Identification and Network of Outer Membrane Proteins Regulating Streptomysin Resistance in Escherichia coli Hui Li, Bao-Cheng Wang, Wen-Jiao Xu, Xiang-Min Lin, and Xuan-Xian Peng* Center for Proteomics, State Key Laboratory of Bio-Control, School of Life Sciences, Sun Yat-Sen University, University City, Guangzhou 510006, People’s Republic of China Received April 23, 2008
Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, ∆tolC, ∆ompT, ∆dps, ∆tolC-R, ∆ompT-R, ∆dps-R and ∆fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli. Keywords: Antibiotics • streptomycin • Escherichia coli • outer membrane proteins • proteomics
1. Introduction During the last half century, most of the major infectious diseases in human and also in a number of nonhuman subjects were more or less brought under control due to the use of antibiotics. However, the increasing incidence of antibioticresistant bacteria came forth because of too frequent and inappropriate use of antibiotics.1,2 The antibiotic-resistant bacteria pose a serious threat to human health today.3,4 Therefore, there is an urgent need to elucidate the mechanisms of bacterial resistance to antibiotics, and to develop novel strategies to control the antibiotics-resistant bacteria.5 Previous reports have indicated that antibiotic resistance can be either intrinsic or acquired.6 The intrinsic resistance of Gram-negative bacteria is largely dependent on the constitutive or inducible changes of active efflux system and porins,7,8 especially the changes at the level of protein expression of outer membrane proteins (OM proteins). The OM proteomes involving in antibiotic resistance have been characterized in Escherichia coli with ampicilin, tetracycline, chloramphenicol or nalidixic acid resistance, in Pseudomonas aeruginosa with ampicilin, kanamycin or tetracycline resistance, in Neisseria * To whom correspondence should be addressed. Dr. Xuanxian Peng, State Key Laboratory of Bio-Control, School of Life Sciences, Sun Yat-Sen University, University City, Guangzhou 510006, People’s Republic of China. Tel, +86-20-3145-2846; fax, +86-20-8403-6215; e-mails,
[email protected],
[email protected].
4040 Journal of Proteome Research 2008, 7, 4040–4049 Published on Web 08/05/2008
gonorrheae with the resistance-induced isogenic mutant (92mu13), and in Staphylococcus aureus with vancomycinintermediate resistance.6,9–15 These studies push forward an understanding of mechanisms of antibiotic resistance. However, little is known about key OM proteins and their roles in the antibiotic resistance. Identification of these key proteins among the altered proteins may provide direct information for the development of new drugs for the control of antibioticresistant microbes.6 On the other hand, identification of these key proteins and their interaction network may provide novel insights into mechanisms of antibiotics resistance. For this purpose, we utilized functional proteomic methodologies to identify and characterize key OM proteins and their interaction network in E. coli with the resistance to streptomycin (SM). As the first step, we utilized 2-DE to identify altered OM proteins in the sarcosine-insoluble fraction of SM-resistant E. coli K12 BW25113 strain (SM-R) which was selected from its original strain (SM-R-O) by the use of 10 sequential propagations in Luria-Bertani (LB) medium with 1/2 minimum inhibitory concentration (MIC) of SM. The changes at the level of protein expression of the altered OM proteins were then validated by Western blotting. Finally, functional characterization of these altered OM proteins and interaction network was performed, respectively, using gene-deleted E. coli K12 BW25113 strains with SM resistance (∆SM-R) and their original strains (∆SMR-O). Our results provide valuable information on the possible 10.1021/pr800310y CCC: $40.75
2008 American Chemical Society
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Figure 1. Comparison of sarcosine-insoluble fractions between SM-R and SM-R-O based on 2-DE analysis. (A) SM-R-O (left) and SM-R (right) grown in LB medium; (B) the enlarged partial 2-DE gels showing altered expression spots; (C and D) histogram displays the changes in spot intensity of them between SM-R (white) and SM-R-O (black), and bars represent spot intensity with relative volume divided by the total volume over the whole image, according to Melanie ImageScan.
