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

Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V.. F or V,,,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 V588F or V631F 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., V588F/V631F and G(593)V/V631F), but additional combinations also occurred involving novel mutations (R816C, H(67)Q, and F563L. First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V588F or V631F 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, E190K and E195K), 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.