Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus

Abstract

The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 microg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.

FIG. 1.

Screening by allele-specific PCR for the G1762T (V588F) mutation. Lanes 1 to 19 show results for selected strains and isolates (from left to right: SCR856, SKB 22, SKB 7, SKB 11, SKB 22, SKB 13, SKB 20, SKB 23, SKB 3, SCR501, SKB 14, SKB 18, SKB 30, SKB 24, SCR1412, SKB 15, SKB 26, SKB 4, and SKB 9). The positive PCR-amplified allele (603 bp) and internal control (764 bp) are indicated by arrows. − and +, absence and presence of G1762T. Lane W contained no template DNA (negative control), and lane M contained 0.5 μg of a 1-kb ladder size marker (Gibco-BRL, Paisley, Scotland).

FIG. 2.

Structure of IleS from S. aureus Oxford. Distribution of mutated residues in isolates showing mupirocin resistance. (A) Overall IleS structure showing bound tRNA^Ile and mupirocin (PDB:1ffy) (17). The backbone fold of IleS is shown as a ribbon, with individual sites of mutation highlighted displaying the residues' side chains (purple) and sequence numbers. The ribbon is colored from N to C terminus by domain as follows: turquoise, N-terminal domain; light green, Rossman fold; gold, CP1/Editing domain; blue, CP2 domain; brown, helical domain; dark green, C-terminal Junction domain; and turquoise, Zn binding domain. The anticodon region of tRNA^Ile is shown in orange. The side chains of residues His₃₉₂ and Tyr₃₉₄ in the editing site are shown in green. (B) Enlarged stereo view of the region of the Rossman fold adjacent to residues 588 and 631. The side chains of these residues in the S. aureus Oxford IleS are shown in purple, and the proposed orientation of the V588F mutation is displayed in blue (arrow). The side chains of some of the residues contributing to the hydrophobic pocket are also shown. The mupirocin molecule is colored by atom type (green, C; red, O) in CPK representation. The Connolly surface for the Oxford IleS is shown in red. Note that the benzene ring of V588F penetrates both this and the van der Waals surface of the mupirocin molecule.