Experimental mouse models were used to compare virulence and reproduction rate of three mouse-adapted variants of the PR8 influenza A virus strain. We observed large differences in pathogenicity in two mouse strains. The PR8M variant was lethal in DBA/2J mice but not in C57BL/6J mice, whereas PR8F and hvPR8 variants were lethal in both mouse strains. High lethality of PR8M in DBA/2J correlated with high viral load at early time points after infection and spread of the virus into alveolar regions. Also, higher viral loads and mortality in mice infected with PR8F resulted in a higher number of infiltrating leukocytes. 3D-protein structure predictions of the HA indicated amino acid sequence alterations which may render the HA cleavage site in PR8F more accessible to host proteases. Infection of C57BL/6J mice with a re-assorted PR8 virus revealed that the HA gene is the main determinant of virulence of the PR8F variant.

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.