Inhibitory Effect Of Kaempferol On Staphylococcus Aureus Sortase A

Staphylococcus aureus(S.aureus)poses a serious threat to the health of people and animals.Adhesion is the first step in infection caused by bacteria.Surface proteins that mediate S.aureu adhesion,including fibronectin binding proteins A,B(FnbpA and FnbpB),fibrinogen binding proteins A,B(ClfA,ClfB),mainly by sortase A(SrtA)Anchored to the cell wall,it plays an important role in the process of bacterial adhesion to host cells and escape the host immune system.Therefore,inhibiting SrtA activity can effectively reduce the virulence of S.aureus.Kaempferol is a type of flavonoid compound derived from plant rhizomes,which has various biological activities such as anti-inflammatory,anti-cancer,antibacterial,and anti-oxidation.The studys found that kaempferol has a good inhibitory effect on S.aureus,but the target is not yet clear.In order to verify whether SrtA is the target of kaempferol,this study used SA USA300 as the test strain and conducted the following studies:1.The srtA gene deletion strains and complemented strains of USA300 strain were constructed by CRISPR/Cas9 technique.The pCasSA-sgRNA plasmid was constructed using pCasSA vector and products of three pairs of srtA gene gRNAs.Then the pCasSA-sgRNA plasmid was modified through deletion S.aureus RN4220strain,and transferred into USA300 strain,and the efficiency of cutting pCasSA-sgRNA plasmid was measured.The homologous sequences in upstream(srtA-L)and downstream(srtA-R)of the srtA gene were amplified by PCR respectively,and their products were ligated by overlapping PCR.Then ligated products inserted into pCasSA-sgRNA plasmid for pCasSA-sgRNA-srtA plasmid construction.Screen and identify srtA gene deletion strains.Using the pLI50 plasmid as a vector,the pLI50-srtA plasmid and the complemented strains were constructed.After chloramphenicol screening,PCR amplification and sequencing identification,the srtA gene deletion strains(?srtA USA300)was obtained.After double digestion and sequencing,it was verified that the pLI50-srtA plasmid was successfully constructed,and the complemented strains(?::srtA USA300)was obtained.2.The effect of kaempferol on the growth of S.aureus.The minimum inhibitory concentration(MIC)of kaempferol against USA300 strain was determined by microdilution method.As a result,the MIC of kaempferol>1024?g/mL.It shows that kaempferol has no antibacterial activity against the USA300 strain.The growth curves of wild-type WT USA300,?srtA USA300,?::srtA USA300,and USA300 strains treated with kaempferol at different concentrations were determined.As a result,the growth curves of different strains were consistent.It shows that kaempferol does not affect the normal growth of the USA300 strain.3.The role of SrtA in the pathogenesis of S.aureus and the protective effect of kaempferol on infected mice.The SPF kunming mice were randomly divided into 5groups,WT USA300 group,?::srtA USA300 group,?srtA USA300 group,and WT-kaempferol group,and were inoculated with 2×10~8 CFU corresponding bacterial solution through the tail vein.In the WT-kaempferol group,2 hours after infection with WT USA300,150 mg/kg of kaempferol was injected subcutaneously twice daily.The control group was injected with 0.1 mL saline.Record the mortality of mice in each group,detect and compare the bacterial load of various organs,take kidney tissues for pathological observation,and determine renal function indexes(creatinine,urea nitrogen)and inflammatory factors(TNF-?,IL-1?)Level.Results,the mortality of mice(10 days)was71.627%,67.5%(P>0.05),30%,25.93%(P>0.05).Visceral tissue load:WT USA300 group and?::srtA USA300 group had similar bacterial load levels(P>0.05),?srtA USA300 was similar to WT-kaempferol group(P>0.05);?srtA USA300 group was significantly lower than WT USA300 group(P<0.01,P<0.05).Pathological observations:WT USA300 group and?::srtA USA300 group mice have atrophy,necrosis,renal interstitial hyperemia,edema,and diffuse hemorrhage in a large area.In the?srtA USA300 and WT-kaempferol groups,the renal tissue lesions were similar,the glomerular volume increased,and the capillary endothelial cells proliferated and swelled.Focal minor bleeding was observed,but the overall lesions were significantly alleviated.Renal function indexes(creatinine,urea nitrogen)and inflammatory factors(TNF-?,IL-1?)levels:WT USA300 group and?::srtA USA300group were consistent(P>0.05).The results of the?srtA USA300 group were comparable to the WT-kaempferol group(P>0.05).The levels of creatinine,urea nitrogen and inflammatory factors TNF-?and IL-1?in the?srtA USA300 group were significantly lower than those in the WT USA300 group(P<0.01).It showed that the results of the WT-kaempferol group and the?srtA USA300 group were highly consistent,both kaempferol and?srtA USA300 reduced the mortality of mice and alleviated renal tissue lesions.4.The effect of kaempferol on the transcription level of S.aureus SrtA and its anchored adhesion proteins fnbA,fnbB,clfA and clfB genes.The qRT-PCR test results showed that kaempferol only down-regulated the srtA gene transcription level.With the increase of kaempferol concentration,the inhibition of srtA gene increased gradually.When the kaempferol concentration was 64?g/mL,the srtA gene was suppressed by 49%,and the inhibitory effect no longer increases with the concentration of kaempferol.However,there was no significant difference in the transcription levels of adhesion proteins fnbA,fnbB,clfA and clfB genes.It was shown that kaempferol reduced the anchorage of cell wall adhesion proteins by inhibiting the transcription levels of SrtA,and ultimately reduces the adhesion efficiency of S.aureus.