Proteomic Analysis of Nalidixic Acid Resistance in Escherichia coli: Identification and Functional Characterization of OM Proteins Xiang-min Lin,† Hui Li,† Chao Wang, and Xuan-xian Peng* Center for Proteomics, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China Received January 30, 2008
The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. To understand the mechanisms of the resistance is extremely important to the control of these bacteria. In the current study, proteomic methodologies were utilized to characterize OM proteome of Escherichia coli with nalidixic acid (NA) resistance. The OM proteins TolC, OmpT, OmpC and OmpW were found to be up-regulated, and FadL was down-regulated in the NA-resistant E. coli strains. The changes at the level of protein expression were validated using Western blotting. Furthermore, the possible roles these altered proteins played in regulation of NA resistance were investigated using genetically modified strains with the deletion of these genes. The results obtained from functional characterization of these genetically modified strains suggest that TolC and OmpC may play more important roles in the control of NA resistance than other OM proteins identified. To gain better understanding of the mechanisms of NA resistance, we also characterized the role of the two-component system EnvZ/OmpR which is responsible for the regulation of OmpC and OmpF expression in response to NA resistance using their genetically modified strains. Our results suggest that OmpF and the EnvZ/OmpR are also important participants of the pathways regulating the NA resistance of E. coli. Keywords: Antibiotics • nalidixic acid • E. coli K-12 • outer membrane proteins • proteomics
1. Introduction Antibiotics have reduced morbidity and mortality of infectious diseases and played an important role in elevating human life expectancy. However, the worldwide emergence of antibioticresistant bacteria poses a serious threat to human health today due to too frequent and inappropriate use of antibiotics.1–3 A line of evidence has indicated that four mechanisms are probably involved in the antibiotic resistance. They are modification or hydrolysis of enzymes, modification of targets, activation of efflux pump systems, and reduction of OM permeability.4 The permeability and the pump systems are mainly controlled by OM proteins in Gram-negative bacteria. The decrease of OM permeability prevents the influx of antibiotics, and the activation of the efflux pump systems pumps the noxious small molecule substances out of cells. Multidrug efflux pump systems have been characterized in Gram-negative bacteria, including Pseudomonas aeruginosa and Escherichia coli since the 1980s.5,6 OM channels related to antibiotic permeability have also been characterized. It has been reported that lack of channel proteins OmpF or OmpC in E. coli resulted in imipenem resistance, and expression of these proteins were decreased in the carbapenem-resistant strain of Enterobacter aerogenes.7,8 In our previous studies, nine, * Corresponding author: Xuan-xian Peng, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China. Tel, +86-20-3145-2846; e-mails, pxuanx@ sysu.edu.cn;
[email protected]. † These authors contributed equally to this work. 10.1021/pr800073c CCC: $40.75
2008 American Chemical Society
eight, and six OM proteins were identified, respectively, from E. coli strains with tetracycline, ampicillin, and chloramphenicol resistance using proteomic methodologies. Both the efflux pump component and OM channels were identified in the studies.9,10 The information obtained from those studies provides novel insights into mechanisms of antibiotic resistance. Nalidixic acid (NA) is a small hydrophobic molecule (MW 232.2) and belongs to a group of broad spectrum antibiotics called quinolones. The mechanism of NA resistance of bacteria is through the inhibition of DNA-gyrase which is required for DNA synthesis. NA-resistant isolates have been reported in clinic.11 These strains originated from most of Gram-negative bacteria including E. coli, Citrobacter freundii, P. aeruginosa, Salmonella, Aeromonas spp., and Shigella spp.11–16 The mutations of subunits of DNA gyrase including gyrA, gyrB and/or parC were detected in these isolates.11,17,18 It has also been reported that expression of OM genes were altered at mRNA levels, and down-regulation of these genes was detected in a thermotolerant, NA-resistant mutant of E. coli using cDNA arrays.19 Increased susceptibility to NA was measured in P. aeruginosa without OM protein omlA.20 However, little is known about OM proteome and the two-component regulating system of these proteins involved in the NA resistance of E. coli. In the present study, 2-D gel based proteomic methodologies were utilized to identify altered OM proteins of E. coli with NA resistance. The changes at the protein expression level detected by 2-D gel electrophoresis were validated by Western blotting. Journal of Proteome Research 2008, 7, 2399–2405 2399 Published on Web 04/26/2008
research articles Furthermore, functional characterization of these altered OM proteins and the related EnvZ/OmpR two-component system were performed using genetically modified E. coli strains. To our knowledge, this may be the first report of OM proteome and the related EnvZ/OmpR two-component system involved in the regulation of NA resistance in E. coli.
