Academic journal article Genetics

Amplification of the Gene for Isoleucyl-tRNA Synthetase Facilitates Adaptation to the Fitness Cost of Mupirocin Resistance in Salmonella Enterica

Academic journal article Genetics

Amplification of the Gene for Isoleucyl-tRNA Synthetase Facilitates Adaptation to the Fitness Cost of Mupirocin Resistance in Salmonella Enterica

Article excerpt

ABSTRACT

Mutations that cause resistance to antibiotics in bacteria often reduce growth rate by impairing some essential cellular function. This growth impairment is expected to counterselect resistant organisms from natural populations following discontinuation of antibiotic therapy. Unfortunately (for disease control) bacteria adapt and improve their growth rate, often without losing antibiotic resistance. This adaptation process was studied in mupirocin-resistant (Mup^sup R^) strains of Salmonella enterica. Mupirocin (Mup) is an isoleucyl-adenylate analog that inhibits the essential enzyme, isoleucyl-tRNA synthetase (IleRS). Mutations causing Mup^sup R^ alter IleRS and reduce growth rate. Fitness is restored by any of 23 secondary IleRS amino acid substitutions, 60% of which leave resistance unaffected. Evidence that increased expression of the original mutant ileS gene (Mup^sup R^) also improves fitness while maintaining resistance is presented. Expression can be increased by amplification of the ileS gene (more copies) or mutations that improve the ileS promoter (more transcription). Some adapted strains show both ileS amplification and an improved promoter. This suggests a process of adaptation initiated by common amplifications and followed by later acquisition of rare point mutations. Finally, a point mutation in one copy relaxes selection and allows loss of defective ileS copies. This sequence of events is demonstrated experimentally. A better understanding of adaptation can explain why antibiotic resistance persists in bacterial populations and may help identify drugs that are least subject to this problem.

DURING growth under nonselective conditions, spontaneous duplications form at rates (10^sup -5^-10^sup -2^/cell/division) that are several orders of magnitude higher than those for base changes (Anderson and Roth 1981; Andersson et al. 2009; Andersson and Hughes 2009; Reams et al. 2010). Further increases in gene copy number appear at about 10^sup -2^/cell/division. Copy-number changes are likely to be the first adaptive response when cell growth is limited by lack of some activity. For instance, copy number of the lacZYA, araBCD, and cat operons increases when growth is limited by ability to use lactose, arabinose, or benzote (Sonti and Roth 1989; Andersson et al. 1998; Reams and Neidle 2003). The frequent gene amplification and growth provides additional mutational targets and thereby enhances the likelihood of a rare point mutation that further improves gene function.

Mutations that provide resistance to an antibiotic usually reduce bacterial fitness (in the absence of antibiotic) due to alteration of an essential function (Andersson and Levin 1999). Compensatory mutations can improve fitness, often without reducing the level of resistance (Andersson 2003, 2006). This has been observed for several antibiotics, including the topical antibiotic mupirocin (pseudomonic acid A) (Hurdle et al. 2004; Paulander et al. 2007a).

Mupirocin is used mainly to inhibit methicillinresistant Staphylococcus aureus or b-hemolytic streptococci during skin infections (impetigo) and to prevent presurgery nasal carriage of S. aureus (Cookson 1998; Edlich et al. 2005; Mori et al. 2005). The antibiotic that is an analog of isoleucyl-adenylate inhibits protein synthesis by binding to class I isoleucyl-tRNA synthetases (IleRS), preventing attachment of isoleucine to its cognate tRNA (Schimmel and Soll 1979; Yanagisawa et al. 1994; Pope et al. 1998; Silvian et al. 1999; Nakama et al. 2001). After clinical introduction of mupirocin in 1985, highly resistant strains of S. aureus were isolated [minimal inhibitory concentration (MIC) > 256 μg/ml]. This resistance was conferred by a plasmid that carried a gene (mupA) encoding a Mup-resistant isoleucyl- tRNA 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 = 8-256 μg/ml) (Yanagisawa et al. …

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