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

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 R ) strains of Salmonella enterica. Mupirocin (Mup) is an isoleucyl-adenylate analog that inhibits the essential enzyme, isoleucyl-tRNA synthetase (IleRS). Mutations causing Mup 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 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.

Antimicrobials as promoters of genetic variation

“…Once these mutations are acquired, selection favours the elimination of the extra copies. Several studies have provided evidence of this process in a variety of genetic systems [88][89][90][91]. In addition, genome-wide overexpression screening for resistance determinants on E. coli revealed that bacterial genomes present a disturbing potential for adaptive amplification [92,93].…”

Novel Pathways for Ameliorating the Fitness Cost of Gentamicin Resistant Small Colony Variants

“…The adverse effect of the resistance mechanism can, however, be suppressed via compensatory events, increasing fitness of the resistant organism and thereby stabilizing the resistance mechanism in the bacterial population in the absence of antimicrobial selection pressure (Andersson and Hughes, 2010). Compensatory genetic events have been extensively studied in the suppression of the adverse effects of target-site resistance mutations (Björkman et al, 1998; Reynolds, 2000; Paulander et al, 2007; Paulander et al, 2010; Brandis et al, 2012). In these cases, selection has mainly been for compensatory events restoring the enzymatic activity that was reduced by the resistance mutation (Björkman et al, 1998; Reynolds, 2000; Paulander et al, 2007, 2010; Brandis et al, 2012).…”

“…Compensatory genetic events have been extensively studied in the suppression of the adverse effects of target-site resistance mutations (Björkman et al, 1998; Reynolds, 2000; Paulander et al, 2007; Paulander et al, 2010; Brandis et al, 2012). In these cases, selection has mainly been for compensatory events restoring the enzymatic activity that was reduced by the resistance mutation (Björkman et al, 1998; Reynolds, 2000; Paulander et al, 2007, 2010; Brandis et al, 2012). Examples include rifampicin resistance mutations in rpoB (Reynolds, 2000; Brandis et al, 2012), streptomycin resistance mutations in rpsL (encoding ribosomal S12 protein) (Björkman et al, 1998) and mupirocin resistance mutations in the ileS (encoding isoleucyl-tRNA synthetase) (Paulander et al, 2007, 2010).…”

Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

“…The precise mechanism of PKR antagonism by RhTRS1 is still unclear; however, it is known to inhibit the PKR pathway at a step after PKR autophosphorylation (14), and this phenotype was maintained in virus populations containing rhtrs1 amplifications (15). Gene amplification is a universal mechanism of rapid adaptation in eukaryotes (16,17), prokaryotes (18,19), and viruses (15,(20)(21)(22), enabling diverse adaptations such as neofunctionalization, antibiotic resistance, and evasion of host restriction factors (reviewed in reference 23). In general, amplification of a gene with weak activity can increase the fitness of an organism through overexpression related to gene dosage effects.…”