2. Materials and Methods 2.1. Bacterial Strains. The bacterial strains used in the current study were E. coli K12 BW25113 and its mutants. E. coli K12 BW25113, ∆ompW, ∆ompT and ∆fadL were kindly provided by NBRP (NIG, Japan): E. coli,21 and ∆ompC and ∆tolC were collections in our laboratory. These E. coli K12 strains were grown in Luria-Bertani (LB) medium at 37 °C. The antimicrobial agents were purchased from a commercial source (AMRESCO, Inc., Parkway, Solon, OH). A NA-resistant strain (NAR) was selected from E. coli K12 BW25113 (NA-R-O) with the use of 10 sequential subcultures in 1/2 MIC concentration of the antibiotic as described previously.22 After the selection, the MIC of NA-R-O was increased from 5 to 40 µg/mL (NA-R). 2.2. Isolation of OM Proteins by Lauryl Sarcosinate Extraction. OM proteins of E. coli were prepared according to a procedure based on lauryl sarcosinate.23 Briefly, the bacteria were grown in LB and then harvested by centrifugation at 4000g for 15 min at 4 °C. Bacterial pellet was washed in 40 mL of sterile saline buffer (0.85% NaCl) three times, resuspended in 10 mL of sonication buffer (50 mM Tris/HCl, pH 7.4) and disrupted by intermittent sonic oscillation for a total of 15 min intervals of 9 s on ice. Unbroken cells and cellular debris were removed by centrifugation at 5000g for 20 min. The turbid supernatant was subjected to ultracentrifugation at 100 000g for 1 h at 4 °C. The pellet was solubilized with 2% (w/v) sodium lauryl sarcosinate (Sigma) at 4 °C and incubated at room temperature for 40 min. Following ultracentrifugation at 100 000g for 1 h at 4 °C, the resulting pellet was dissolved in sterile pure water and stored at -80 °C for use. 2.3. 2-DE Proteomics. 2-DE was performed according to a procedure described previously.23 Briefly, OM protein extracts containing 20 µg of proteins were dissolved in lysis solution (8 M urea, 2 M thiourea, 4% CHAPS and 80 mM DTT). Isoelectric focusing (IEF, 0.02 cm × 7 cm) was carried out using pH 3-10 carrier ampholyte for total 8000 Vh. After equilibration for 15 min in 50 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, and 5% β-mercaptoethanol, the gels were transferred to the second-dimension electrophoresis using 12% acrylamide gel and stained with Coomassie blue-R250. 2-DE gels were scanned in ImageScan and analyzed with ImageMaster 5.0 software (Amersham Bioscineces, Sweden). Altered spots were standardized and then compared based on their volume percentages in the total spot volume over the whole gel image. Significantly changed spots were selected by rate increased/decreased g 2-fold or complete appearance and disappearance. Protein spots of interest were cut from the gels for mass spectrometric analysis. All MALDI analysis was performed by a fuzzy logic feedback control system (Reflex III MALDI-TOF system, Bruker) equipped with delayed ion extraction. Peptide mass fingerprintings were searched using the program Mascot (Matrix Science, London, U.K.) against the NCBI database; E. coli species database was defined as a matching species; one missed cleavage per peptide was allowed and the mass tolerance was 150 ppm. The protein subcellular locations were determined by Program PSORTb version 2.0 (http://www.psort.org/psortb/). 2400
