JAC Advance Access originally published online on October 24, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1145-1153; doi:10.1093/jac/dkl413
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Effect of fluoroquinolone exposure on the proteome of Salmonella enterica serovar Typhimurium
1 Department of Food and Environmental Safety, Veterinary Laboratories Agency Addlestone, Surrey, KT15 3NB, UK 2 Antimicrobial Agents Research Group, Division of Immunity and Infection University of Birmingham, Birmingham, B15 2TT, UK
*Corresponding author. Tel: +44-1932-357827; Fax: +44-1932-357595; E-mail: n.g.coldham{at}vla.defra.gsi.gov.uk
Received 6 July 2006; returned 8 August 2006; revised 5 September 2006; accepted 14 September 2006
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Objectives: The physiological response of Salmonella enterica serovar Typhimurium to fluoroquinolone antibiotics was investigated using proteomic methods.
Methods: Proteomes were prepared from strain SL1344 following treatment of broth cultures with ciprofloxacin (0.03 and 0.008 mg/L; 2x and 0.5x MIC) and enrofloxacin (0.03 mg/L) and from a multiple antibiotic resistant (MAR) mutant. Protein expression was determined by two-dimensional HPLC-MSn and also after exposure to ciprofloxacin by two-dimensional gel electrophoresis (2D-GE).
Results: The number of proteins (mean ± SD) detected by 2D-GE derived from control cultures of the wild-type strain was significantly (P < 0.05) reduced from 296 ± 77 to 153 ± 36 following treatment with ciprofloxacin (0.03 mg/L). Raised expression (P < 0.05) of 17 proteins was also detected, and increases of up to 8-fold (P < 0.0001) were observed for subunits of F1F0-ATP synthase, TolC and Imp. Analysis by two-dimensional HPLC-MSn provided higher proteome coverage with 787 ± 50 proteins detected, which was reduced (P < 0.005) to 560 ± 14 by ciprofloxacin (0.03 mg/L). Increased expression of 43 proteins was observed which included those detected by 2D-GE and additionally the efflux pump protein AcrB. The basal expression of the AcrAB/TolC efflux pump was elevated in the MAR mutant compared with the untreated wild-type and augmented following treatment with ciprofloxacin (0.03 mg/L). F1F0-ATP synthase and Imp were only elevated in the mutant when treated with ciprofloxacin.
Conclusions: These studies suggest that increased expression of AcrAB/TolC was associated with resistance while other increases, such as in F1F0-ATP synthase and Imp, were a response to fluoroquinolone.
Keywords: proteomics , fluoroquinolones , S. enterica
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Ciprofloxacin and enrofloxacin are fluoroquinolone antibiotics used for the treatment of systemic Salmonella infections in man1 and food-producing animals.2 The primary target for fluoroquinolones is GyrA, a subunit of DNA gyrase, which is a tetramer composed of two A and two B subunits. The GyrA subunit is responsible for DNA cleavage and re-ligation, whereas the GyrB component transduces energy from ATP hydrolysis for conformational change of DNA. Binding of fluoroquinolones to GyrA produces a trapped intermediate of enzyme, drug and substrate, which prevents the progression of replication forks and transcription.1,2 In Escherichia coli, this DNA damage induces the SOS response, which includes production of RecA, an activator of the DNA repair system.3 The ParC subunit of DNA topoisomerase IV is a secondary target of fluoroquinolones. DNA topoisomerase IV is also composed of two subunits, ParC and ParE, and mediates the relaxation of duplex DNA. The energy for this conformational change is again provided by ATP. Previous studies have suggested at least three mechanisms of bacterial cell killing2 including those dependent and independent of DNA and RNA synthesis. The primary mechanism of fluoroquinolone action is independent of the SOS response and does not require active protein synthesis.3 Induction of the SOS response following exposure to quinolones is also responsible for extensive bacterial cell filamentation.3
