Inhibitory Effect And Mechanism Of Eriodictyol On Staphylococcus Aureus Sortase A

Methicillin-resistant Staphylococcus aureus（MRSA）is a multidrug-resistant bacterium resistant not only to methicillin but also to otherβ-lactam antibiotics（e.g.,oxacillin or cefoxitin）and members of other antibiotic families.MRSA is an important human commensal and opportunistic pathogen that causes a broad spectrum of human diseases ranging from moderate skin infections to more severe endocarditis,bacteremia,sepsis,osteomyelitis,blood infections,and pneumonia.Although the incidence rate of MRSA has been declining in many countries in recent years,high mortality rate associated with MRSA remains a great threat in clinical practice and imposes a considerable economic burden.The development of novel antibiotics remains an indispensable strategy for the treatment of MRSA infections;however,the development of new antibiotics is difficult,slow,and far behind the development of drug-resistant strains.Therefore,it is imperative to identify alternative strategies to combat increasingly powerful and evolving bacteria.S.aureus utilizes a vast array of virulence factors to survive and thrive ineither normal host environments or under extreme conditions,including surface-associated adhesins and secreted proteinaceous toxins.The secreted proteinaceous toxins and enzymes can damage host cells and tissues,are involved in the clearance of the host immune system,and promote bacterial spreading in the host.Targeting S.aureus virulence by small molecule inhibitors is a promising approach because it disarms bacteria to ameliorate infections but does not exert selective pressure on the bacteria thus reducing the risk of the development of antibiotic resistance.Several surface-associated adhesins,such as ClfA/ClfB,fibronectin-binding protein（FnBPs）and collagen adhesin（CAN）,are conducive to the pathogenesis of S.aureus infection.These essential virulence factors of S.aureus are covalently anchored to the bacterial surface by sortase A（SrtA）.Therefore,SrtA is considered an ideal druggable target for the development of novel anti-infective drug.In this research,we screened SrtA inhibitors from a variety of natural compounds,including alkaloids,tannins,flavonoids,quinones,steroids and terpenoids.Among them,eriodictyol can significantly inhibit the activity of SrtA at low concentrations,and their IC₅₀ values are lower than that of most previously reported SrtA inhibitors.Importantly,the results of minimum inhibitory concentration（MIC）and growth curve showed that eriodictyol did not affect the growth of bacteria at the concentration required for repression of SrtA,which avoided the possibility of drug resistance to a certain extent,and could be used as an innovative means to combat drug-resistant bacteria and virulence.Subsequently,we further verified the effects of eriodictyol on SrtA and its related virulence phenotypes in vitro.The results showed that eriodictyol can inhibited the adhesion of S.aureus to fibrinogen,reduced the formation of biofilm and the anchoring of staphylococcal protein A（SpA）on cell wall.The results of fluorescence quenching and local surface plasmon resonance（LSPR）experiments demonstrated a strong interaction between eriodictyol and SrtA.Molecular docking and molecular dynamics simulation further revealed that eriodictyol interact with SrtA through different amino acid residues,and the total free energy is-12.4 kcal/mol.In addition,eriodictyol can reduced the adhesion-dependent invasion of A549 cells by S.aureus.Subsequently,the inhibition effect in vivo were further explored in a mouse pneumonia model.Eriodictyol can reduce the virulence of S.aureus by inhibiting SrtA,significantly improve the survival rate of mice,reduce the colonization of S.aureus in lung tissue,improve the damage of lung tissue,and block the development of infection in a mouse pneumonia model.Overall,the results indicated that eriodictyol can attenuate MRSA virulence and prevent the development of resistance by inhibiting SrtA,suggesting that eriodictyol may be a promising lead compound for the control of MRSA infections.The combination of anti-virulence drugs and antibiotics is one of the current strategies against multi drug resistant pathogens.Anti-virulence therapies could be used synergistically with traditional antimicrobials,allowing less of each agent to be used.Additionally,with less of the therapeutic agent there will be less of an opportunity for the pathogen to develop resistance,increasing the time for which both types of therapy can be used.Chessboard dilution test and time-kill curve assay were used to confirm the synergistic effect of eriodictyol and cefoxitin,and the fractional inhibitory concentration index（FICI）was 0.3125.In addition,eriodictyol effectively enhanced the activity of cefoxitin in a mouse model of MRSA-induced pneumonia.These results indicate that eriodictyol is a highly effective inhibitor of S.aureusSrtA,which can inhibit its biological activity through direct interaction with SrtA,significantly reduce the pathogenicity of MRSA,and have a significant therapeutic effect on its infection of pneumonia in mice.In addition,the combined use of eriodictyol and cefoxitin can improve the anti MRSA effect of cefoxitin in vivo and in vitro,which provides a new strategy for the effective control of S.aureus infection.