Multiple mechanisms to ameliorate the fitness ... - Wiley Online Library

Molecular Microbiology (2007) 64(4), 1038–1048

doi:10.1111/j.1365-2958.2007.05713.x

Multiple mechanisms to ameliorate the fitness burden of mupirocin resistance in Salmonella typhimurium Wilhelm Paulander,1 Sophie Maisnier-Patin1 and Dan I. Andersson2* 1 Department of Bacteriology, Swedish Institute for Infectious Disease Control and Microbiology, Tumor and Cell Biology Center, Karolinska Institute, S-171 82 Solna, Sweden. 2 Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75123 Uppsala, Sweden. Summary We examined how the fitness costs of mupirocin resistance caused by mutations in the chromosomal isoleucyl–tRNA synthetase gene (ileS) can be ameliorated. Mupirocin-resistant mutants were isolated and four different, resistance-conferring point mutations in the chromosomal ileS gene were identified. Fifty independent lineages of the lowfitness, resistant mutants were serially passaged to evolve compensated mutants with increased fitness. In 34/50 of the evolved lineages, the increase in fitness resulted from additional point mutations in isoleucine tRNA synthetase (IleRS). Measurements in vitro of the kinetics of aminoacylation of wild-type and mutant enzymes showed that resistant IleRS had a reduced rate of aminoacylation due to altered interactions with both tRNAIle and ATP. The intragenic compensatory mutations improved IleRS kinetics towards the wild-type enzyme, thereby restoring bacterial fitness. Seven of the 16 lineages that lacked second-site compensatory mutations in ileS, showed an increase in ileS gene dosage, suggesting that an increased level of defective IleRS compensate for the decrease in aminoacylation activity. Our findings show that the fitness costs of ileS mutations conferring mupirocin resistance can be reduced by several types of mechanisms that may contribute to the stability of mupirocin resistance in clinical settings.

Accepted 21 March, 2007. *For correspondence. E-mail Dan. [email protected]; Tel. (+46) 18 4714175; Fax (+46) 18 4714673.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd

Introduction Mupirocin is a topical antibiotic that is primarily used for eradicating Staphylococcus aureus during nasal colonization and for treating skin infections (impetigo) caused by S. aureus and beta-haemolytic streptococci (Cookson, 1998; Watanabe et al., 2001; Edlich et al., 2005; Mori et al., 2005). Mupirocin binds to the class I isoleucyl– tRNA synthetase (IleRS) from several eubacteria and prevents protein synthesis by inhibiting the aminoacylation of isoleucine (Ile) to the cognate tRNA (Yanagisawa et al., 1994; Pope et al., 1998; Silvian et al., 1999). The aminoacylation reaction proceeds in two steps: first Ile is activated in presence of ATP resulting in formation of isoleucyl–adenylate (Ile-AMP) which in the second step is transferred to the 3′ terminus of its cognate tRNA to yield isoleucyl–tRNA (Schimmel and Soll, 1979). Protection of IleRS from tryptic digestion when ATP or mupirocin is bound, together with data from the crystal structures of IleRS in complex with the antibiotic, show that mupirocin acts as an analogue of Ile-AMP thereby blocking the binding of the activated amino acid to the catalytic cleft of the Rossman fold domain of IleRS (Yanagisawa et al., 1994; Silvian et al., 1999; Nakama et al., 2001). The moiety of mupirocin that resembles the hydrophobic sidechain of Ile is recognized by amino acids in the isoleucine binding pocket in the aminoacylation catalytic site while the pyran ring and the C1 to C3 of the antibiotic mimics the interactions of the ribose and adenine moieties of Ile-AMP with IleRS. Mupirocin resistance in clinical isolates of S. aureus results from two different mechanisms: either the uptake of a plasmid carrying the mupA gene, encoding an isoleucyl–tRNA synthetase sharing 30% identity with the native enzyme (Hodgson et al., 1994), or by point mutations in the chromosomally encoded ileS gene (Gilbart et al., 1993; Antonio et al., 2002). High level of mupirocin resistance [minimum inhibitory concentration (MIC) > 500 mg ml-1] is conferred by presence of the plasmid-encoded IleRS, while single amino acid substitutions in the native S. aureus or Escherichia coli IleRS confer a lower level of resistance (MIC = 8–256 mg ml-1) (Yanagisawa et al., 1994; Antonio et al., 2002; Hurdle et al., 2004). In S. aureus, the level of chromosomally encoded resistance to mupirocin can be further increased after acquisition of additional mutations in ileS (Hurdle

Mupirocin resistance in Salmonella typhimurium 1039 Table 1. Mupirocin-resistance mutations in S. typhimurium LT2. Generation time (min)

Strain number

Amino acid change

MIC (mg ml-1)

In absence of mupirocin

In presence of mupirocin (100 mg ml-1)