The primary response to fluoroquinolone exposure has been investigated at the transcriptional level using microarray technology. Induction of the major SOS genes, including recA, recN and sulA, was a common observation following treatment of E. coli with norfloxacin4 and ofloxacin5 and Haemophilus influenzae with ciprofloxacin.6 Substantial changes in the transcriptional profile were detected following treatment with fluoroquinolones, particularly for repressed genes, which included those involved in metabolism, transport of small molecules, protein synthesis and outer membrane proteins.5,6 These effects were also dependent upon both the concentration and period of exposure to fluoroquinolone.4,6 Parallel studies investigating both protein expression by two-dimensional gel electrophoresis (2D-GE) and mRNA by microarray revealed qualitatively similar expression data but with some quantitative differences.6 Exposure of E. coli to ofloxacin has been shown to induce several genes of stress regulons including soxS and csrA.5 Other transcriptional profiling studies in E. coli have investigated specific stress responses including the effects of the superoxide generating agent paraquat, an inducer of the soxRS system, and salicylate, an inducer of the mar locus.7 Modulation of 112 genes was observed in response to paraquat and 134 to salicylate. Constitutive expression of MarA, the activator of the mar locus, in E. coli has been shown to up-regulate transcription of 47 genes, including those encoding the efflux pump protein acrA and tolC, but acrB transcription was unchanged. Down-regulation of 17 genes by MarA, including ompF, was also reported.8
Coordinated regulation of protein effector expression is a key feature of innate reduced susceptibility to multiple antibiotics.9 The chromosomal multiple antibiotic resistance locus (mar) of E. coli, in cooperation with other regulatory loci, plays a pivotal role in innate reduced susceptibility (circa 4-fold) to some unrelated antibiotics and certain disinfectants.10 Overexpression of the AcrAB-TolC efflux pump contributes to multiple antibiotic resistant (MAR) in E coli,11 and has also been associated in conjunction with mutations in gyrA with resistance to fluoroquinolones in Salmonella enterica.12,13 The AcrAB efflux pump of E. coli and S. enterica belongs to the resistance/nodulation/cell division (RND) family and consists of a proton antiporter (AcrB)10 and a membrane fusion protein (AcrA).14,15 These two proteins associate with an outer membrane channel protein, such as TolC, to form a functional efflux pump unit providing selective molecular translocation of solutes from the periplasm to the external environment.11 Reduced expression of porin proteins located in the outer cell membrane may act synergistically with efflux pumps to reduce penetration of antibiotic into the bacterial periplasm.16 Whilst much is understood about the mechanisms of efflux, little is known of the secondary responses enabling the physiological adaptation of Salmonella to fluoroquinolones. In the present study, protein expression was investigated initially by 2D-GE, and subsequently by two-dimensional HPLC-MSn (2D-HPLC-MSn) for wider proteome coverage, to determine the physiological response and potential protein effectors of innate resistance.
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Materials
IPG strips (pH 4–7) were obtained from Bio-Rad (Hemel Hempstead, UK), and polyacrylamide gels (12–14%) and reagents for the second-dimension gel electrophoresis were from Amersham Biosciences (Little Chalfont, UK). GelCode colloidal Coomassie stain was from Perbio Science (Cheshire, UK), sequence grade trypsin was obtained from Promega (Southampton, UK) and HPLC solvents were from Merck (Lutterworth, Leicester, UK). Picotip capillary HPLC columns were procured from New Objectives Inc. (Woburn, MA, USA) and packed with Jupiter Proteo reverse phase (C12) material from Phenomenex (Macclesfield, UK). Ciprofloxacin and enrofloxacin were provided by Bayer HealthCare AG at analytical standard grade and stated purities of 99.8% and 99.7%, respectively. Chemicals were obtained from Sigma (Poole, UK).
