engleski

Aminoglycoside resistance methyltransferase NpmA: functional characterization and interaction with ribosomal RNA.

Some antibiotic producing bacteria as well as a growing number of clinical pathogens contain different rRNA methyltransferases (MTases) from Arm and Kam family, which methylate a specific nucleotide in 16S rRNA, thereby rendering bacteria resistant to aminoglycoside antibiotics. Recently a novel plasmid mediated MTase NpmA has been detected in multiple aminoglycoside resistant clinical strain of Escherichia coli. NpmA methylates the N1 position of A1408 in the A site of 16S rRNA and is a member of Kam family. In this study we focused on the functional characterization of the NpmA MTase. By site-directed alanine mutagenesis we investigated 12 evolutionary conserved amino acids and their effect on enzyme activity and bacterial resistance to aminoglycoside antibiotics. We first determined the minimal inhibitory concentration for kanamycin. By comparing the in vivo activity of mutant variants with the action of the wild (wt) type enzyme we got the first insight into the localization of the catalytic site and cofactor S-adenosyl-methionine binding site. Using methylation assay in vitro we determined the enzyme activity on substrate 30S rRNA and, as expected, the enzyme activity of several mutants was significantly lower. Primer extension analysis of 16S rRNA methylated by the wt NpmA confirmed that methylation occurred at A1408 in 16S rRNA, while in several mutants it was decreased or completely absent, in agreement with other functional tests. We also investigated how the presence of the enzyme affects the biological cost. In growth competition assays we examined the ability of cells carrying the NpmA to compete with the cells lacking the enzyme. The results showed that the presence of the npmA gene represents a competitive advantage only in growth conditions containing aminoglycoside antibiotics. Predicting the structure of NpmA in complex with cofactor and substrate would be a great benefit for predicting potential inhibitors. Our results could therefore set a basis in possible finding of a specific inhibitor of NpmA MTase, which would in turn block this new type of clinical aminoglycoside resistance.

Antibiotic resistance; Methyltransferase; NpmA; Aminoglycosides; mutagenesis

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