JB124 (susceptible wild type) JB1850 JB1853 JB1855 JB1872

– WV630-631L H594Y F596L W443R

16–24 > 1024 > 1024 > 1024 > 1024

18 59 33 30 75

No growth 74 80 72 138

et al., 2004). These secondary mutations result in a decreased fitness that can be partly restored by at least five different point mutations in the ileS gene (Hurdle et al., 2004). With regard to the development of mupirocin resistance in clinical isolates of S. aureus, depending on the clinical setting trends with either increasing or decreasing frequencies of resistance have been reported (Watanabe et al., 2001; Yun et al., 2003; Lobbedez et al., 2004; Yang et al., 2006). A recent investigation of the frequency of resistance of S. aureus strains isolated from long-term care facilities showed that 11% of the isolates were resistant, with an approximately equal distribution between high and low level resistance (Yoo et al., 2006). The fitness cost of mutational antibiotic resistance results from disturbances of cellular functions and enzymes and is usually expressed as a reduced growth rate in antibiotic-free environments. The presence of such costs suggest that if antibiotic use is reduced, resistance would decrease in frequency because the more fit susceptible bacteria will out-compete the resistant ones (Andersson, 2006). However, compensatory mutations that restore the activity of the altered cellular functions without reducing the level of resistance may allow highly resistant clones to be maintained in the population (Björkman et al., 1998; Björkman and Andersson, 2000; Reynolds, 2000; Björkholm et al., 2001; Maisnier-Patin et al., 2002; Besier et al., 2005; Nilsson et al., 2006). The aims of this study were to determine in Salmonella enterica var. Typhimurium LT2 (S. typhimurium throughout the text), a bacterium more amenable to genetic analysis than S. aureus: (i) the effect of mupirocin resistance due to mutations in the chromosomally encoded ileS gene on fitness and on tRNA aminoacylation and (ii) the rate and mechanisms for restoring fitness by evolving the low-fitness resistant lineages in growth medium with or without mupirocin. In absence of antibiotic, most lineages restored fitness by acquisition of compensatory mutations in ileS that concomitantly caused a reduction in the level of resistance. For lineages evolved in the presence of mupirocin different and fewer intragenic compensatory mutations were found or fitness improvement resulted from gene amplification. The combined data on IleRS structure, kinetics, bacterial fitness and genetics are used to discuss the long-term stability of antibiotic

resistance. Such data might also allow us to describe and predict the rate and trajectory of the emergence of different antibiotic resistances, including the chromosomally encoded mupirocin resistance. In addition, studies of compensatory mechanisms are important for our understanding of many fundamental evolutionary processes such as the evolution of intra- and inter-molecular interactions, genetic networks and developmental circuits, new species and extinction processes (Poon and Otto, 2000; Poon and Chao, 2005; Poon et al., 2005; Ferrer-Costa et al., 2007; Haag, 2007).

Results Mutations conferring resistance to mupirocin Resistant mutants were selected on Luria–Bertani agar (LA) plates containing 500 mg ml-1 mupirocin, which corresponds to 31 times the MIC of the wild typesusceptible S. typhimurium strain (16 mg ml-1), and they appeared after 4–8 days of incubation (median frequency of 1 ¥ 10-9). All resistant clones had a MIC > 1024 mg ml-1(Table 1). Sequencing of the predicted target gene ileS from 12 independent resistant clones identified four types of resistance mutations (Table 1). As these four mutations were repeatedly isolated it is likely that we have identified most of the potential single-step mutations in IleRS that confer a MIC > 500 mg ml-1. Three mutations were single nucleotide changes leading to an amino acid substitution (W443R, H594Y and F596L) and one mutation was a deletion of three nucleotides resulting in a concomitant amino acid deletion and substitution (WV630-631L). The mutated residues lie within the IleAMP/mupirocin binding pocket at the aminoacylation site, and are near the resistance mutations previously identified in S. aureus (Fig. 1) (Silvian et al., 1999; Nakama et al., 2001; Antonio et al., 2002; Hurdle et al., 2004). The residues H594 and F596 are part of the binding site of mupirocin or Ile-AMP and replacement of these particular amino acids might weaken interactions between mupirocin and IleRS (Nakama et al., 2001). The residues W443 and WV630-631L are located further away from the antibiotic-binding site but close to conserved regions

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 1038–1048

1040 W. Paulander, S. Maisnier-Patin and D. I. Andersson Fig. 1. Location of the residues that confer resistance to mupirocin on the structure of the IleRS of Staphylococcus aureus (PDB accession number 1FFY). The signature motifs of the catalytic domain (red) and mupirocin (black) are indicated. The mutated amino acids residues are symbolized by blue spheres for the resistance mutations isolated in Salmonella typhimurium and by purples spheres for mutations isolated in Staphylococcus aureus. All molecular representations were produced by using PyMOL (http://pymol.sourceforge.net).

