Effect Of Ermb Mutation On Its Methylation Activity And Minimum Antibacterial Concentration

Since the first generation macrolide antibiotic erythromycin was introduced,it has been widely used because of its good therapeutic effect on gram-positive bacteria.However,due to abuse,most antibiotics can not be completely absorbed by the body,and up to 85%of antibiotics are excreted in the original form or metabolites.Over time,the growing problem of bacterial resistance has followed.The important mechanisms that cause high levels of resistance to macrolides are ribosomal methylation encoded by the ermB gene,macrolide exfoliation encoded by the mefA gene,and mutations in rRNA and ribosomal proteins.The ribosomal methylation encoded by ermB is the most common erythromycin resistance mechanism in China,Sri Lanka and South Korea.Studies have shown that erm can promote the methylation or dimethylation of bacterial ribosomes,changes in the structure of bacterial ribosomes after methylation,and hiding the spatial sites that bind to macrolides,resulting in macrolides.Antibiotics can not bind to bacteria and cause drug resistance.However,the detailed mechanism by which ERM methyltransferase mediated macrolides resistance is not clear.In particular,which amino acid residues in ERM methyltransferase mediated ERM methylation,the relationship between the degree of methylation and the minimum inhibitory concentration of wild ERM methyltransferase mutants,and the contribution of mutated amino acids to macrolides resistance were not reported at home and abroad.The laboratory's preliminary study isolated six strains of Streptococcus galactidis(S.Gallolyticus subsp.Pasteurianus),it was found that the high level of macrolide resistance in these isolates was caused by the joint induction of macrolide resistance genes ermB and ermT.On the basis of bioinformatics analysis,32 mutant strains of ermB have been constructed in previous studies.This study uses microbiological and pharmacological methods to conduct experiments based on the methods recommended by the American Clinical and Laboratory Standards Committee(CLSI)M07-A9.The MIC values of macrolides and lincolamide antibiotics were detected by agar plate method and trace amounts of Routangxishifa and bacteria containing these mutants.Erythromycin(Ery)and clarithromycin(Cla)were selected as representative drugs of macrolide antibiotics,and lincomycin(Lin)was selected as representative drugs of lincomide antibiotics;The wild type ermB and two MICs were selected to increase the value of wild type,the two MICs were not significantly changed compared to wild type,and the three MICs decreased compared to wild type.A total of 8 strains were extracted and purified.The MS2 protein of maltose label was extracted and purified.The ribosome 23S rRNA was extracted by the MS2 system affinity purification ribosome reported by Rachel Green Laboratory,and the methylation activity of wild ermB and mutant strains was detected.The methylation activity of ribosomes was explored using biochemical means.The findings are as follows:1.Using agar plate method rough sieve detection ermB,ermB mutation strain MIC valueWith reference to the method recommended by the American Clinical and Laboratory Standards Committee(CLSI),a rough screening of ermB and ermB mutant strains with minimum antibacterial concentration(MIC)was conducted using agar plate method.The results of the rough sieve experiment showed that:Compared with the wild ermB to erythromycin MIC(512?g/mL),the four mutants were more sensitive.They were E58A,D60E,R139A and K165A respectively;Compared with the wild ermB to the claymycin MIC(512?g/mL)value,the four mutants are more sensitive.They are D60E,R76A,R139A and K165A.Compared with the wild ermB to the Lincomycin MIC(512?g/mL)value,the four mutants are more sensitive.They are E35A,D60E,E116A and E127A.2.The determination of ermB,ermB mutants with trace of RoutangxishifaFirst,the reliability of the drug prepared by the quality control bacterium Staphylococcus aureus ATCC 29213 was verified,and the drug that meets the standards could be tested.Using the above three antibiotics,the antibiotics are diluted by double dilution and the concentration gradients(?g/mL)are 1024,512,256,128,64,32,16,8,4,2,1 and 0.5.The results of fine sieve showed that 4 mutants were more sensitive than wild ermB to erythromycin MIC(1024?g/mL).Compared with the wild ermB to