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Lin et al. 2.4. Western Blotting. Western blotting on 1-DE and 2-DE gels was performed as previously described.24 Rabbit antisera to TolC, OmpW, FadL, OmpT, OmpC or OmpF were obtained from Wenta Bio Sci Tech Corp (Ji’an, China), in which the specificity of anti-TolC and OmpW was validated in our previous publications.10,25 Briefly, 1-DE and 2-DE gels were transferred to NC membranes for 3 h at 100 mA in transfer buffer (48 mM Tris, 39 mM glycine and 20% methanol) at 4 °C. The membranes were blocked with 5% skim milk and then incubated with rabbit antisera to TolC, OmpW, FadL, OmpT, OmpC or OmpF as the primary antibodies. The horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody was used as the secondary one. Antibody-tagged protein spots were detected by DAB. The 2-DE Western blotting was performed on two NC membranes, one from NA-R and the other from NA-R-O, by adding antibodies one by one, which may guarantee the reliability because there were up-regulation and down-regulation of target OM proteins in each of the NC membranes. 2.5. Investigation of Antimicrobial Susceptibility Using MIC Assay. MICs of NA-R-O, NA-R and these mutants were measured by a 2-fold standard broth microdilution method.9 Each strain was grown in a 5 mL culture at 37 °C to an optical density at 600 nm of about 0.5 and then diluted in MH to 5 × 105 cfu/mL. In triplicate wells of a 96-well microtiter polystyrene tray, 10 µL of bacterial suspension was added to series of 100 µL MH media containing 2-fold-increasing concentrations of NA. The cultures were incubated at 37 °C for 16-18 h. The lowest concentration at which bacterial growth was largely prevented as compared with the control plate was determined as the MIC of the strain. 2.6. Investigation of Antimicrobial Susceptibility Using Bacterial Survival Capability Assay. Bacterial survival capability assay was performed as described previously with a modification.10 In brief, inoculums of these strains tested were separately cultured in LB medium overnight, and the cultures were diluted 1:1000 into 5 mL of fresh LB medium. Six tubes were tested for each of these bacteria. Half of them contained different concentrations of NA, and the other was used for control without NA. Bacterial growth at 8 h was determined by measurement of OD (600 nm) of the cultures. Survival ability was characterized by comparison between experimental and control groups, and was termed an inhibiting rate. Difference in inhibiting rates was investigated with SPSS program. The experiment was repeated at least three times.
3. Results 3.1. Determination of MICs of NA-R-O and NA-R. E. coli K12 BW25113 were subcultured in LB media with 1/2 MIC of NA and without NA for 10 serial passages. After these passages, MICs of these bacteria cultured in the media without (NA-RO) and with (NA-R) NA were 5 and 40 µg/mL, respectively. The MIC of NA-R was increased 8-fold compared to that of NA-RO. Therefore, the strain NA-R was the NA-resistant bacterium of NA-R-O. 3.2. Analysis of Sarcosine-Insoluble Fraction Using 2-DE Proteomics. To investigate OM proteins in response to NA resistance, a subproteomic approach was utilized to identify altered proteins in sarcosine-insoluble fractions between NA-R and NA-R-O. Approximately 50 protein spots were visualized in each of these gels which were stained with CBB R-250 (Figure 1A and B). Out of the 50 spots, 11 showed significant changes at the level of protein expression. These protein spots were
OM Proteins & EnvZ/OmpR for NA Resistance
research articles NA-R with respect to NA-R-O. In this way, one extra spot of TolC and FadL, and two extra spots of OmpT and OmpW were detected by this combined 2-DE and Western blotting approach but not by 2-DE alone, suggesting higher sensitivity of 2-DE immunoblotting in protein detection. Generally, the results obtained from 2-DE and Western blotting studies were consistent with those obtained from the 2-DE alone.
