Mechanistic Study Of ErmB Leading To Macrolide Resistance

Macrolides have been widely used in clinic to treat infections caused by Gram-positive bacteria.However,with the wide and long time usage of this type of antibiotics,bacterial resistance has emerged and become increasingly severe.Erythromycin resistant methylase(erm)is one of the leading causes to high macrolide resistance.It has been proposed that erm catalyzes the mono-or dimethylation of A2058 in 23 S rRNA.After methylation,the antibiotic binding site in 23 S rRNA was hidden,thus preventing antiobiotic binding and leading to resistance to macrolide,lincosamide and streptogramin B.However,the methylation mechanism and its contribution to antibiotic resistance of erm still remain to be addressed.Previously our lab has discovered the presence of both ermB and ermT in Streptococcus gallolyticus subsp.pasteurianus isolated from the brains and spleens of ducklings from natural outbreaks.The inducible expression of the two genes is responsible for the high macrolide resistance in the isolates.Meanwhile,we also found that different isolates contain single nucleotide polymorphism(SNP)in ermB gene and exhibit amino acid changes and different MIC values to macrolides.In order to explore the methylation mechanism of ermB and its contribution to macrolide resistance,in this study,the expression constructs for ermB and ermT were optimized and ermB was chosen to be the focus of this study.Based on the SNPs in ermB genes in natural isolates along with bioinformatical analysis,32 ermB mutants were constructed and screened using micro-dilution method.Six mutants were selected and their methylation activities and MIC values to erythromycin,clarithromycin and lincosamide were measured.The results are as follows: 1.Construction of expression clones of erm B and erm T and the purification of erm B,erm T and 23 S rRNAermT was cloned into pET28 a and pET15 b,respectively,while ermB was constructed into pET21 b and pET28 a separately.Based on the results of the protein expression and purification,the ermB and ermT clones in pET28 a were chosen for this study.Purification was performed for ErmB,ErmT and MBP-MS2 as well as S.gallolyticus subsp.pasteurianus 23 S rRNA using affinity purification.2.Comparison of the methylation activities of erm B and ermTThe methylation activities of ermB and ermT were examined using the 3H-signal transferred from the methyl donor 3H-adomet to the substrate 23 S rRNA.The results indicated that both ErmB and ErmT can methylate S.gallolyticus subsp.pasteurianus 23 S r RNA and that ErmB appears to exhibit lower activity than ermT.3.Construction of erm B mutantsIn line with previous SNP information in ermB genes from S.gallolyticus subsp.pasteurianus isolates,ermB was chosen as the focus of this study.Sequence alignment of all erm genes were performed,and 32 mutants were constructed.4.Screening of erm B mutants under the MIC condition of WT erm BAll 32 ermB mutant strains were screened under the MIC condition of the wild type(WT)strain.In comparison to WT strain,while mutant strains No.40,69-2,83-1,139 exhibited reduced MICs to erythromycin and mutant strains No.40,69-2,116-1,139 showed lower MICs to lincosamide,mutant strains No.58-1,58-2,60-2,83-1,139,165 displayed reduced MICs to clarithromycin.5.MIC measurement of six mutant strains using micro-dilution methodBased on the screening results,six mutant strains were selected for MIC measurements.While mutant strain No.83-1exhibited MIC values of 512 ?g/mL,64 ?g/mL,512 ?g/mL to erythromycin,clarithromycin and lincosamide,strain No.139 demonstrated MIC values of 512 ?g/mL,64 ?g/mL and 128 ?g/mL,respectively.Meanwhile,strain No.165 displayed MICs of 1024 ?g/mL,128 ?g/mL,128 ?g/mL to these three antibiotics,and strain No.40 showed MICs of 512 ?g/mL,128 ?g/mL,256 ?g/mL,respectively.In addition,strain No.37 showed 1024 ?g/mL,256 ?g/mL,512 ?g/mL and strain No.39 exhibited 1024 ?g/mL,512 ?g/mL,1024 ?g/mL to three antibiotics.6.Methylation activities of WT and mutant ermBThe methylation activities of WT and mutant ermB were monitored using 3H-adomet as methyl donor and 23 S rRNA as substrate.The results suggested that ermB mutant No.37 and 39 exhibited higher activities than WT enzyme,while mutant No.40,83-1,139,165 demonstrated lower activities than WT enzyme.7.Construction of phylogenetic tree of erm BThe sequences of ermB from different species were analyzed and the phylogenetic tree was constructed using the Mrbays3.1.2 software.Conclusion: ermB-pET28 a and ermT-pET28 a resulted in better protein expression than other clones.Among 32 ermB mutants,mutant strain No.40,69-2,83-1,139 exhibited lower MICs than WT strain to erythromycin,while mutant strain No.40,69-2,116-1,139 demonstrated reduced MICs than WT strain to lincosamide,and mutant strain No.58-1,58-2,60-2,83-1,139,165 had decreased MICc than WT strain to clarithromycin.ErmB mutant No.37 and 39 exhibited higher acticities than WT ermB,while mutant No.40,83-1,139,165 displayed lower activites than WT ermB,and the trend is in agreement with MIC measurement.These data suggested that amino acid K40,D83,R139 and K165 are critical for ermB methylation and further exert effect on macrolide resistance.Whether these amino acids participate in substrate binding or catalysis still remains to be addressed.This study provided insight to methylation mechanism of erm,and also evidence for the first time to correlate MICs and methylation,pinpointing a new direction to address macrolide resistance problem.