Bacterial culture and protein extraction
Proteomes were prepared from S. enterica serovar Typhimurium (SL1344) and an isogenic MAR mutant produced by selection with tetracycline as described previously.17 The MICs of ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid and tetracycline were 1/4; 2/8; 0.015/0.06 and 4/8 mg/L for the parent SL1344/MAR mutant, respectively. The MIC values of these antibiotics were determined using the BSAC agar doubling dilution method. The effects of ciprofloxacin and enrofloxacin on the proteome of these strains were investigated in replicate (n = 3) cultures by inoculation of Luria–Bertani broth without glucose (100 mL) with an aliquot (4 mL) of an overnight culture containing stationary phase cells. Ciprofloxacin (0.0312 and 0.0078 mg/L representing 2x and 0.5x MIC of SL1344) and enrofloxacin (0.0312 mg/L) or vehicle (water, 0.1 mL) alone were added to the cultures when the optical density (
600 nm) reached 0.1 ± 0.01. Preliminary studies revealed that the addition of fluoroquinolone at bacterial cell densities of less than 0.1 (600 nm) caused growth arrest and a subsequent failure to collect sufficient protein in fluoroquinolone-treated cultures for analysis. Similar concentrations of fluoroquinolone, approximating to the appropriate MIC value, and incubation protocol have been used in other studies.6 After a further 90 min of incubation, with the cultures in the logarithmic growth phase (OD 0.6–0.7), the cultures were cooled rapidly by plunging in ice. The bacteria were maintained at 4°C and washed with PBS (100 mL) by centrifugation and the proteomes were extracted from bacterial cells by sonication. Proteome was extracted with a lysis buffer containing urea (5 M), thiourea (2 M), CHAPS (2% w/v), SB 3–10 (2% w/v), pharmalytes (0.5% v/v), DTT (100 mM) and Tris (40 mM).18 The protein concentration of the extracts was determined using the Bradford assay.
Microbial cell viability and morphology
The viability of bacterial cell cultures following treatment with ciprofloxacin was determined by the method of Miles and Misra.19 The samples were diluted in PBS and cultured on agar plates and incubated overnight at 37°C. The viability of ciprofloxacin-treated cultures is expressed as a percentage of untreated controls. The morphology of bacterial cells was assessed by Gram staining.
2D-GE
Proteins in the proteome were separated by 2D-GE as described elsewhere.18 A series of preliminary studies were conducted to determine optimal levels of protein loading (0.05–1 mg). High protein loadings (1 mg) were associated with horizontal streaking and poor separation in the first dimension. Proteins were separated in the first dimension using pH 4–7 IPG strips. Analytical gels were loaded with 0.1 mg of protein and stained using PlusOne (GE Healthcare) silver staining kits with omission of glutaraldehyde. Higher protein loadings of silver-stained gels produced a darker background and problems with spot detection. Evaluation of protein abundance, as a function of spot volume, and comparison of protein expression between control and ciprofloxacin-treated extract gels was made using Phoretix 2D Advanced (version 6.1) software. Expression analysis was performed with background subtraction using the non-spot mode and normalization to total spot volume. All protein spot volumes were multiplied by 100 and compared (six control gels and three gels each following 0.03 and 0.008 mg/L ciprofloxacin) for statistical significance using the Student's t-test. Observed masses for proteins were interpolated by comparison of their mobilities with those of molecular weight markers. Coomassie-stained preparative gels with a protein loading of 0.5 mg were prepared for identification of protein spots. Coomassie staining was selected for preparative gels as there was not sufficient sensitivity at the mass spectrometer for the routine detection of tryptic peptides from silver-stained gels. For identification, protein spots were subdivided into four quarters and digested using a standard in-gel procedure. Tryptic peptide extracts were analysed and proteins identified using mass-based sequencing by capillary-HPLC-MSn as described elsewhere.20
2D-HPLC-MSn
The proteome extracts were analysed by 2D-LC-MSn as described previously20 with some minor modifications: the quantity of tryptic peptides separated in the first dimension by strong cation exchange chromatography was equivalent to that derived from 0.05 mg of protein and the number of samples collected from strong cation exchange chromatography for analysis in the second dimension by HPLC-MSn was decreased from 20 to 15 fractions. Mass spectrometer data files (Xcalibur .raw files; n = 15) for each extract replicate were independently processed for peptide identification using TurboSEQUEST (Bioworks 3.1 package, ThermoFinnigan) against the Salmonella Typhimurium strain SL1344 database obtained in FASTA format from the NCBI. Trypsin was selected as the proteolytic enzyme with up to two missed cleavages and a floating modification for methionine oxidation of ±16 amu. Peptide identifications were filtered with a minimum of a single peptide identification per locus and proteomes compiled using the DTASelect algorithm.21 The peptide identifications were filtered by DTASelect using threshold cut off Xcorr values of 2, 2 and 2.3 for [M+H]+, [M+2H]2+ and [M+3H]3+ ions, respectively, to provide a false positive rate of 1% at the 95% confidence level. The relative abundance of the proteins was compared using the spectrum count,22 following published guidelines,23 and denotes the number of peptide counts (‘hits’) detected for each protein. Expression analysis was limited to only those proteins common to all three replicates (n = 3) from control (replicates 1–3) or fluoroquinolone-treated (replicates 4–6) cultures. Proteomes following exposure to fluoroquinolone (ciprofloxacin 0.03 and 0.008 and enrofloxacin 0.03 mg/L) were compared using Microsoft Access to yield five groups that are each presented in separate Excel worksheets: group 1, proteins found in controls (flasks 1–3); group 2, proteins found following fluoroquinolone treatment (flasks 4–6); group 3, proteins found in controls only (flasks 1–3); group 4, proteins found after fluoroquinolone treatment only (flasks 4–6); and finally, group 5, those common to controls and fluoroquinolone treatment (flasks 1–6). The statistical significance of percentage changes in protein expression in group 5 was determined using a two-tailed Student's t-test.