the claymycin MIC(512?g/mL)value,6 mutants are more sensitive;Compared with the wild ermB to Lincomycin MIC(1024?g/mL),3 mutants are more sensitive.Among them,the MIC values of the mutated strain R139A against erythromycin,claymycin,and lincomycin were 32?g/mL,16?g/mL and 512?g/mL respectively;The MIC values of the mutated strain K165A against erythromycin,claymycin,and lincomycin were 64?g/mL,32?g/mL and 512?g/mL respectively;The MIC values of the mutated strain D60E against erythromycin,claymycin and lincomycin were 128?g/mL,128?g/mL and256?g/mL,respectively;The MIC values of mutated strains K40A and E58D were basically increased;The MIC values of the mutated strains K188A and K196A remained basically unchanged.3.Study on the expression conditions of ermB proteinThe conditions of expression of ermB protein were investigated.The selected temperatures were 25°C and 37°C and IPTG concentrations were 0.4 mmol/L,0.8mmol/L,1.0 mmol/L and 1.2 mmol/L respectively.The results showed that at a temperature of 37°C,Regardless of the concentration of IPTG,the proteins are almost all expressed in precipitation and have less expression in the upper Qing Dynasty.At a temperature of 25°C and an IPTG concentration of 1.0 mmol/L,the upper clear expression was relatively high,so a subsequent protein extraction with a concentration of1.0 mmol/L was performed at 25°C.After the determination of the MIC value of three antibiotics,each MIC value was compared with the MIC value of wild ermB.The wild type ermB and two MICs were selected to increase the wild type,the two MICs were not significantly changed,and the three MICs decreased the wild type.A total of 8 mutant strains were extracted and purified.12%of SDS-PAGE identification.4.Extraction S.Gallolyticus subsp.Pasteurianus 23S rRNA and purificationUsing the method reported by Rachel Green Laboratory to use MS2 system affinity to purify ribosomes.First isolate and purify MBP-MS2 protein,concentrate it for S.Gallolyticus subsp.Extraction of pasteurianus 23S rRNA.A lot of broken S.Gallolyticus subsp.Pasteurianus and ribosomal purification buffer B undergo a density gradient centrifugation and precipitate.The precipitate is diluted and added to a column of maltose label bound with MBP-MS2 protein.The ribosomal elution buffer D is eluted and the elution is collected.After concentration,12%of the urine gel is identified.5.Detection and analysis of methylase activity of ermB and ermB mutantsMethylase activity of wild ermB and ermB mutants was detected by using ~3H labeled S adenosine methionine as a methyl donor and removing streptococcal ribosome 23S rRNA as a methylation substrate.The results showed that the mutated strains D60E,R139A and K165A were lower than wild ermB methylase;The methylase activity of mutated strains K40A and E58D was higher than that of wild ermB.Mutant strains K188A,K196A.The methylase activity and the wild ermB methylase activity were slightly reduced but basically equivalent;Conclusion:Compared with the MIC value of wild ermB,the mutated strains of macrolide erythromycin MIC were:E58A,D60E,R139A and K165A;The mutated strains with reduced values for macrolides:D60E,R76A,R139A,K165A and E201A;The mutated strains with reduced value of Lincomycin MIC were:E35A,D60E,E116A and E127D.The enzyme activity of wild ermB and mutant strains D60E,R139A,K165A,K40A,E58D,K188A and K196A were measured respectively.The results showed that the enzyme activity of mutated strains D60E,R139A and K165A was less than that of wild ermB,and the enzyme activity of mutated strains K40A and E58D was greater than that of wild ermB.The enzyme activity obtained by mutated strains K188A and K196A was slightly smaller than that of wild ermB,and the result was basically the same as the change trend of MIC value.Therefore,the mutations of the 60th,139th and 165th amino acids have a greater impact on ermB methylase activity and indirectly affect the resistance of macrolides.In this study,the mechanism of action of erm gene(ermB)in macrolide resistance and the binding of ermB protein to ribosome 23S rRNA to macrolide resistance were revealed by measuring the value of MIC and ermB methylation activity and comparing the intrinsic relationship between the two.It provides a new direction to solve the resistance of macrolides caused by ribosome methylation and provides a theoretical basis for the research and development of antibacterial additives.