roles of these altered OM proteins, and their network in regulating SM resistance in E. coli. To our knowledge, this is the first report of OM proteome and its interactional network involved in the regulation of SM 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 genetic modified strains with gene deletion. E. coli K12 BW25113, ∆fadL, ∆ompT, ∆ompW, ∆lamB and ∆dps were kindly provided by NBRP (NIG, Japan). E. coli,16 and ∆tolC was the collection in our laboratory. The antimicrobial agent was purchased from a commercial source (Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. China). SM-R was selected by the use of 10 sequential propagations in LB medium with 1/2 MIC of SM of SM-R-O as described previously.9 The resulting MIC was 4-fold higher than that of SM-R-O. Meanwhile, six
∆SM-R-O: ∆tolC, ∆fadL, ∆ompT, ∆ompW, ∆lamB and ∆dps were sequentially propagated in the same condition. The resulting strains were termed ∆SM-R. They were ∆tolC-R, ∆fadL-R, ∆ompT-R, ∆ompW-R, ∆lamB-R and ∆dps-R. All bacteria were cultured in LB medium at 37 °C. 2.2. Isolation of Bacterial Membrane Proteins and OM Proteins. Bacteria were cultured in LB medium overnight, and then diluted at 1:100 using fresh LB medium. After the bacteria were cultured for 1.0 OD, they were harvested, washed in sterile saline, resuspended in 10 mL of sonication buffer (50 mM Tris/ HCl, pH 7.4) and disrupted by intermittent sonic oscillation for a total of 35 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 resulting pellet, membrane proteins, was solubilized in appropriate sterile pure water for Western blotting analysis. For isolation of OM proteins, the Journal of Proteome Research • Vol. 7, No. 9, 2008 4041
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Table 1. Identification of Altered Proteins by PMF in Streptomycin-Resistant Strains spot no. accession name
subcellular location
character description
MW(Da)/pI
3 4 6 9 11
multidrug efflux and protein export TOLC_ECOLI multidrug efflux and protein export ACEA_ECOLI Isocitrate lyase ACEA_ECOLI Isocitrate lyase OMPT_ECOLI Protease OMPT _ECOLI Protease LAMB_ECOLI Maltose-inducible porin
Up-Regulation Integral OM 53967/5.46 protein Integral OM 53967/5.46 protein Cytoplasm 47200/5.44 Cytoplasm 47200/5.44 OM 35540/5.76 OM 35540/5.76 OM 49941/4.85
5
FADL_ECOLI
OM
1 2
7 8 10 12 13 14
TOLC_ECOLI
long-chain fatty acid transport protein OMPT _ECOLI Protease OMPT _ECOLI Protease OMPW_ECOLI a receptor for colicin S4 DPS_ECOLI DNA protection during starvation protein DPS _ECOLI DNA protection during starvation protein DPS _ECOLI DNA protection during starvation protein
volume difference (SM-R-O/SM-R)
7
25%
60
0.405 ( 0.148/1.083 ( 0.263
12
36%
114
0.212 ( 0.070/0.764 ( 0.325
10 8 13 10 9
33% 24% 52% 36% 38%
109 81 176 121 70
0.252 ( 0.303/1.5120 ( 0.232 0.566 ( 0.073/1.646 ( 0.132 0.694 ( 0.091/1.626 ( 0.322 0.653 ( 0.346/0.158 ( 0.463 0.238 ( 0.075/0.579 ( 0.339
Down-Regulation 48742/5.09
8
26%
62
1.882 ( 0.119/0.624 ( 0.150
OM OM OM unknown
35540/5.76 35540/5.76 22928/6.03 18564/5.72
12 12 6 8
50% 48% 28% 53%
unknown
18564/5.72
7
53%
unknown
18564/5.72
9
45%
pellet was solubilized with 2% (w/v) sodium lauryl sarcosinate (Sigma) at 4 °C according to a procedure described previously.17 The concentration of the extracted membrane proteins and OM proteins was determined using the Bradford method.18 2.3. 2-D Gel Electrophoresis. 2-DE was performed according to a procedure described previously.17 Twenty micrograms of proteins from each sample was used for the gel electrophoresis. The samples were first dissolved in the 2D sample buffer (8 M urea; 2 M thiourea; 4% CHAPS and 80 mM DTT, 0.5% carrier ampholyte), and then separated by the first linear isoelectric focusing (IEF, 0.2 cm × 7 cm) and the seconddimension electrophoresis using 12% acrylamide gels. After the gel electrophoresis, the gels were stained with Coomassie blueR250. 