The Establishment Of Erm Methylation System And Activities Of ErmB Mutants From Resistant Strains

Macrolide, lincosamide and streptogramin B(MLSB) have been extensively used for treating infections caused by Gram-positive bacteria. MLSB antibiotics inhibit protein biosynthesis by targeting the peptidyl transferase center within the 50 S ribosomal subunit. However, with the application of antibiotics, bacterial resistance problems have emerged. The most widespread mechanism of bacterial resistance to MLSB antibiotics is the modification of the target site exhibited by a family of r RNA methyltransferases designated Erm. Yet the detailed mechanisms of r RNA methylation by Erm still remain to be addressed. From S. gallolyticus subsp. pasteurianus isolated from veterinary clinic, our laboratory has identified the macrolide resistance mediated by erm genes and Erm B mutants associated with erm B single nucleotide polymorphism. The present study aims to establish the Erm methylation system and to investigate the methylation activities of Erm T, wild type and mutant Erm B associated with erm B SNP, in order to provide biochemical basis for the macrolide resistance of S. gallolyticus subsp. pasteurianus isolates from veterinary clinic. The results are as follows:1. The establishment of Erm methylation system:Plasmids of pc I857, p LK35 and p MBP-MS2 were obtained from r Rachel Green Laboratory in United States. After transformation into E. coli cells, these plasmids were extracted and sequenced. The 23 S r RNA of S. gallolyticus subsp. pasteurianus was cloned into the plasmid p LK23SMS2 and corrected by site-directed mutagenesis. The plasmid pc I857 was transformed into DH5α competent cells, and the resulting DH5α cells harboring pc I857 were prepared as competent cells. The p LK23SMS2 plasmid was introduced into the DH5α carrying the pc I857 by electroporation. The expression of S. gallolyticus subsp. pasteurianus 23 S r RNA was induced at 42 ℃. The S. gallolyticus subsp. pasteurianus 23 S r RNA was prepared by affinity purification using purified MBP-MS2. Also, Erm T, wild type and mutant Erm B were purified by His-tag purification.2. Methylation activity of Erm B and Erm T :In vitro enzyme activity assay using 3H-adomet as methyl donor was used to monitor the radioactivity of 23 S r RNA after methyltransfer by Erm methylases. While both Erm B and Erm T can methylate S. gallolyticus subsp. pasteurianus 23 S r RNA, Erm T exhibited higher activity than Erm B.3. Methylation site of Erm B and Erm T:Sequence alignment of the E. coli and S. gallolyticus subsp. pasteurianus 23 S r RNAs was performed using NCBI blast tool, and A2057 in S. gallolyticus subsp. pasteurianus 23 S r RNA was predicted to align with A2058 in the E. coli 23 S r RNA. The A2057 was mutated to G, and the A2057 G mutant 23 S r RNA was purified and used to test Erm methylation activity. The result showed that the activity on A2057 G mutant is 10 fold lower than that on wild type 23 S r RNA. This suggested that The A2057 may be the target site of Erm B and Erm T in S. gallolyticus subsp. pasteurianus 23 S r RNA.4. Methylation activities of Erm B mutants:The Erm B mutants I22V、D181G、V182A and V226 I were constructed by site-directed mutagenesis, and the mutant activities were assayed by enzyme kinetic experiment. The result showed that the activities of mutants I22V、D181G、V182A were comparable with wild type Erm B, while the V226 I mutant activity is higher than other mutants. These data were consistent with the MIC values measured in our lab.5. Phylogenetic tree of Erm methylases:The phylogenetic tree of Erm methylases was constructed by Mrbayes3.1.2. It indicated that Erm T, Erm C and Erm Y shared 77% of sequence homology, while Erm T and Erm B exhibited 52% of sequence homolgy.Conclusion: Both Erm B and Erm T can methylate S. gallolyticus subsp. pasteurianus 23 S r RNA, possibly at A2057. Among all Erm B mutants caused by erm B SNPs, the activities of mutants I22V、D181G、V182A were comparable with wild type Erm B, while V226 I exhibited higher methylation activity than other mutants. These data were in agreement with MIC data collected in our lab, and provided biochemical insight to macrolide resistance in the clinical isolates of S. gallolyticus subsp. pasteurianus.