Inhibitory Effect And Mechanism Of Myricetin On Staphylococcus Aureus ClpP

Staphylococcus aureus（S.aureus）is a common opportunistic pathogen,which can cause endocarditis,osteomyelitis,necrotizing fasciitis and other diseases.Due to the evolution of S.aureus and the abuse of antibiotics,drug-resistant strains of S.aureus are emerging.Methicillin resistant Staphylococcus aureus（MRSA）is a very pathogenic clinical common strains,and more than 90%of them have the characteristics of multi-drug resistance,resulting in high infection rate and mortality.At present,the spread and prevalence of MRSA has become an extremely serious global public health safety problem.Therefore,it is of great significance to develop drugs based on new drug targets,which can effectively control infection and avoid bacterial resistance.Virulence factors of S.aureus are very important for the occurrence and development of infection.They can not only promote the adhesion and invasion of S.aureus to host cells and tissues,but also help it escape the host’s immune defense.With the increasingly serious problem of drug resistance of S.aureus,as well as the in-depth understanding of its pathogenic mechanism,the important role of virulence factors in the infectivity and pathogenicity of S.aureus has become more and more clear,so the study of anti virulence is particularly important.The strategy of anti virulence is to inhibit the key virulence factors of bacteria,reduce their pathogenicity,and use the body’s own immune system to eliminate bacteria.Because this strategy will not kill bacteria,it will not cause a huge pressure on the survival of bacteria,leading to the emergence of drug resistance.Casein hydrolase ClpP is a highly conserved serine protease that exists in all kinds of organisms.In the past decades,it has been widely studied as a new antibacterial target.The hydrolysis of ClpP protease plays an important role in the pathogenicity of S.aureus.It can not only affect the formation of S.aureus biofilm,but also participate in its adhesion process.In addition,the hemolytic ability of S.aureus with clpP knockout was greatly reduced.Because ClpP plays an important role in the pathogenic process of S.aureus,it is of great practical significance to screen ClpP protease inhibitors and explore its mechanism of action.In the present study,we constructed a ClpP inhibitor screening system using Suc-LY-AMC as a specific fluorogenic substrate of ClpP,more than 500 TCM monomers currently available in the laboratory were screened by fluorescence resonance energy transfer（FRET）.It was found that myricetin was able to effectively inhibit the activity of ClpP without inhibiting the growth of S.aureus USA300 and exhibited little cytotoxicity;Further experimental results showed that myricetin could effectively reduce the expression of multiple virulence factors in S.aureus USA300and Newman,and especially had a significant inhibitory effect on the hemolytic ability of S.aureus USA300 and Newman;the results of thermal shift assay（TSA）and cellular thermal shift assay（CETSA）indicated that myricetin was able to bind to ClpP in vitro and in vivo and reduced its thermal stability.The dissociation constant of myricetin and ClpP was determined by surface plasma resonance（SPR）,it was 6.356×10^-4 M,indicating that there was a binding between myricetin and ClpP.The mode of action of them was investigated by molecular docking and molecular dynamics simulation,indicating that there was a binding between myricetin and ClpP.Docking results were further verified by point mutation analysis and FRET,the results show that Gln-47 and Met-31 were the key amino acids involved in the binding of the myricetin to ClpP through.Finally,we constructed a murine MRSA infection pneumonia model and proved that myricetin could effectively reduce the mortality rate as well as the bacterial load in the lungs of mice,significantly ameliorate the infection and pathological changes in the lungs of mice caused by S.aureus.Taken together,myricetin is able to effectively protect mice against S.aureus induced pneumonia infection by inhibiting the activity of S.aureus ClpP and thereby effectively attenuating the virulence of MRSA,demonstrating that myricetin holds promise as a candidate compound for the development of innovative drug to control MRSA infections.