The Study On Antimicrobial Effect And Mechanism Of Simvastatin And Antimicrobial Peptide DEFB1/LYZ1

Methicillin-resistant Staphylococcus aureus(MRSA)can cause many diseases in livestock and poultry and cause great economic losses to livestock and poultry breeding industry.Penicillin binding protein 2a(PBP2a)is one of the main causes of MRSA resistance.It is anchored on the membrane of MRSA,and oligomerized in the functional membrane microdomains(FMM)of the membrane to play a role.In addition,MRSA also synthesizes wall teichoic acids(WTA)through cell membrane protein transport to further thicken cell walls and enhance resistance.Studies have shown that both statins and antimicrobial peptides can affect MRSA resistance,but the specific mechanism is unclear.Therefore,in this study,simvastatin and antimicrobial peptide DEFB1/LYZ1 were used as the main materials,and technical methods such as gene knockout,bacteriostatic test and animal test were carried around the cell membrane FMM stability of MRSA and the inhibition of drug resistance.The following research progress has been made:(1)Simvastatin restored MRSA’s sensitivity to antibiotics.The inhibitory effects of simvastatin at different concentrations on MRSA strains BA01611 and Mu50 were determined.The results showed that 50μM simvastatin did not affect the growth of MRSA.After treatment with 50μM simvastatin,the minimum inhibitory concentration(MIC)of oxacillin against MRSA decreased from 64 ～ 256μg/m L to < 1μg/m L.The results of BN-PAGE and Western Blot showed that simvastatin made PBP2 a of MRSA lose its pentamer state from the cell membrane and completely lost.In this section,simvastatin significantly inhibited the oligomerization of PBP2 a and restored the sensitivity of MRSA to oxacillin.(2)Simvastatin affects MRSA resistance independently of FMM flotillin and staphyloxanthin.The flotillin encoding gene flo A of FMM in BA01611 and Mu50 was knocked out.BN-PAGE and Western Blot analysis showed that PBP2 a of knockout strains was still pentamer.The MIC,co-incubation and bactericidal curve results of β-lactam antibiotics to Wild-type and knockout strains showed that resistance to knockout strains did not change.In the mouse experiment,the mortality rate of both wild-type and knockout strains of BA01611 was 100% after three days in mice infected separately and not treated with oxacillin in both groups.After oxacillin treatment,the survival rate of mice increased to12.5% and 18.8%,but there was no significant difference.And there was no significant difference in the bacterial load in the liver and kidney of mice in each group.In addition,the function gene crt M of staphyloxanthin in Mu50 was knocked out,and the determination of MIC showed that the resistance of knockout strain was not changed.But after 50μM simvastatin treatment,the expression levels of staphyloxanthin in BA01611 and Mu50 were down-regulated by 4.34 times and 2.31 times.The experiment in this section shows that simvastatin affects MRSA resistance by inhibiting PBP2 a oligomerization,which is not caused by the dysfunction of Flo A or staphyloxantnin,but may have a more complex mechanism.(3)The study on antibacterial function of DEFB1 and LYZ1.The LYZ1 was successfully cloned into the prokaryotic expression vector p ET-28 a,and was induced in the expression host E.coli BL21(DE3)and purified to obtain the antimicrobial peptide LYZ1 at a concentration of 0.9 mg/m L.The antimicrobial peptide DEFB1 was chemically synthesized.The results of bacteriostatic zone test showed that LYZ1 and DEFB1 had bacteriostatic activities against S.aureus,S.hemolyticus and S.epidermidis.The inhibition curves of DEFB1 and LYZ1 at different doses against MRSA strain Mu50 showed that the growth status of Mu50 decreased with the increase of DEFB1 and LYZ1 doses.And LYZ1 also inhibited the formation of Mu50 biofilm.The results of combination drug test showed that DEFB1 had synergistic antibacterial effect to Mu50 with oxacillin,nafcillin and cefazolin.Transmission electron microscopy showed that the cell morphology of Mu50 was obviously damaged by the combined use of DEFB1 and oxacillin.The experiment in this section indicated that DEFB1 and LYZ1 had good antibacterial activity and combined drug value against MRSA.In summary,simvastatin has weak bactericidal ability when used alone,but can depolymerize PBP2 a of MRSA from the cell membrane,and restore MRSA’s sensitivity to β-lactam antibiotics.This phenomenon has nothing to do with the dysfunction of FMM’s flotillin Flo A or staphyloxantnin,but simvastatin does affect the expression of staphyloxantnin,which may have a more complex mechanism of action on MRSA.In addition,antimicrobial peptides DEFB1 and LYZ1 had antibacterial activity against Staphylococcus,especially against MRSA strain Mu50,and DEFB1 had synergistic antibacterial activity with β-lactam to against Mu50.By exploring the inhibitory effect and mechanism of simvastatin and DEFB1/LYZ1 on MRSA,to provide theoretical basis for the prevention and treatment of various animal diseases caused by MRSA in livestock and poultry industry.