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Effect of ciprofloxacin on growth, viability and protein content
Growth curves, cell morphology, percentage viability and quantity of protein in the extracts were assessed as gross markers of ciprofloxacin action. Microbial cell density, estimated by measurement of the optical density at 600 nm, was significantly reduced (P < 0.05) after 60 and 90 min of incubation with 0.03 mg/L ciprofloxacin but not with 0.008 mg/L. Morphological examination by Gram staining of cultures exposed to 0.03 mg/L ciprofloxacin revealed gross cell elongation and filamentation (data not shown). Bacterial cell viability (mean ± SD) was reduced to 42 ± 12% (NS) and 0.8 ± 0.8% (P < 0.05) following 90 min of treatment with 0.008 and 0.03 mg/L ciprofloxacin, respectively. The concentration of protein (mean ± SD) in extracts was 3.16 ± 0.15 mg (circa 3 x 1010 cells) from control cultures, and 3.56 ± 0.65 mg (NS) and 1.71 ± 0.06 mg/mL (P < 0.001) following treatment with 0.008 and 0.03 mg/L ciprofloxacin, respectively.
Characterization of the proteome
The identities of 50 of the most abundant proteins determined from a composite (n = 3) of silver-stained second-dimension electrophoretograms were determined. A representative 2D gel from untreated microbial cells is illustrated in Figure 1. Their functional annotation, protein name, theoretical mass and pI are presented in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. A minimum of two tryptic peptides were matched to any protein and the lowest coverage was 8%. The proteins identified in the proteome included mainly cytosolic proteins and some cell envelope and outer membrane proteins, and those involved in a wide range of cellular functions including motility, transport, enzymes involved with energy production, protein synthesis and chaperones. Their presence was consistent with many of those reported to be present in extracts prepared by others from Salmonella Typhimurium18 and E. coli21 and included TolC and OmpX that have been reported to be associated with MAR.8 The 100 most abundant proteins in the proteome of Salmonella Typhimurium determined by 2D-HPLC-MSn have been published elsewhere.20
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Effect of ciprofloxacin by 2D-GE
Reproducible quantification of the protein concentration in microbial cell extracts is an essential prerequisite for expression analysis by 2D-GE and 2D-HPLC-MSn. In the present study, the Bradford method provided between and within coefficients of variation for repeat analyses of an extract of 11.3% (n = 9) and 2% (n = 6), respectively. The limit of protein detection was 25 mg/L, which was an order of magnitude below the typical concentration found in diluted extracts sampled for protein quantification. Representative two-dimensional electrophoretograms of protein extracts derived from control and ciprofloxacin-treated (0.03 mg/L) cells are provided in Figures 1 and 2, respectively. The number of spots detected by Phoretix was 296 ± 77 (n = 6) in control gels and 182 ± 47 (n = 3; NS) following treatment with 0.008 mg/L ciprofloxacin; the number of spots was significantly (n = 3; P < 0.05) reduced to 153 ± 36 by 0.03 mg/L ciprofloxacin.
