Study On The Activity And Mechanism Of Action Of Harmine Against Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is a widely distributed food-borne pathogen which can cause contamination of aquatic products,vegetables and meat products and cause food poisoning.At the same time,S.aureus can lead to a variety of zoonotic diseases,such as suppurative pneumonia,sepsis,and tissue infections,which poses a great threat to public health and safety.It is reported that S.aureus can secrete a variety of virulence proteins,which is the main reason for its pathogenicity.Among them,?-hemolysin is the most important virulence factor.It can not only destroy the immune system and arouse an inflammatory response,but also induce monocytes to produce inflammatory factors and apoptosis.In addition,S.aureus can form biofilms which produce a protective effect on the bacteria enclosed inside.The biofilms reduce sensitivity of the bacteria to antibiotics and bactericides and improve the drug resistance of the bacteria.Harmine(HM)is a natural substance extracted from the seeds of the camellia plant,a tricyclic ?-carboline alkaloid,which has a broad spectrum of antibacterial activity,and especially shows good antibacterial effect on streptococci.This study explored the inhibitory effect of HM on S.aureus biofilms and?-hemolysin,as well as the mechanism of inhibition.The following four aspects of research work were studied:(1)Study on the inhibitory effect of HM on S.aureus.The drug susceptibility test results showed that the minimum inhibitory concentrations(MICs)of HM on S.aureus were in the range of 64-128 ?g/mL.For the standard strain of S.aureus ATCC29213,the MIC value was 128 ?g/mL.By plotting the growth curve of S.aureus under different time and different concentrations of HM,the growth inhibition of S.aureus by HM was dynamically reflected.In addition,the destruction effect of HM oncell membrane of S.aureus was studied by UV absorption experiment.The results showed that HM can cause damage to S.aureus cell membranes in a dose-dependent manner.When the concentration is 128 ?g/mL for 1 h,the content leakage rate is as high as 73%.(2)Study on the inhibitory effect and mechanism of HM on the biofilm of S.aureus.The minimum inhibitory concentration of HM on S.aureus biofilms ranges from 128 to 256 ?g/mL.For the standard strain S.aureus ATCC 29213,the minimum inhibitory concentration of biofilm value is 256 ?g/mL.It was verified that HM has an inhibitory effect on S.aureus biofilm through crystal violet adhesion experiment and fluorescence microscope observation.The higher the concentration,the stronger the inhibitory effect.Based on this,the release amounts of polysaccharide intercellular adhesin(PIA)and extracellular DNA(eDNA)during the biofilm formation were measured.The results showed that HM showed inhibitory effect on both,and it was related to the concentration.When the concentration was increased to128-256 ?g/mL,the amount of PIA synthesis decreased by 68% to 74%,and the amount of eDNA released decreased by about 75%.(3)Study on the inhibitory effect of HM on S.aureus a-hemolysin.By conducting hemolytic activity experiment,it was found that HM can inhibit the hemolytic activity of ?-hemolysin in a dose-dependent manner.When the HM concentration was 64 ?g/mL,the hemolytic capacity decreased sharply to 32.54% of the control group.Western Blot experiment showed that HM under sub-inhibitory concentration can inhibit the expression of ?-hemolysin protein.Real-time RT-PCR experiment analyzed the inhibitory effect of HM,and the results also showed a quantitative dependence.(4)Study the molecular docking of HM and S.aureus ?-hemolysin.By selecting11 adjacent polymerization key sites with high probability,the molecular docking calculation of HM and ?-hemolysin was performed in sequence.The calculation results showed that HM is most likely to bind to the huge hole formed at the N-terminus of ?-hemolysin with the two ends connecting to the N-terminus and?-sandwich domain respectively.This docking posture can inhibit the ability of?-hemolysin to deform itself and the others to bind to this site,thereby inhibiting the formation of ?-hemolysin heptamer and reducing its hemolytic activity.The above experimental datas clarified the inhibitory effect and mechanism of HM on S.aureus biofilm and ?-hemolysin,and laid a foundation for further development of HM as a natural and efficient food preservative and antibacterial drugs.