2.4. Image Analysis and Protein Identification by Peptide Mass Spectrometry. 2-DE gels were scanned in ImageScan (Amersham Biosciences, Sweden) and analyzed with ImageMaster 5.0 software (Amersham Biosciences). Altered spots were standardized and 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. Protein spots of interest were cut from the gel for mass spectrometric analysis according to a procedure described previously.17 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. 2.5. Western Blotting. OM protein and membrane protein samples were separated with 1-DE or 2-DE gels, and were then transferred to a NC membrane for 1 h at 70 V in the transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol) at 4 °C. Western blotting was carried out using a standard procedure. Rabbit antisera to TolC, FadL, OmpT, OmpW, LamB, or Dps were purchased from Wenta Bio Tech Corp., Ji’an, China. The specificity of anti-TolC, FadL, OmpT and OmpW was validated as described in our previous publications.6,12 The antisera were used as the primary antibodies for 1-D and 2-DE 4042
no. of peptides sequence recovered coverage% score
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159 0.709 ( 0.184/0.268 ( 0.282 151 0.309 ( 0.060/0.646 ( 0.112 65 3.826 ( 0.241/1.553 ( 0.095 95 14.299 ( 0.295/4.850 ( 0.098 92 32.431 ( 0.172/14.254 ( 0.169 102
2.022 ( 0.217/0.686 ( 0.189
Western blotting. Antibody-tagged protein bands and spots were detected by DAB and scanned in an AGFA white-light scanner at a resolution of 400 by 200 nm. Following background subtraction, the membrane patterns were matched to each other by visual comparison. Altered spots were compared and their intensities were measured based on the spot volumes. 2.6. Testing of Antimicrobial Susceptibility by MIC and Survival Capability Assays. MIC assay and survival capability assay were performed as described previously.6 MIC was measured using a 2-fold standard broth microdilution method. For survival capability assay, E. coli strains were first cultured for 16 h to saturation. The cultured media were then diluted at 1:1000. The diluted bacteria were cultured in 5 mL of LB medium containing SM at different concentrations. These cultures were shaken at 200 rpm for 6 h and the OD values were then measured at 600 nm. The experiments were performed in triplicates.
3. Results 3.1. Analysis of Sarcosine-Insoluble Fraction from SM-R and SM-R-O Using Subproteomic Approach. To investigate an altered OM proteome in response to SM resistance, a subproteomic approach was utilized to identify differential expressed proteins of sarcosine-insoluble fractions from SM-R and its control SM-R-O. SM-R was obtained by 10 sequential subcultures of SM-R-O using 1/2 MIC of SM. The MIC of SM-R was 25 µg/mL which was 4-fold higher than that of SM-R-O. SM-R and SM-R-O were separately cultured until 1.0 of OD value was reached at 600 nm. The sarcosine-insoluble fractions from SM-R and SM-R-O were extracted and then separated by 2-DE. Approximately 60 protein spots were visualized in each of the gels stained with CBB R-250 (Figure 1A). Fourteen of the 60 spots showed significant changes at the level of protein expression. These 14 protein spots were labeled from spots 1 to 14. Figure 1B was an expanded view of the spots in the gels. Spots 1-4, 6, 9, and 11 were found to be up-regulated, and spots 5, 7, 8, 10, and 12-14 were down-regulated in SM-R with respect to SM-R-O (Figure 1C,D). Table 1 showed the identities
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Figure 2. Validation of altered proteins by 1-DE, and 2-DE and Western blotting combination. (A) Testing for antibody specificity; (B) 1-D Western blotting; (C) 2-DE and Western blotting analysis; (D and E) histogram displays the analysis in intensity of 2-DE Western blotting between SM-R (white) and SM-R-O (black), and bars represent spot volume.
