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. Secondstep 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.

Predicting the virulence of MRSA from its genome sequence

“…Many of the interactions fell on straight lines, suggesting that a small number of genetic loci containing SNPs may be interacting with numerous other loci. From these we identified five genes that interacted with more than 20 other loci with high statistical significance: the ileS gene encoding isoleucyltRNA synthetase (Hurdle et al 2004); the mreC gene involved in (Kyburz et al 2010); an uncharacterized gene on the beta-haemolytic converting phage (Bae et al 2006); the phytoene dehydrogenase gene, which is a key enzyme in the carotenoid biosynthetic pathway (Mijts et al 2005); and a small, putative, regulatory RNA molecule (ssr100) (Anderson et al 2006). Interestingly, the SNP in ileS has been shown previously to be responsible for conferring mupirocin resistance [V(588)F] (Hurdle et al 2004), suggesting this may have pleiotropic effects on gene expression.…”

Molecular Docking Study of Aminoacyl-tRNA Synthetases with Ligand Molecules from Four Different Scaffolds

“…These compounds were developed based on the structural backbone of commercial antibiotic, mupirocin and the isoleucyl-adenylate reaction intermediate with isoleucyl moiety in the tail part. 15,16 To investigate binding mode of aaRS with the developed derivatives, molecular docking simulations of four different scaffolds were carried out within the aminoacylation sites of eight different aaRSs (seven from E. coli and one from S. aureus).…”

Amplification of the Gene for Isoleucyl–tRNA Synthetase Facilitates Adaptation to the Fitness Cost of Mupirocin Resistance inSalmonella enterica

“…Mupirocin-resistance mutations in the chromosomal ileS gene cause a severe reduction in fitness due the impairment of the IleRS enzyme (Hurdle et al 2004;Paulander et al 2007a). Full activity can be restored to the mutant enzyme (Mup R ) by secondary mutations in the ileS gene.…”

“…Compensatory mutations can improve fitness, often without reducing the level of resistance (Andersson 2003(Andersson , 2006. This has been observed for several antibiotics, including the topical antibiotic mupirocin (pseudomonic acid A) (Hurdle et al 2004;Paulander et al 2007a).…”

“…This resistance was conferred by a plasmid that carried a gene (mupA) encoding a Mup-resistant isoleucyltRNA synthetase with 30% identity to the chromosomal IleRS enzyme (Hodgson et al 1994). Point mutations in the chromosomally encoded ileS gene were shown to confer a lower level of resistance (MIC ¼ 8-256 mg/ml) (Yanagisawa et al 1994;Antonio et al 2002;Hurdle et al 2004). Both types of mupirocin-resistant S. aureus were isolated at equal frequency from patients in longterm care facilities (Yoo et al 2006).…”