engleski

Mutational analysis of the ribosomal A site reveals the binding pattern for the 16S rRNA A1408 methyltransferases from the clinical pathogen and a natural producer of aminoglycosides

The aminoglycosides represent a class of antibiotics that are often prescribed for the treatment of various infections caused by both Gram-positive and Gram-negative bacteria. They are natural substances derived from Streptomyces spp. or Micromonospora spp. or synthesized in vitro. Aminoglycoside-producing bacteria have evolved a self-protecting system that inhibits the aminoglycoside binding to their target by methylating specific ribonucleotides in antibiotic-binding sites of the ribosome. Enzymes that are involved in this process are members of 16S rRNA G1405 or A1408 methyltransferase subfamilies and were until recently only been found in the natural producers of aminoglycoside antibiotics. However, a number of occurrences of highlevel resistance to aminoglycosides in the clinic has been reported in recent time and a few novel G1405 and A1408 methyltransferases have been isolated from clinical pathogens. In this work, we studied the ribosomal A site binding pattern of two of the A1408 methyltransferases, KamB from the natural producer of aminoglycosides and NpmA, a novel methyltransferase isolated from a clinical pathogen. Both enzymes confer high-level resistance to aminoglycoside antibiotics by methylating the A1408 nucleotide in the 16S rRNA of the small ribosomal subunit. In our work we used a specialized E. coli system, in which all rrn operons were inactivated, and ribosomal RNA was transcribed from a vector- based rrn operon. We constructed single nucleotide mutations in the part of the operon corresponding to the A site of 16S rRNA. We introduced actively expressing enzymes in these cells and monitored their ability to grow in media supplemented with various concentrations of kanamycin. We determined minimal inhibitory concentration of kanamycin for these cells and analyzed the target nucleotide A1408 methylation with primer extension. Our results show that some of the point mutations introduced into 16S rRNA reduce the ability of both KamB and NpmA methyltransferases to effectively methylate the target nucleotide, A1408. We also observed a slight difference between the 16S rRNA binding patterns for these enzymes, suggesting that even though they have the same mode of action they might not bind to their substrate in the same manner. Our results presented here will be of great assistance in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.

16S rRNA A1408; Aminoglycoside resistance; NpmA

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