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The identity and expression levels of 37 protein spots that were evident following treatment with 0.03 and 0.008 mg/L ciprofloxacin at the Coomassie staining level were determined. The protein spot reference number, protein name, functional annotation (NCBI database), and expression in control and ciprofloxacin-treated (0.008 and 0.03 mg/L) proteomes is presented in Table 1. Several proteins, particularly the products encoded by ompA, were found in multiple forms (spots 18–23). Several proteins including TolC, Imp AtpA, AtpC and AtpD had increased expression as the dose of ciprofloxacin was raised.
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Effect of ciprofloxacin and enrofloxacin by 2D-HPLC-MSn
The proteome was further investigated by 2D-HPLC-MSn to provide better coverage, including efflux pump proteins, which have been associated with resistance to fluoroquinolones, but were not detected in the proteome by 2D-GE. This approach also enabled both detection and identification of proteins with decreased expression levels, which was not possible by 2D-GE as protein identification was limited to the Coomassie staining level. The effects of ciprofloxacin (0.03 and 0.008 mg/L) and enrofloxacin (0.03 mg/L) on proteomes following analysis by 2D-HPLC-MSn are summarized in Table 2. The number of proteins detected in untreated cultures (controls), following treatment with fluoroquinolone, in controls only, following treatment with fluoroquinolone only, and those common to both controls and treated is summarized in Table 2. The number of proteins with significantly (P < 0.05) increased or decreased levels of expression is also provided in Table 2. The effect of the fluoroquinolones on relative protein expression, as denoted by the spectrum count, is provided in full in Tables S2 (ciprofloxacin 0.03 mg/L), S3 (ciprofloxacin 0.008 mg/L) and S4 (enrofloxacin 0.03 mg/L) [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The effect of fluoroquinolones on the expression of selected proteins (consistent with those presented in Table 1) relative to untreated controls is summarized in Table 3.
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The expression of proteins including F1F0-ATP synthase and AcrAB-TolC was also evaluated by 2D-HPLC-MSn in a MAR mutant and following treatment with ciprofloxacin (0.03 mg/L). This analysis of the MAR mutant was explicitly targeted to determine whether certain effects, such as increased F1F0-ATP synthase expression, were specific to fluoroquinolone treatment or a more general phenomenon of innate resistance. The expression of AtpA, AtpC, AtpD, AtpF, AtpG, AtpH and Imp was not significantly increased in the untreated MAR mutant (data not shown) compared with the untreated parent. However, many of these proteins and AcrB and TolC were significantly (P < 0.05) increased in the MAR mutant following treatment with ciprofloxacin (0.03 mg/L) compared with untreated MAR mutant cultures (Table 3). By contrast, the expression of the efflux pump proteins AcrAB-TolC was significantly (P < 0.05) increased in the untreated MAR mutants compared with the wild type and increased further following treatment with ciprofloxacin (Figure 3).
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In the first instance, the proteome was evaluated by the traditional approach of 2D-GE to determine proteome coverage, in terms of both the number and identity of those proteins detected. The objective of these experiments was to establish the range of proteins present, especially within the context of those that have been previously associated with antibiotic resistance, such as efflux pumps.24,25 Previous studies have reported a similar wide range of proteins with diverse function in extracts prepared from Salmonella Typhimurium18 and E. coli.21 Several efflux proteins of potential interest including AcrA and AcrB were not detected by 2D-GE. AcrA has a pI (pI 8.4; 42 kDa) above the range of the IPG strip employed (4–7), whereas high mass proteins, such as AcrB (pI 5.41, mass 114 kDa), were poorly represented on the gels, possibly due to solubility problems. In the present study 2D-HPLC-MSn26 provided wider proteome coverage as demonstrated by the number of proteins detected and thereby enabled a more comprehensive evaluation of changes in protein expression in response to treatment with fluoroquinolones. Additionally, the efflux pump proteins AcrA and AcrB were readily detected by 2D-HPLC-MSn. Consequently, further studies investigating protein expression in the MAR mutant and following exposure of the wild-type to enrofloxacin were conducted using 2D-HPLC-MSn. The number of proteins detected with significantly increased expression by 2D-HPLC-MSn (Table 2) was higher than that found by 2D-GE (Table 1). Of those proteins detected in the proteome by 2D-GE following exposure to fluoroquinolone, four were not detected by 2D-HPLC-MSn. Comparison of expression analysis by both analysis methods revealed broadly similar changes in expression of most proteins but with some exceptions (MopA and DnaK). Clearly, both approaches have merits, and the observation that the expression of similar proteins was increased by both methods of proteome analysis provided an important level of assay validation. Furthermore, both approaches revealed a significant reduction in the total number of proteins detected following treatment with fluoroquinolone, which suggests perturbed protein synthesis.