Figure 1. Analysis of sarcosine-insoluble fraction in response to NA based on 2-DE maps. (A) 2-DE map of NA-R-O grown in LB medium without NA; (B) 2-DE map of NA-R grown in LB medium without NA; (C) enlarged partial 2-DE gels showing altered expression spots; (D and E) histogram displays the changes in spot intensity of them between NA-R (white) and NA-R-O (black), and bars represent spot intensity with relative volume divided by the total volume over the whole image, according to ImageMaster 5.0 software description.
named from 1 to 11. The spots 1, 2, and 6-11 were found to be up-regulated and the spots 3-5 were down-regulated in the NA-R strain (Figure 1C-E). The spots of interest in the 2-DE gels were distained and washed, reduced and alkylated, digested in-gel with trypsin, extracted from the gels, mixed with 1 µL of matrix solution and then subjected to PMF by MALDI-TOF/MS for protein identification. Table 1 showed the identities of these spots. The 11 spots were identified as seven unique proteins. They were TolC (up spots 1, 2), AceA (down spot 3), FadL (down spots 4, 5), OmpT (up spots 6, 7), OmpC (up spot 8), PspA (up spot 9), and OmpW (up spots 10, 11). Results obtained from PSORTb program suggest that TolC, FadL, OmpT, OmpC and OmpW were OM proteins, and PspA was an inner membrane protein, and AceA was a cytoplasm protein. 3.3. Characterization of Differentially Expressed OM Proteins Using Combined 2-DE and Western Blotting Approach. In the current study, our aim is to determine the OM proteins involved in the NA resistance. Of the five OM proteins identified, TolC, FadL, OmpT and OmpW showed more than one spots in 2-DE gels. Thus, we utilized 2-DE Western blotting rather than 1-DE Western blotting to further characterize these spots (Figure 2). Up-regulation of three TolC spots, four OmpT spots, three OmpW spots, and down-regulation of two FadL spots and an unchanged spot of FadL were detected in
3.4. Functional Characterization of Altered OM Proteins in Genetic Modified Strains. To further investigate the capability of these altered OM proteins in response to NA resistance, two antimicrobial susceptibility assays were performed using their gene deletion mutants. They were MIC assay and survival capability assay. Figure 3A was a summary of MICs of NA-R-O and NA-R as well as the strains with the deletion of the OM genes identified. The strains were ∆tolC, ∆fadL, ∆ompT, ∆ompC, and ∆ompW. Deletion of tolC or ompC resulted in a significant decrease in MICs of the mutants which were 12.5 and 2.5 µg/mL, respectively. However, MICs in the mutants with deletion of fadL, ompT or ompW remained without change. These results may suggest the importance of TolC and OmpC in response to NA resistance. Survival capability assay was also performed to investigate the ability of NA-R-O, NA-R and these mutants in response to NA resistance (Figure 3B-D). Eight concentrations of NA were utilized for the studies. They included a series of 2-fold dilutions ranging from 0.078 µg/mL (1/64 MIC of NA-R-O) to 2.5 µg/mL (1/2 MIC of NA-R-O), and then 3.33 µg/mL (4/6MIC of NA-R-O) and 4.167 µg/mL (5/6 MIC of NA-R-O). Having survived in media with 1.25 µg /mL NA or lower, NA-R-O grew a bit faster than NA-R, but NA-R grew faster than NA-R-O when 2.5 µg/mL of NA or above was used. Only about 2/3 and 1/3 OD values were measured in NAR-O with respect to NA-R when 3.333 and 4.167 µg/mL NA were utilized, respectively (Figure 3B). Compared with NA-R-O, these mutants showed significantly elevated or decreased growth. The higher the NA concentrations that were used, the more significant the changes detected were. Specifically, ∆tolC and ∆ompC grew significantly slower when 0.625-4.167 and 2.5-4.167 µg/mL of NA were used, respectively, and showed only 13.7% and 40.8% of growth of NA-R-O cultured with 4.167 µg/mL of NA. On the contrary, ∆ompT and ∆ompW showed elevation of growth in the culture with 0.625-4.167 and 0.078-4.167 µg/mL of NA, respectively, and showed 128.2% and 116.7% growth of NA-R-O cultured with 4.167 µg/mL of NA. ∆fadL showed either increased or decreased growth, respectively, in the culture with