of these spots which were obtained by mass spectrometric analysis.17 The 14 spots were identified as seven uniquely proteins. They were TolC (up spots 1, 2), AceA (up spots 3, 4), FadL (down spot 5), OmpT (up spots 6, 9 and down spots 7, 8), OmpW (down spot 10), LamB (up spot 11), and Dps (down spots 12-14). Among these proteins, TolC, FadL, OmpT, OmpW and LamB are OM proteins, AceA is a cytoplasm protein and Dps is a location-unknown protein based on PSORTb Program analysis. 3.2. Characterization of Differentially Expressed OM Proteins Using 1-D or 2-D Gel Electrophoresis and Western Blotting. In the present study, we focused on OM proteins involved in SM resistance. The changes at the expression level of proteins such as TolC, FadL, OmpT, OmpW, and LamB and Dps were validated using 1-DE or 2-DE and Western blotting (Figure 2). Up-regulation of TolC, OmpT, LamB, and down-regulation of FadL, OmpW, Dps detected by 2-DE in SM-R were first confirmed by 1-DE and Western blotting (Figure 2B). To validate the changes of isoforms or other modifications of the altered proteins observed by 2D gel, we also performed Western blotting of 2D gels. Compared with the results obtained from 2-DE alone, the combined approach of 2-DE and Western blotting displayed two additional spots of FadL, OmpW and LamB with the equal changes, either increased or decreased; and the equal number spots of TolC
and Dps. In addition, there were four up-regulated spots of OmpT that were detected (Figure 2C-E). Generally, the results obtained from combined 2-DE and Western blotting analysis are consistent with those obtained from 2-DE alone. 3.3. Functional Characterization of Altered OM Proteins Using ∆SM-R-O. We next performed functional characterization of the altered OM proteins using genetic modified strains with deletion of the altered genes (∆SM-R-O). Specificity of the gene deleted strains was validated by specific antibodies (Figure 3A). The functional analysis carried out using two antimicrobial susceptibility assays: MIC assay and survival capability assay. Figure 3B was a summary of MICs of ∆tolC, ∆fadL, ∆ompT, ∆ompW, ∆lamB and ∆dps. Deletion of TolC, OmpT, LamB or Dps resulted in significantly either decrease or increase in MICs of the gene-deleted strains (>2-fold), respectively, whereas the absence of FadL or OmpW had no effect on the MICs of the modified strains. Therefore, the results from functional characterization of ∆tolC, ∆ompT or ∆dps were consistent with the results obtained from 2-D gel analysis of SM-R alone. The survival capability assay was also carried out to further investigate anti-SM abilities of the ∆SM-R-O strains. These bacteria were cultured in tubes with series of 2-fold dilution of SM concentrations from 3.125 to 0.39 µg/mL. The resulting cultures were assayed by measurement of optical density at 600 nm and the survival capability was determined by Journal of Proteome Research • Vol. 7, No. 9, 2008 4043
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Figure 3. Histogram of MIC assay by broth microdilution method. (A) Genetic modified strains with the gene deletion of the altered OM proteins validated by specific antibodies; (B) MICs of SM-R-O and ∆SM-R-O used in this study; (C) MICs of SM-R and ∆SM-R used in this study; (D) folds of MIC by division of ∆SM-R over ∆SM-R-O.