involved in Ile binding (blue shaded spheres, Fig. 1) (Schmidt and Schimmel, 1995). Fitness of the resistant mutants Growth rates of the resistant mutants were determined in liquid medium [Luria–Bertani (LB)] and in LB containing 100 mg ml-1 of mupirocin. The resistant mutants had as compared with the wild type (fitness set to 1.0) a relative fitness of 0.24–0.60 in LB and 0.13–0.24 in LB with mupirocin (Table 1, Fig. 2). The resistance mutations F596L and H594Y had the smallest effect on fitness whereas the W443R mutant grew slowly both with and without mupi-

Fig. 2. Fitness of wild-type (wt, squares), mupirocin-resistant (R, squares) and compensated (circles) strains evolved in LB (E) or LB containing 100 mg ml-1 of mupirocin (E + M). The relative growth rate is given as the ratio of the susceptible (wild-type) strain growth rate divided by the growth rate of the resistant or compensated strain. Growth rates were measured in LB (filled symbols) and LB containing 100 mg ml-1 of mupirocin (open symbols). Wild-type fitness in LB was set to 1.0. The amino acid substitutions in IleRS that confers mupirocin resistance are indicated under the x-axis. The strains (numbers in parenthesis) that did not grow in medium containing mupirocin are indicated below the dotted line.

rocin present (Table 1, Fig. 2). In control experiments where a plasmid-borne wild-type ileS gene was introduced to the resistant mutants the growth rate was restored to the susceptible parent strain, demonstrating that the fitness reductions observed were caused by the ileS mutations (not shown).

Compensatory evolution of mupirocin-resistant mutants We determined if the fitness costs of resistance could be reduced by compensatory mutations. Fifty independent lineages of the mupirocin-resistant mutants were evolved: 10 lineages of each mupirocin-resistant mutant were evolved in the absence (five lineages) or presence (five lineages) of mupirocin with the exception of the resistant mutant F596L from which a total of 20 lineages were evolved (10 in absence of antibiotic and 10 in presence of antibiotic). Appearance of spontaneous mutants with faster growth rate was regularly screened for by visual examination of bacterial colony size on LB agar plates as previously described (Maisnier-Patin et al., 2002). Fast-growing mutants generally appeared faster in LB than in LB containing mupirocin (80–360 generations versus 260–520 generations). Mutation rates for compensation in LB were estimated from the fitness of the resistant and compensated mutants, the population sizes during serial passage and the number of generations required for fixation of the compensated mutants (Maisnier-Patin et al., 2002). For the four resistant mutants examined, the compensatory mutation rates varied between 2 ¥ 10-9 and 6 ¥ 10-9 per cell per generation. As shown in Fig. 2, all the evolved mutants had increased their fitness in LB compared with the ancestral resistant strains. In LB the relative fitness of the evolved mutants varied between 0.66 and 1 and fitness improve-


Mupirocin resistance in Salmonella typhimurium 1041 Table 2. Spectrum of compensatory mutations identified in 50 independently passaged S. typhimurium lineages. Compensatory change in evolved lineages Resistance mutation

Amino acid

Mutation

Number of lineages (50 total)

Presence of mupirocin during serial passage

MIC (mg ml-1)

W443R W443R W443R W443R W443R H594Y H594Y H594Y F596L F596L F596L WV630–631L W443R W443R W443R W443R H594Y H594Y H594Y H594Y H594Y H594Y H594Y F596L F596L F596L F596L WV630–631L WV630–631L WV630–631L WV630–631L WV630–631L WV630–631L WV630–631L WV630–631L

E103A S193A A252D I413S V631G Unknown Y594C V631G A650V L195Q A252T V529A V185M A417V I444S V631G I444S V631G G448C G595C T634S S643A – Unknown Unknown I66L – Unknown Unknown A198V P336L G337V H338Q A417V P340L I413S Q420H –

A307->C T576->A C754->A T1237->G, T1891->G Unknown A1780->G T1891->G C1948->T T583->A G753->A T1585->C G552->A C1249->T T1330->G T1891->G T1330->G T1891->G G1341->T G1781->T C1900->G T1926->G ileS amplification Unknown Unknown A195->C ileS amplification Unknown Unknown C592->T C1006->T G1009->T C1013->A, C1249->T C1018->T T1237->G G1259->C ileS amplification

1 1 1 1 1 2 5 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 2 4 4 1 1 1 1 1 1 1 1 2