NA that was below or above 0.625 µg/mL. These results may suggest that TolC, OmpC and possibly FadL played some roles in NA resistance directly, but not OmpT and OmpW. 3.5. Involvement of EnvZ/OmpR Two-Component System in the Regulation of NA Resistance. Up-regulation of OmpC in response to NA resistance was detected in the current study. A line of evidence has indicated that OmpC and OmpF are regulated through the EnvZ/OmpR signal transduction pair. Therefore, it would be interesting to investigate possible roles of EnvZ, OmpR, OmpC and OmpF in NA resistance further. As the first step, we measured MICs of the strains with the deletion of these genes. Figure 4A is a summary of MICs of ∆ompC, ∆ompF, ∆ompR and ∆envZ. Deletion of ompR or ompC resulted in significantly increase or decrease in MICs of their genedeleted strains, respectively (40 or 2.5 µg/mL of NA). However, deletion of EnvZ and ompF genes had no effect on the MICs of their genetic modified strains. Second, we performed survival capability using these genetically modified strains in order to Journal of Proteome Research • Vol. 7, No. 6, 2008 2401
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Table 1. Identification of E. coli OM Proteins Shown in Figure 1 by PMF Searching and Subcellular Locations by Program PSORTb Version 2.0 (http://www.psort.org/psortb/) spot no.
accession name
character description
subcellular location
MW/pI
peptides matched
sequence coverage%
Mosco w score
volume difference (NA-R-O/NA-R)
Up-Regulated 1 TOLC_ECOLI
OM
53967/5.46
13
40%
138
0.181 ( 0.017/0.346 ( 0.062
2
OM
53967/5.46
13
40%
138
0.117 ( 0.005/0.269 ( 0.009
OM OM OM IM OM OM
35540/5.76 35540/5.76 40343/4.58 25362/5.4 22928/6.03 22928/6.03
13 13 8 9 6 6
36% 36% 35% 30% 28% 28%
176 121 125 80 65 65
0.694 ( 0.091/1.340 ( 0.178 0.310 ( 0.060/0.659 ( 0.087 0.223 ( 0.057/0.4052 ( 0.118 0/0.620 ( 0.208 2.826 ( 0.294/9.098 ( 0.269 0.641 ( 0.124/1.573 ( 0.312
Cytoplasm 47200/5.44 OM 48742/5.09
10 8
33% 26%
109 62
0.852 ( 0.203/0.307 ( 0.093 1.81 ( 0.071/0.7424 ( 0.140
9
22%
64
1.575 ( 0.175/0.388 ( 0.235
6 7 8 9 10 11
multidrug efflux and protein export TOLC_ECOLI multidrug efflux and protein export OMPT_ECOLI Protease OMPT_ECOLI Protease OMPC_ECOLI Omp 1B PSPA_ECOLI Phage shock protein A OMPW_ECOLI a receptor for colicin S4 OMPW_ECOLI a receptor for colicin S4
Down-Regulated 3 ACEA_ECOLI 4 FADL_ECOLI 5
FADL_ECOLI
Isocitrate lyase long-chain fatty acid transport protein long-chain fatty acid transport protein
OM
48742/5.09
investigate the roles of ompC, ompF, ompR and envZ in the NA resistance (Figure 4B). We normalized bacterial growth values in medium without NA as percentages. The normalized growth percentages of ∆ompC, ∆ompR, ∆envZ, and NA-R, were significantly lower, and higher than those of NA-R-O in a series of NA concentrations, respectively. These changes correlated with the increase of NA concentrations used. However, no significant change in the growth percentage for ∆ompF strain was observed as compared to that of the control except for a slight faster growth in medium with 1.875 µg/mL of NA. Expression levels of OmpC and OmpF in NA-R-O, NA-R, ∆ompC, ∆ompF, ∆envZ and ∆ompR were also investigated using Western blotting (Figure 4C). With respect to NA-R-O,
up-regulation and down-regulation of OmpC were observed, respectively, in NA-R and ∆ompF, and ∆envZ and ∆ompR cultured with or without NA. Meanwhile, OmpC level in ∆envZ and ∆ompR was significantly lower in cultures with 1/2 MIC NA than the cultures without NA. In detail, no or trace OmpC was detected when ∆envZ and ∆ompR survived in medium with 1/2 MIC of NA. On the other hand, up-regulation and down-regulation of OmpF were appreciated in ∆ompC and ∆envZ, and NA-R and ∆ompR, respectively, compared to NAR-O. Level of OmpF in NA-R-O, ∆ompC and ∆envZ was significantly lower in media with 1/2 MIC NA than that without NA. Interestingly, no OmpF was detected in NA-R and ∆ompR. These results on the OmpC and OmpF expression were consistent with those obtained from 2-DE analysis, and might suggest an important role of EnvZ/OmpR two-component system in the regulation of NA resistance.