division of OD values of the cultures with SM over those without SM. The survival rate of ∆lamB cultured in 0.39 or 1.562 µg/mL of SM was significantly decreased (P < 0.05) or increased (P < 0.01), respectively. Increased survival rates (P < 0.05) were found in ∆dps cultured in 0.781 and 1.562 µg/mL of SM. Decreased survival rates (P < 0.01) were detected in ∆tolC cultured in 0.781 µg/mL of SM (Figure 4A). Generally, the results obtained from survival capability assay are in consistence with those obtained from MIC assay. In summary, the results obtained from functional characterization of ∆tolC, ∆ompT and ∆dps were consistent with the changes of TolC, OmpT and Dps, respectively, at the level of protein expression, which was detected by 2-DE analysis of SM-R. However, this consistence was not observed in ∆fadL, ∆ompW and ∆lamB, suggesting that TolC, OmpT and Dps might be functionally and directly linked to SM resis4044
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tance, and TolC and OmpT might positively and Dps negatively contribute to the regulation of the SM resistance. 3.4. Functional Characterization of Altered OM Proteins Using ∆SM-R. Since altered OM proteins play important roles in the regulation of SM resistance, we hypothesized that these OM proteins may also be able to contribute to the selection of SM-resistant strains. ∆tolC, ∆fadL, ∆ompT, ∆ompW, ∆lamB or ∆dps was cultured separately in LB media containing 1/2 SM MIC of SM-R-O. After 10 sequential propagations, the ∆SM-R strains were generated. The resistant strains were named ∆tolCR, ∆fadL-R, ∆ompT-R, ∆ompW-R, ∆lamB-R, and ∆dps-R respectively. Figure 3C is a summary of MICs of the genetic modified SM-R strains and ∆SM-R which was used as a control. The MICs of ∆fadL-R, ∆dps-R and the other five were 100, 50, and 25 µg/mL, respectively. Compared with their corresponding ∆SM-R-O, ∆fadL-R, ∆tolC-R, ∆ompT-R, ∆ompW-R, ∆lamB-R,
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Figure 4. (Continued). Journal of Proteome Research • Vol. 7, No. 9, 2008 4045
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Figure 4. Testing for survival capability. (A) ∆SM-R-O and SM-R-O; (B) ∆SM-R and SM-R; (C) Ratios of survival capability by division of ∆SM-R over ∆SM-R-O cultured in 0.781 µg/mL of SM; (D) ratios of survival capability by division of ∆SM-R over ∆SM-R-O cultured in 3.125 µg/mL and 1.562 µg/mL of SM; (E) folds of C; (F) folds of D.
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Figure 5. Western blotting analyses of samples from bacterial membrane protein extracts for the effects of the absence of each of the six altered OM proteins on the expression of other five proteins. The changes at the level of protein expression were investigated by detection of each of the altered six OM proteins in six pairs of ∆SM-R and ∆SM-R-O strains, and marked by OD intensity. (A) TolC; (B) FadL; (C) OmpT; (D) OmpW; (E) LamB; (F) Dps.
and ∆dps-R showed elevated MICs by 16-, 8-, 4-, and 2-fold, respectively (Figure 3D). The MIC of SM-R was elevated 4-fold, which was in the middle of those obtained from the six ∆SM-R tested. Obviously, the strains with distinct changes in folds of MIC showed more important ability in the selection of SMresistant strains than others. Therefore, among the altered OM proteins, FadL, TolC and OmpT may play more important roles in the selection of resistant strains. Survival capability assay was also carried out to investigate the anti-SM abilities of the ∆SM-R strains. These bacteria were cultured in a dilution series of SM in duplicate. The resulting cultures were assayed by measurement of optical density at 600 nm. Survival rates of ∆SM-R were significantly higher than those of SM-R strains in the cultures with 0.781, 1.562, 3.125, 4.683, 6.25 µg/mL of SM. In the cultures with 12.5 µg/mL of SM, ∆fadL-R and ∆lamB-R grew faster than SM-R, and ∆tolC-R, ∆ompT-R, ∆ompW-R, and
Table 2. Changes at the Level of Protein Expression in ∆SM-R with Respect to ∆SM-R-O TolC
∆tolC-R ∆fadL-R ∆ompT-R ∆ompW-R ∆lamB-R ∆dps-R
V -
FadL
OmpT
OmpW
LamB
Dps
-
V V
v V
V v V V
v v v V
v V V v
v V v
v v
v
∆dps-R had significantly lower survival capability than SM-R (Figure 4B). These results suggest that the deletion of the six OM proteins may result in the SM resistance, and the important roles of these proteins in the regulation of SM resistance. Similarly, TolC and OmpT were found to play a positive role, and Dps a negative role in the selection of SM resistant strains. FadL, like Dps, was also found to play a negative role in the Journal of Proteome Research • Vol. 7, No. 9, 2008 4047
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Table 3. Comparison in Changes at the Level of Protein Expression in ∆SM-R Detected by Western Blotting and Those in SM-R Detected by 2D Gel Electrophoresisa TolC
∆tolC-R ∆fadL-R ∆ompT-R ∆ompW-R ∆lamB-R ∆dps-R
V V VV V V
FadL
OmpT
OmpW
LamB
Dps
v
VV VV
vv v -
VV VV VV
vv vv vv v -
vv vv
VV -
vv vv
-
a Comparison was based on alterations obtained from ∆SM-R in Table 2 and those from SM-R in Figure 1. v, The protein has no change between ∆SM-R and ∆SM-R-O in Western blotting, but down-regulation in SM-R with respect to SM-R-O in 2-DE. vv, The protein is up-regulated in ∆SM-R with respect to ∆SM-R-O in Western blotting, but down-regulation in SM-R with respect to SM-R-O in 2-DE. V, The protein has no change between ∆SM-R and ∆SM-R-O in Western blotting, but up-regulation between SM-R and SM-R-O in 2-DE. VV, The protein is down-regulated in ∆SM-R with respect to ∆SM-R-O in Western blotting, but up-regulation in SM-R with respect to SM-R-O in 2-DE. -, The protein has no change between ∆SM-R and SM-R as well as between their controls.