– – – – – – – – – – – – + + + + + – – + + + – + + + – – + – – + + – +

48–64 16–24 32 64–128 16–24 32 128–256 128–192 192 64 252–384 128–192 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 512–768 > 1024

ment was highest in resistant lineages carrying the W443R mutation. As a control five susceptible parental (wild-type) lineages were cycled for 440 generations. These lineages did not show any fitness increase or mutations in ileS, indicating that the mutations identified (see below) were not generally increasing fitness, as then they should also have been selected in the susceptible

strain. Of the 25 LB-cycled lineages that had increased fitness, seven maintained the same resistance level (MIC > 1024 mg ml-1) as the parental strain, while 18 showed a substantial reduction in MIC (Table 2 and Fig. 3). Susceptibility to mupirocin of five of these 18 mutants became close or equal (MIC = 32 mg ml-1) to wild-type S. typhimurium (Table 2). As expected, all linFig. 3. Fitness of wild-type (wt), mupirocin-resistant (R) and compensated clones evolved in LB (E) or LB containing 100 mg ml-1 of mupirocin (E+M) as a function of the MIC values. The relative growth rate is given as the ratio of the susceptible (wild-type) strain growth rate divided by the growth rate of the resistant or compensated strain. Growth rates were measured in LB (panel A) and LB containing mupirocin (panel B). Wild-type fitness in LB was set to 1.0. The strains (numbers in parenthesis) that did not grow in medium containing mupirocin are indicated below the dotted line.


1042 W. Paulander, S. Maisnier-Patin and D. I. Andersson Fig. 4. Location of the compensatory mutations in IleRS on the structure of the IleRS of Staphylococcus aureus (PDB accession number 1FFY). The signature motifs of the catalytic domain (orange), the binding site for Ile (light blue), the conserved regions of the editing domain (grey) and mupirocin (black) are indicated. Mutated amino acid residues are symbolized by red spheres (compensatory mutations) and blue spheres (resistance mutations).

eages evolved in LB with mupirocin (100 mg ml-1) maintained a MIC of > 1024 mg ml-1. There was a weak negative correlation between fitness and MIC when the compensated mutants were grown in the absence of mupirocin whereas with mupirocin present fitness and MIC were positively correlated (Fig. 3). Finally, for all evolved lineages, including those serially passaged in presence of mupirocin, fitness was generally higher in LB than in LB with mupirocin (Fig. 2). Compensatory mutations that improve fitness of mupirocin-resistant mutants Among the 50 lineages, compensatory mutations located within the ileS gene were found in 34 lineages but for the remaining 16 lineages no additional ileS mutation was found. The intragenic mutations were mostly found in the lineages evolved in LB (22/25) while for lineages evolved in presence of mupirocin fewer mutations (12/25) were located in the ileS gene (Table 2). A total of 24 different single nucleotides substitutions and three double substitutions were identified in the ileS gene. As the double substitutions were a combination of the isolated single ones, this corresponds to 23 identified amino acid substitutions at 22 positions. Most compensatory mutations were isolated once with the exception of V631G that was found in nine independent evolved lineages, carrying either the resistance mutation F596L (five times) or W443R (four times). As most mutations were only recovered once it is likely that IleRS is not saturated with all existing compensatory mutations (Maisnier-Patin et al., 2002). Gene dosage analysis by Southern hybridization and real-time polymerase chain reaction (PCR) of the 16 lineages, which showed no addi-

tional mutation in ileS, revealed that in seven of them the copy number of ileS was increased two- to sixfold. Using recA as an internal control, the relative amounts of ileS in the compensated strains were twofold (DA9150), sixfold (DA9155), twofold (DA9163), twofold (DA9167), fourfold (DA9169), twofold (DA9171) and fourfold (DA11024) higher than the parental mupirocin-resistant strain and wild-type S. typhimurium. We found that ileS expression measured by real-time RT-PCR correlated well with ileS amplification levels, except for one strain (DA9150) in which ileS expression was four times higher than amplification. By providing an increased amount of the impaired IleRS expressed from an expression vector, we confirmed that an increased gene dosage of the mutated IleRS enzymes increased fitness of mupirocin-resistant S. typhimurium (data not shown). Localization of the compensatory mutations We used the crystal structure of S. aureus IleRS to localize the compensatory mutations found in the S. typhimurium enzyme. The 22 positions of amino acid substitutions found among the 34 lineages with intragenic compensatory mutations clustered at two main domains of the enzyme (red spheres, Fig. 4). Ten mutated residues (I66, I444, G448, V529, Y594, G595, V631, T634, S643 and A650) were located in the Ile-AMP binding-site pocket in close proximity to the resistance mutations (blue spheres, Fig. 4). The residue E103 mapped further away from the catalytic site but still close to a region previously identified as a binding site for Ile (blue shade, Fig. 4) (Schmidt and Schimmel, 1995). The remaining 12 mutant residues (V185, S193, L195, A198, A252, P336, G337,


(0.13) (0.079) (0.29) (0.25) (0.77) W443R, S193T W443R, I413S, V631G W443R, A252D W443R, V631G W443R, A417V DA8978 DA8980 DA8983 DA9013 DA9015


NA, not applicable because the strain does not grow in presence of 100 mg ml-1 mupirocin. ND, an accurate determination could not be performed because of the high Km.