4. Discussion
Figure 2. Confirmation of altered proteins using 2-DE Western blotting. (A) Testing for antibody specificity; (B) enlarged partial 2-DE Western blotting showing alteration of TolC, FadL, OmpT and OmpW in response to NA resistance; (C) histogram displays the analysis in intensity of them between NA-R (white) and NAR-O (black), and bars represent spot volume. 2402
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Antibiotic-resistant mechanism is an intriguing scope with the increasing drug resistance worldwide. NA-resistant bacteria have frequently been reported, but information regarding OM proteome and its roles in the regulation of NA resistance is not available. Our previous reports investigated antibioticresistant OM proteomes in response to tetracycline, ampicillin and chloramphenicol using linear9 and nonlinear 2-DE.10 The 2-DE-PAGE patterns of the linear gradient gels were different from those of the nonlinear gradient gels, but the OM proteins detected in both gels were almost identical.9,10 Functional proteomic approach based on linear 2-DE was applied to this study. Eleven spots representing 7 altered proteins were determined. Out of the seven proteins, TolC, FadL, OmpT, OmpC and OmpW are OM proteins. Up-regulation of TolC, OmpT, OmpC and OmpW, and down-regulation of FadL and OmpF were confirmed using Western blotting. Moreover, significant decreases of MIC and survival capability in ∆tolC and ∆ompC were observed, whereas no MIC change and elevated survival capability in ∆fadL, ∆ompT, ∆ompW and ∆ompF were detected. These findings may suggest that TolC and OmpC are important to NA resistance. TolC is a part of the well-characterized AcrAB-TolC drug efflux system in E. coli that is energetically driven by proton antiport, which is recently
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Figure 4. Investigation of EnvZ/OmpR two-component system in regulation of NA resistance. (A) MICs of NA-R-O, NA-R, ∆ompC, ∆ompF, ∆envZ, and ∆ompR. The detailed values of MIC are showed on the right side. (B) Survival capabilities of NA-R-O, NA-R, ∆ompC, ∆ompF, ∆envZ, and ∆ompR. (C) Western blotting analysis of samples from NA-R-O, NA-R, ∆ompC, ∆ompF, ∆envZ, and ∆ompR.
Figure 3. Histogram displays MICs and survival capabilities. (A) MICs of NA-R-O, NA-R, ∆ompT, ∆fadL, ∆ompW, ∆tolC and ∆ompC. The detail values of MIC are shown on the right side. (B) Survival capabilities of NA-R-O, NA-R. (C and D) Survival capabilities of NA-R-O, ∆ompC, ∆ompT, ∆tolC, ∆fadL, and ∆ompW. Concentrates of NA range first from 0.078 to 2.5 µg/mL (equal to 1/64 to 1/2 MIC of NA-R-O) and then from 3.33 to 4.167 µg/mL (equal to 4/6 and 5/6 MIC of NA-R-O). Each bar shows the average and standard error of three separate experiments, and the asterisks denote the datum points that differed significantly (P < 0.05) between the NA-R-O and related strains by the paired Student’s t test.