Following fluoroquinolone exposure, the greatest increase in protein expression was observed with enrofloxacin and specifically in the alpha-, beta-, delta- and epsilon-subunits of the F1F0-ATP synthase that dominated the proteome and OmpC. The F1F0-ATP synthase protein complex is partly embedded in the cell membrane and catalyses the synthesis of ATP in the terminal step of oxidative phosphorylations in bacteria. In prokaryotes, this protein complex may operate in reverse as an ATPase to generate the transmembrane proton electrochemical gradient required for molecular translocations27 and other activities including flagella-mediated locomotion.28 Several efflux pump systems, including those of the RND family, utilize proton motive force to create a transmembrane proton gradient (
) that provides the energy to drive drug efflux activity.27 Transport studies in E. coli lacking F1F0-ATP synthase activity29 have implicated this ubiquitous protein in provision of the proton motive force for efflux pump activity. Other enzymes, including NADHdhI and cytochrome d oxidase, also translocate protons across the cytoplasmic membrane during electron transport.30 Elevated expression of the F1F0-ATP synthase complex in response to treatment with ciprofloxacin would be consistent with increased ATP hydrolysis and proton translocation across the membrane to provide the proton motive force for ciprofloxacin efflux.12,16 This contention is supported by studies in E. coli where the activity of MdfA was abrogated in the unc mutant which lacks functional F1F0-ATP synthase.29 However, this hypothesis was not confirmed by analysis of the MAR mutant. MAR can be achieved by the activity of efflux pumps16 such as AcrAB-TolC in E. coli and S. enterica.27 Although such increased efflux activity would require increased proton motive force to drive the pumps and maintain homeostasis, there was no change in the expression of F1F0-ATP synthase in the MAR mutant, compared with the parent, in the absence of fluoroquinolone. Increased expression of certain F1F0-ATP synthase subunits (and Imp) were only associated with fluoroquinolone exposure in both the wild-type and MAR strain. This would suggest that raised expression of F1F0-ATP synthase in response to fluoroquinolone exposure is due to other factors unrelated to proton motive force provision. Inhibition of DNA synthesis by fluoroquinolones has been shown to induce the SOS response,3–6 and in the present study, analysis by 2D-HPLC-MSn also revealed significantly (P < 0.05) increased expression of RecA following treatment (0.03 mg/L) with ciprofloxacin and enrofloxacin. Secondary biochemical perturbations, arising from inhibition of DNA gyrase and DNA topoisomerase, are highly probable, particularly given the incubation time of 90 min. These may include increased expression of F1F0-ATP synthase, perhaps by a feedback loop, since both enzymes are dependent on ATP for conformational change of DNA. Decreased expression of F1F0-ATP synthase activity has been demonstrated by microarray studies following treatment with norfloxacin4 and salicylate7 in E. coli. Reduced expression of F1F0-ATP synthase genes following treatment with norfloxacin4 was thought to reflect a non-specific general decline in metabolism; also the incubation time with antibiotic in this microarray study was different to that used in the present proteomic investigation.