28.8 17.6 63.9 55 172.5 (0.59) (0.26) (0.44) (0.29) (0.41) 1.99 0.86 1.47 0.99 1.38 (4.6) (3.3) (1.5) (1.2) (0.5) 0.069 0.049 0.023 0.018 0.008 (0.45) (0.84) (0.5) (0.41) (0.47) 0.8 1.5 0.9 0.73 0.84 (0.5) (0.5) (0.26) (0.18) (0.47) 2.52 2.5 1.31 0.89 2.36 (0.72) (0.60) (0.52) (0.44) (1.0) 2.02 1.70 1.45 1.23 2.82 NA NA NA 0.53 0.61

– H594Y W443R JB124 JB1853 JB1872

1.04 0.96 0.90 0.85 0.81

3.36 (1) 1.15 (0.34) 0.2 (0.06) 0.015 (1) 0.28 (19) ND 1.79 (1) 0.75 (0.42) 1.26 (0.7) 5 (1) 0.6 (0.12) 0.19 (0.038) 2.79 (1) 0.81 (0.29) 0.15 (0.05) 1 0.25 0.14

kcat tRNAIle (s-1) (relative kcat) Km tRNAIle (mM) (relative Km) LB+Mup LB

Relative fitness

Amino acid substitution(s) in IleRS Strain number

Table 3. In vitro kinetic data of IleRS aminoacylation.

To assess the compensatory effect of the second-site mutations found in ileS, we performed in vitro aminoacylation kinetics. IleRS variants carrying resistance mutations (W443R and H594Y), compensatory mutations (S193A, A252D, A417V, V631G and V631G+I413S in combination with the resistance mutation W443R) and the wild-type enzyme were purified to 70–90% homogeneity. In the aminoacylation reaction, tRNA or ATP concentrations were varied and aminoacyl–tRNA formation was measured in the linear phase to determine the respective Km and Vmax values. The wild-type S. typhimurium IleRS showed a Km (tRNA) for tRNAIle of 2.8 mM and a kcat (tRNA) of 5 s-1, resulting in a catalytic efficiency kcat (tRNA)/Km (tRNA) of 1.8 mM-1 s-1(Table 3), similar to that previously determined for E. coli (Shepard et al., 1992; Xu et al., 1994). IleRS purified from the resistant mutant strains H594Y and W443R showed a decrease in Km (tRNA) and kcat (tRNA) by 3- to 19-fold and 8- to 26-fold respectively. The resulting kcat (tRNA)/Km (tRNA) ratio (R-factor) was reduced 30% to 60% for the resistant mutants as compared with the susceptible strain (Table 3). IleRS from the five different compensated mutants showed, as compared with the resistant mutants, an increase in both kcat (tRNA) and Km (tRNA) whereas the aminoacylation efficiency (kcat (tRNA)/Km (tRNA)) was increased for some compensated mutants and decreased for others. When examining the growth rates of the different strains and the kinetic parameters kcat (tRNA), Km (tRNA) or kcat (tRNA)/Km (tRNA) of the respective IleRS enzymes, a correlation with fitness was seen only for kcat (tRNA) (Table 3). Thus, the resistant mutants showed a reduced kcat (tRNA) (0.2–0.6 s-1) as compared with the wild-type (5 s-1) and the compensated mutants showed a partial restoration of kcat (tRNA) (0.9–2.5 s-1). The ATP titrations showed that the Km (ATP) values for the resistance mutants H594Y and W443R were increased at least 20-fold compared with the sensitive wild-type strain. Additional compensatory mutations in IleRS decreased Km (ATP) back to wild-type level (Table 3). Furthermore, the kcat (ATP) was decreased for the resistant mutants three- to 17-fold and the compensated

kcat/Km (mM-1 s-1) (relative kcat/Km)

Wild type and mutant IleRS in vitro aminoacylation kinetics

1 0.60 0.24

Km ATP (mM) (relative Km)

kcat ATP (s-1) (relative kcat)

kcat/Km (mM-1 s-1) (relative kcat/Km)

H338, P340, I413, A417 and Q420) mapped at a large insertion in the Rossman fold domain called the connective polypeptide 1 domain (CP1). CP1 contains two distinct but overlapping subsites for the pre- and posttransfer editing pathways (Schmidt and Schimmel, 1994; Hendrickson et al., 2002). Five of the compensatory mutations (A252, P336, G337, H338 and P340) were located near or within the highly conserved regions (see grey shade in CP1, Fig. 4) involved in the editing pathways (Hendrickson et al., 2002; Fukunaga et al., 2004; Fukunaga and Yokoyama, 2006).

