during the binding of PN with PLY,SLY and LLO,arginine and tyrosine contribute energies only during the binding of PN with PLY and SLY.Hydrogen bond was generated during the binding process of PN with PLY and SLY,and the stability over than 60%,indicating that hydrogen bond was critical for the complex system stabilization.In addition,we found that the meta-position hydroxyl of PN was critical for the formation of hydrogen bond based on an analysis of radial distribution function.The reliability and credibility of these results were confirmed by site-specific mutagenesis and fluorescence quenching assays.