In Gram-negative bacteria active efflux systems are a common mechanism of reduced susceptibility to fluoroquinolones that may confer clinical resistance, particularly when associated with mutation in gyrA.24 Reduced accumulation of antibiotics in S. enterica serovar Typhimurium, including ciprofloxacin, has been associated with increased expression of the efflux pump protein AcrB.13 Similarly, inactivation of the acrAB locus in E. coli and S. enterica produces hypersusceptibility to fluoroquinolones.31 The expression of AcrB and TolC was significantly increased following treatment with ciprofloxacin and enrofloxacin. In contrast to the subunits of F1F0-ATP synthase, which were only increased following fluoroquinolone exposure, AcrAB and TolC were also significantly increased in the untreated MAR mutant and further raised following treatment of the mutant with ciprofloxacin. This would suggest the operation of different control mechanisms for the expression of AcrB and TolC to F1F0-ATP synthase. Transcription and molecular genetic studies in E. coli have revealed regulation of acrB, acrAB and tolC by the marRAB operon.8,32 Other stress response loci including rob and soxRS regulate the expression of similar genes.7,32 These stress response loci control the expression of genes with common functional themes to resist environmental stress. Increased expression of acrA but not acrB has been reported in response to treatment with paraquat7 or following constitutive expression of MarA8 in E. coli. In the present proteomic study statistically significant increased expression of AcrB but not AcrA was observed following exposure to ciprofloxacin and enrofloxacin. The expression values for AcrA did not reach statistical significance from the control but increased expression of AcrA and AcrB was found in the MAR mutant compared with the wild type. Other approaches for quantitative proteomics utilizing stable isotopes are available which may provide better sensitivity for detecting small changes in protein expression. The AcrAB efflux pump has been shown to play an important role in the active efflux of [14C]ciprofloxacin and resistance to fluoroquinolones.13,31 The expression of other efflux pumps including AcrD, AcrE and AcrF has been detected by 2D-HPLC-MSn in protein extracts of Salmonella Typhimurium prepared from specific knockout mutants, which was consistent with transcriptomic data,33 but they were not detected in the present studies. Increased expression of a TolC homologue in Pseudomonas putida KT2440 has been reported using 2D-GE approaches following exposure to herbicides34 and phenol-induced stress.35
Increased expression of the outer membrane proteins OmpA and OmpC was also observed on exposure to fluoroquinolone. OmpA is the most abundant outer membrane protein of E. coli with 105 copies per cell.36 Although currently annotated as a putative hydrogenase, this protein has been associated with a wide range of functions.37 A study38 indicated that OmpA is a flexible temperature-responsive protein which creates large open pores in the outer membrane of E. coli at physiological temperatures (>34°C) and mediates the transfer of hydrophilic molecules across the outer membrane. The detection of multiple forms of this protein is consistent with previous reports and due to procedural artefacts during analysis by 2D-GE39 or post-translational modifications.6 OmpC is also a porin and allows the passive diffusion of low molecular weight hydrophilic molecules. In E. coli, the expression of the porin OmpF was reduced following activation of the mar locus by MarA8 and reduced transcription of OmpF and OmpC was observed following treatment with salicylate, an inducer of this locus.7 Increased expression of the organic solvent tolerance protein encoded by the ostA gene (also known as imp) was also observed. Studies with E. coli indicate that Imp has an essential role in the biogenesis of the outer membrane providing a targeting/usher system for various components including lipopolysaccharide.40,41 Increased transcription of impA was observed in H. influenzae in response to ciprofloxacin (0.03 mg/L).6 The explanation for increased expression of these outer membrane proteins is unclear but again may be associated with secondary effects arising from fluoroquinolone treatment that are also evident at the macroscopic level by the extensive filamentation observed following treatment with ciprofloxacin.
In conclusion, the present studies indicate increased and decreased expression of a wide range of proteins some consistent with a diverse secondary response to treatment with fluoroquinolones. This included proteins associated with innate mechanisms of resistance, such as AcrAB-TolC, and others including subunits of F1F0-ATP synthase which may be due to the direct effects of fluoroquinolones.
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None to declare
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Tables S1–4 are available as Supplementary data at JAC Online http://jac.oxfordjournals.org/).
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This study was supported by the Department for Environment, Food and Rural Affairs, UK (project OD 2011).
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Gmuender H, Kuratli K, Padova KD, et al. (2001) Gene expression changes triggered by exposure of Haemophilus influenzae to novobiocin or ciprofloxacin: combined transcription and translation analysis. Genome Res 11:28–42.
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Pomposiello PJ, Bennik MHJ, Demple B. (2001) Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183:3890–902.
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Barbosa TM and Levy SB. (2000) Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA. J Bacteriol 182:3467–74.
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Okusu H, Ma D, Nikaido H. (1996) AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple antibiotic resistance (Mar) mutants. J Bacteriol 178:306–8.
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