224 (1) 4.1 (0.018) –

Mupirocin resistance in Salmonella typhimurium 1043

1044 W. Paulander, S. Maisnier-Patin and D. I. Andersson mutants showed a partial restoration of the kcat (Table 3).

(ATP)

Discussion Determining the impact of antibiotic resistance on bacterial fitness is highly relevant both from a medical and evolutionary perspective because it can explain and help predict the rate and trajectory of resistance development under different growth conditions (Levin et al., 1997; 2000; Andersson and Levin, 1999; Levin, 2001; Andersson, 2006). In this study, we investigated how mupirocin resistance affects fitness and IleRS activity in S. typhimurium and how the reduction in fitness could be compensated. The highly conserved nature of the IleRS enzyme makes a comparison between the alterations found in S. typhimurium and the clinically more relevant S. aureus meaningful (Chalker et al., 1994; Hodgson et al., 1994). We identified four resistance mutations in IleRS that conferred a MIC of >1024 mg ml-1 and a reduction in growth rate (relative fitness between 0.24 and 0.63) in LB growth medium (Table 1). The large reduction in fitness in the resistant mutants shown here, and demonstrated also in mice and Caenorhabditis elegans (Paulander et al., 2007) underlines the impact of isoleucine aminoacylation rate on bacterial fitness. As the ratelimiting supply of one amino acid will essentially control the incorporation of all other amino acids, the lowered isoleucine aminoacylation rate will reduce the overall rate of protein synthesis (Elf and Ehrenberg, 2005). The saturating concentration of tRNA used for the in vitro kinetics was 4–8 mM, well below the intracellular tRNAIle concentration, which depending on growth rate varies between 11 mM and 25 mM (Dong et al., 1996). This implies that in vivo wild-type IleRS is saturated with tRNA and functions at kcat (tRNA). In support of this idea, for the susceptible, resistant and compensated strains, fitness correlated only with kcat (tRNA) but not with Km (tRNA) or kcat (tRNA)/Km (tRNA) (Fig. 3). This also agrees with the observation that for aminoacylation reactions performed by class I synthetases the rate-limiting step is the release of the aminoacylated tRNA and not tRNA binding (Zhang et al., 2006). Furthermore, the interaction with ATP is disturbed in the resistant mutants (increased Km (ATP) and decreased kcat (ATP)) and in the compensated mutants both the Km (ATP) and kcat (ATP) values were partially restored towards the susceptible wild-type enzyme. Thus, the alterations in the interaction between mutant IleRS and tRNA and/or ATP can account for both the fitness cost and the mechanism of compensation of mupirocin resistance. An additional indirect fitness effect could result from a potential increase in the level of deacetylated tRNAIle in the resistant mutants that via activation of stringent factor RelA and increased production of the global regulator (p)ppGpp would down-

regulate transcription of ribosomal proteins and stable RNAs, resulting in a reduced growth rate (Magnusson et al., 2005). A substantial fraction (12/23) of the compensatory amino acid substitutions were located in the CP1 editing domain (Fig. 4). The primary contact of IleRS with the tRNA 3′ end is mediated through the CP1 domain, which is believed to be indirectly required for amino acid activation (Zhang et al., 2006). There are several potential mechanisms for how the compensatory mutations in the CP1 domain could restore aminoacylation activity, including increased translocation from the active site to the CP1 domain or an increased rate of Ile-tRNA release. Analysis of the compensatory mutations found in CP1, when separated from the resistance mutations, might help to define how the editing domain interacts with the catalytic domain during the different aminoacylation steps. For seven of the 16 extragenically compensated mutants the copy number of the ileS gene was increased (two- to sixfold). This increase in gene dosage is likely to increase the level of the defective enzymes and accordingly increase the aminoacylation rate. Gene dosage compensation was only seen for the relatively fit resistant mutants (H594Y, F596L and WV630-631L), which is expected because an increased amount of IleRS with a relatively high aminoacylation rate can restore fitness more efficiently than an increased level of a very inefficient IleRS (i.e. W443R). Gene amplification is an important mechanism of adaptation in response to various selective pressures (Romero and Palacios, 1997; Reams and Neidle, 2004). Thus, when overexpression of a gene product confers a fitness advantage, bacteria with spontaneously formed gene amplifications might be selected in the population. For example, in E. coli and S. typhimurium 2- to 100-fold amplification of specific genes may confer resistance to antibiotics (Edlund and Normark, 1981), increase the growth rate on various limiting carbon sources (Sonti and Roth, 1989; Reams and Neidle, 2003; Roth et al., 2006) or compensate for the effect of deleterious mutations (Nilsson et al., 2006). Another possible mechanism of compensation could be an increased amount of tRNAIle. However, this seems less likely because an increase in tRNA concentration without an increased aminoacylation rate of IleRS would lead to induction of the stringent response and slowed growth. In addition, the in vitro kinetic data also suggest that an increased tRNAIle concentration would not increase the overall reaction rate (Table 3). We conclude that at least three mechanisms exist that can compensate for the deleterious resistance mutations: (i) amino acid substitutions in the ATP binding domain of IleRS, (ii) amino acid substitutions in the CP1 editing domain of IleRS, and (iii) increased IleRS levels due to amplification of the ileS gene.