structurally resolved.26 Both the central core of this ternary complex and in particular channels through the individual proteins are considered for the export of different small compounds. This function is also demonstrated by a study using the mutants of the AcrAB-TolC system acrB.27 The expression of OmpC and OmpF is regulated by EnvZ-OmpR or CpxA-CpxR components which belong to two distinct twocomponent systems.28,29 In the present study, down-regulation of OmpC in ∆ompR and ∆envZ, and of OmpF in ∆envZ was
appreciated when they survived in the cultures with 1/2 MIC NA. Levels of OmpC in ∆OmpR and ∆envZ, and of OmpF in ∆ompR were significantly lower than those in NA-R-O. Upregulated OmpC and OmpF were detected, respectively, in ∆ompF and ∆ompC but not in NA-R-O. On the other hand, inhibited growth of ∆envZ, ∆ompR, and ∆ompC was observed when they survived in media with NA. These results suggest that EnvZ and OmpR contribute to the bacterial resistance to NA. A line of evidence indicates that the porin channel size of OmpF is larger than that of OmpC and thus is more favored for antibiotics.30,31 Expression of OmpC and OmpF usually goes in opposite directions. It has been reported that the loss of OmpF increased the resistance to norfloxacin, tetracycline, cephalothin and cefoxitin, and slightly to chloramphenicol, while the loss of OmpC only increased the resistance to cephalothin and cefoxitin, but had little effect on the resistance to norfloxacin, chloramphenicol and tetracycline.32 The lack of expression of the analogues of the OmpC and OmpF in E. aerogenes strain results in decreased susceptibility to Meropenem and cefepime to imipenem resistance.8 Hirakawa et al. reported that ompR gene conferred high deoxycholate resistance and low-level SDS and fosfomycin resistance in a comprehensive study on drug resistance mediated by overexpression of the two-component signal transduction systems in Journal of Proteome Research • Vol. 7, No. 6, 2008 2403
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E. coli. However, little is known about role of EnvZ/OmpR in antibiotic resistance. The results obtained from our study suggest that EnvZ/OmpR two-component system may be involved in the regulation of NA resistance in E. coli, and the mechanism of NA resistance mediated by the two component system and downstream OmpC is different from that of β-lactamases resistance. In the present study, we also found that ∆ompT and ∆ompW grew significantly faster in medium with NA, whereas ∆fadL grew significantly slower at NA concentrations from 0.078 to 0.313 µg/mL, but not in media with 0.625 µg/mL NA or higher (1.250-4.167 µg/mL NA). Of the three OM proteins, FadL and OmpW have 14-stranded and 8-stranded β-barrel structures with a hydrophobic channel as long-chain fatty acid transport and a passage of small hydrophobic molecules, respectively.34,35 It has been reported recently that the FadL homologues were involved in the transport of xenobiotics and aromatic hydrocarbons, such as styrene, xylene, cumene and toluene which are hydrophobic compounds.36–39 Mutation of fadL results in resistance to antimicrobial compounds.27 In the current study, decreased FadL and elevated OmpW were detected in NAresistant E. coli, though both are hydrophobic channels. The loss of fadL resulted in diverse changes of bacterial growth in response to antimicrobial activity, depending on the dose of NA used. These results may suggest NA transportation mediated by the channel proteins may be regulated at multiple levels. In summary, E. coli OM proteome in response to NA resistance was first reported here. Of these OM proteins, TolC and OmpC may play more important roles in the regulation of NA resistance. In addition, EnvZ/OmpR two-component system was found for the first time to be involved in the regulation of NA resistance in E. coli. These findings may provide novel insights into mechanisms of NA resistance.
Acknowledgment. This work was sponsored by grants from “863” project (2006AA09Z432), NSFC project 30530610, Guangzhou Key Project 2006Z3-E0251 and Guangdong NSF key project (7117645).
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Journal of Proteome Research • Vol. 7, No. 6, 2008 2405
