Analysis of mupirocin resistance and fitness in Staphylococcus aureus by molecular genetic and structural modeling techniques

Abstract

Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V(588)F or V(631)F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V(588)F or V(631)F mutations in IRS, but a new mutation (G(593)V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V(588)F/V(631)F and G(593)V/V(631)F), but additional combinations also occurred involving novel mutations (R(816)C, H(67)Q, and F(563)L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V(588)F or V(631)F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A(196)V, E(190)K, and E(195)K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.

FIG. 1.

Amino acid sequence alignment of three regions in the S. aureus (S.a) IRS enzyme involved in mupirocin resistance, with the IRS enzymes of Escherichia coli K-12 (E.c; GenBank accession no. SYECIT), M. thermoautotrophicum Marburg (M.a; SYEXI), and Thermus thermophilus (T.t; P56690). The consensus sequences involved in ATP binding are boxed. Important amino acids involved in isoleucine binding are underlined. First- and second-step mutations (superscripts 1 and 2) in the S. aureus IRS are shown in boldface above the wild-type amino acid.

FIG. 2.

Correlation between fitness of mupirocin-resistant mutants evaluated by doubling time and competitive fitness (W).

FIG. 3.

Survival of mupirocin-resistant mutants in a murine wound abscess model.

FIG. 4.

Changes occurring in mupirocin susceptibility (A) and doubling time (B) during passage of unfit second-step mupirocin-resistant mutants.

FIG. 5.

Detail of mupirocin-binding region in S. aureus IRS. Residues that have been mutated are shown in green, and the mutation is indicated in italics. Nucleoside residue G68, in Ile tRNA, is also included.

FIG. 6.

Structures of mupirocin (structure 1), the reactive intermediate (structure 2), and the sulfonamide-based mimic (structure 3).

FIG. 7.

Overlay of the X-ray crystal structure of IRS from T. thermophilus (green) containing the reactive intermediate mimic (yellow) on the modeled reactive intermediate (Ile-AMP) bound within the IRS of S. aureus.

FIG. 8.

Comparative views of bound structures of mupirocin (top), reactive intermediate (red, bottom), and superposition of structures (middle) in the IRS of S. aureus.

FIG. 9.

Detail of the acylated intermediate binding region in IRS of S. aureus. Residues that have been mutated are shown in green, and the mutation is indicated in italics.