Mupirocin resistance in Salmonella typhimurium 1045 To assess how mupirocin affected the resistant strains, their fitness was determined with mupirocin levels (100 mg ml-1) well below their MICs (> 1024 mg ml-1) in the growth medium. This concentration of mupirocin further decreased fitness, as compared with the antibiotic-free medium, showing that the resistance mutations did not completely prevent binding of mupirocin to IleRS but only reduced drug affinity (Table 1). The fitness differences observed during growth in medium with and without mupirocin lead us to examine the trajectories of compensatory evolution in the two environments. The lineages that were compensated in the absence of mupirocin mostly acquired intragenic compensatory mutations that increased drug affinity (reduced MIC) and restored fitness by increasing the rate of aminoacylation (Table 3). In contrast, lineages that compensated with mupirocin present (where the above types of mutations are selected against), acquired mostly extragenic compensatory mutations that maintained low mupirocin affinity and concomitantly increased the IleRS aminoacylation rate to restore fitness (Table 3). As a consequence, when only the intragenic compensatory mutations that restore aminoacylation and maintain a low affinity for mupirocin are viable, the relative fraction of extragenic compensatory mutations found in the selection is expected to increase. These findings show that depending on the concentration of mupirocin different types of compensatory mutations will be selected, which could potentially also be of importance in clinical settings. An important question is why chromosomal ileS mutations with high-level mupirocin are not found clinically (Antonio et al., 2002; Yang et al., 2006; Yoo et al., 2006). One possibility, supported by these data and previous analysis of S. aureus (Hurdle et al., 2004), is that the fitness costs are so severe that the mutants never can become established in a patient. An alternative explanation is that the isolation procedure for S. aureus used in clinical settings is suboptimal for detection of strains with potential chromosomal ileS mutations. Thus, the substantial reduction in growth rate seen for the chromosomal mutants suggests that it might take longer for the high level resistant clones to appear than the incubation time normally used to detect S. aureus in clinical samples. In conclusion, our analysis shows that compensation in vitro to reduce the fitness cost of mupirocin resistance is both common (23 types of intragenic and two types of extragenic compensatory mutations were found, Table 2) and efficient (Fig. 3). As a consequence of the large mutational target for compensation, the mutation rate for compensation is as high as 2 ¥ 10-9-6 ¥ 10-9 per cell per generation, suggesting that compensation might occur in a mupirocin-resistant bacterial population within an infected individual (Björkholm et al., 2001). Whether we can extrapolate these data obtained for S. typhimurium

under laboratory conditions to predict the likelihood that compensatory evolution is also occurring in low-fitness, mupirocin-resistant S. aureus strains during growth in patients is at present unclear. Circumstantial evidence for the occurrence of compensation among clinical isolates comes from previous studies which show that among low-level resistant S. aureus strains, some clones carry in addition to the resistance mutations as many as four additional point mutations in the ileS gene (Antonio et al., 2002; Yang et al., 2006). These extra mutations are located at many different positions, including six in the CP1 domain of IleRS where we have identified several compensatory mutations (Fig. 4). Thus, it is conceivable that the mutational changes seen in these S. aureus isolates represent both resistance mutations that increase MIC and reduce IleRS aminoacylation rates as well as compensatory mutations that reduce the MIC and restore IleRS kinetics. Finally, even though this is a plausible hypothesis it remains to be demonstrated that the suspected compensatory mutations indeed appear during bacterial growth in the patient and that they specifically compensate the effect of the preceding resistance mutations (Andersson, 2003). Experimental procedures Strains, media and genetic methods All strains used in this study are derivates of Salmonella enterica var. Typhimurium LT2 (Table S1). Bacteria were grown in LB broth with or without addition of 100 mg ml-1 mupirocin (GlaxoSmithKline) at 37°C. Transfer of the ileS gene was performed by P22 transduction of a linked Tn10 following standard procedures (Davies et al., 1980).

Mupirocin resistance Selection for resistant mutants was performed by plating 109 cells from each of several independent cultures on LA plates supplemented with 500 mg ml-1 of mupirocin. After 4–8 days of incubation at 37°C, one colony from each culture was re-streaked on LA plates containing mupirocin and frozen at -70°C in LB containing 10% DMSO. To determine the mutation frequency of mupirocin resistance, 3 ¥ 1010 bacteria from 30 independent cultures were plated on LA supplemented with 500 mg ml-1 of mupirocin. The mutation frequency was calculated as the ratio of the number of mupirocin-resistant mutants divided by the total number of cells (determined by plating appropriate dilutions of the cultures on LA). Determination of the mutation rate for the compensatory mutations was calculated as described previously (Maisnier-Patin et al., 2002). MICs were determined by using E-test as described by the manufacturer (Biodisk).

Strain evolution and fitness measurements Several independent lineages of the resistant strains JB1850, JB1853, JB1855 and JB1872 (Table S1) were evolved to


1046 W. Paulander, S. Maisnier-Patin and D. I. Andersson improve fitness. Lineages were grown overnight in liquid medium and serially passaged with a population bottleneck of 3 ¥ 106 bacteria into either 1 ml LB or LB containing 100 mg ml-1 mupirocin. Every 50 generations, the cultures were plated on LA plates to estimate the proportion of cells with improved fitness (determined by an increase in colony size). When the cultures contained mainly cells with improved fitness, i.e. > 50% cells showed a large colony size, one single fast growing colony was picked and saved at -70°C. Growth rates were determined by measuring optical density (OD) at 600 nm of bacteria grown in LB medium at 37°C as a function of time by using a BioscreenC reader (Labsystems). The relative fitness of the strains was calculated as the ratio of tgen(wt)/tgen(mutant).

PCR, DNA sequencing, Southern hybridization and real-time PCR Primers used for PCR, sequencing and real-time PCR can be found in Table S2. The PCR products were purified with a GFX PCR, DNA and gel band purification kit (Amersham Pharmacia Biotech). The sequencing reactions were performed with Big dye terminator cycle sequencing ready reaction kit V1.1. (Applied Biosystems), and sequences were read with an ABI prism 3100 genetic analyser. For RNA isolation the bacteria were grown in LB into mid-exponential phase OD 0.4 (600 nm), the Isolation of RNA was performed with SV total RNA isolation system (Promega). The Conversion of RNA into cDNA was made using the high-capacity cDNA reverse transcription kit that includes an RNase inhibitor (Applied Biosystems). Real-time quantitative PCR reactions were performed with an ABI PRISM 7900 Sequence Detection System using the power SYBR green kit (Applied Biosystems). The results were analysed with the RQ manager 1.2 program (Applied Biosystems). Southern blots were performed using ECL random-prime labelling and detection system according to the manufacturer’s instructions (Amersham Biosciences).

Cloning and protein purification The ileS genes were amplified by PCR (primers in Table S2) and cloned into a pET101/D-TOPO vector (Invitrogen). Expression of IleRS was induced in BL21 star (DE3) cells with 1.0 mM IPTG when the culture reached OD600 = 0.4. After 4 h of induction, the cells were centrifuged, resuspended in buffer A (50 mM Tris-HCl pH 7.9, 10 mM MgCl2, 50 mM KCl and 2 mM dithiothreitol) and ruptured by sonication for 5 ¥ 20 s. IleRS was subsequently purified as described earlier (Shepard et al., 1992). The fractions collected from high-performance liquid chromatography using a HiTrap Q HP column (Amersham Biosciences) that contained purified IleRS were pooled and concentrated using a Microsep centrifugal device 50 K (Pall life science) and frozen at -70°C in 15% glycerol and 0.1 mM phenylmethanesulphonyl fluoride.

Activity measurements (IleRS and steady-state enzyme kinetics) The steady-state activity assays were performed with timecourses in the linear phase of product formation and based

on the protocol from Pope et al. (1998) with some modifications. Assays were performed in 50 ml volumes at 25°C and included the following components: tRNA (Roche) MRE 600, 0.2–8 mg ml-1, ATP (Amersham Biosciences) 0.25 mM-5 mM, L-isoleucin (Sigma Aldrich) 1 mM L-[4,5-3H] Isoleucin (Amersham Biosciences) (specific activity of 3.26Tbq using 1 mCi ml-1) mixed in buffer A. At different time intervals, the reaction was quenched with 1 ml of ice-cold 7% trichloroacetic acid (TCA) (Merck) and incubated on ice for 10 min. The TCA-insoluble material was trapped onto GF/C filters (Whatman) pre-soaked with 7% TCA, subsequently washed with 2 ¥ 5 ml 7% TCA, 2 ¥ 5 ml of 70% ethanol, and dried. The filters were then placed in plastic vials with 5 ml of scintillation fluid (Quicksafe N, Zinsser Analytic) and counted in a Beckman scintillation counter (LS-3801) at the standard settings for tritium. The data were fit to the Michaelis-Menten equation using the Graphpad PRISM software (version 4).

Acknowledgements This work was supported by grants to DIA from the Swedish Research Council, the Swedish Institute for Infectious Disease Control and Uppsala University. We would like to thank Beston Hamasur and Mats Andersson for advise and help with setting up the protein purification and Otto Berg for calculations of mutation rates.

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Supplementary material The following supplementary material is available for this article: Table S1. List of Salmonella typhimurium LT2 strains used in this study. Table S2. List of primers used for PCR, sequencing, and cloning. This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.13652958.2007.05713.x (This link will take you to the article abstract). Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