selection of SM-resistant strains. Comparison of survival capability between ∆SM-R-O and ∆SM-R was performed using SM at the concentrations of 0.781, 1.562 and 3.125 µg/mL because these three concentrations were overlapped in the detection. Ratios were obtained by the division of survival rates of ∆SM-R over those of ∆SM-R-O (Figure 4C,D), and elevated folds were achieved by the division of the ratios of ∆SM-R over those of SM-R (Figure 4E,F). Notably, the ratios of ∆tolC-R/ ∆tolC, ∆fadL-R/∆fadL and ∆ompT-R/∆ompT were 868.3, 958.4 and 968.5 at 3.125 µg/mL, respectively. The corresponding elevated folds were 19.2, 21.2, and 21.5. These results indicate that deletion of TolC, OmpT and FadL may result in more significant changes in bacterial survival capability than the other proteins in the selection of SM-resistant strains. 3.5. Network of Altered OM Proteins. To investigate whether an interaction network of the altered OM proteins exists, we isolated membrane proteins of ∆SM-R and ∆SM-R-O, and investigated the effects of the absence of each of the six altered OM proteins on the expression of other five proteins using Western blotting (Figure 5). Table 2 was a summary of the Western blotting results. Increased, decreased and unchanged protein expression in ∆SM-R with respect to ∆SM-R-O was marked as elevation (v), decrease (V) and unchangeableness (-) of the proteins. Furthermore, the changes obtained from ∆SM-R with respect to ∆SM-R-O above were compared with those obtained from SM-R with respect to SM-R-O in 2-DE gels. Table 3 was a summary of the comparison results. The changes were classified into five types. They were weak up-regulation (v), up-regulation(vv), weak down-regulation (V), down-regulation (VV) and unchangeableness (-). As shown in Table 3, the opposite changes of most altered OM proteins were found in ∆SM-R compared with those in SM-R. These results suggest an interaction among the altered OM proteins and a regulating network of these proteins may exit. The effect of the absence of TolC, FadL, OmpT, OmpW, LamB or Dps on the expression of other five proteins was summarized in Figure 6. The upregulation, or deletion of TolC, LamB and OmpT resulted in the elevation of Dps and OmpW, OmpW, and Dps and FadL, respectively; deletion of TolC, Dps or LamB resulted in the elevation of OmpW; deletion of Dps and FadL resulted in elevation of FadL, OmpT, OmpW, and LamB and LamB and Dps, respectivrly. Interestingly, expression of TolC was not affected by the absence of other proteins, suggesting that TolC 4048
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Figure 6. Effect of absence of tolC, fadL, ompT, ompW, lamB or dps on the other five protein expression. The start and end of arrows, respectively, indicate ∆SM-R and altered proteins. (A) Deletion of genes results in up-regulation of proteins; (B) deletion of genes results in down-regulation of proteins.
may be an independent and upstream protein in the network. In addition, it was also found that Dps was the only protein that interacted with all other five proteins. The deletion of Dps resulted in increased expression of FadL, OmpT, OmpW and LamB and the absence of TolC, FadL and OmpT resulted in increased expression of Dps (Figure 6A). These results may suggest the central role of TolC and Dps in the network. In this interaction network, it was also found that the downregulation or absence of TolC, FadL, OmpT, OmpW negatively regulated the expression of LamB and OmpT, OmpT, LamB and OmpW, TolC, and FadL and LamB, respectively. Compared with other proteins, Dps was more independent. Only LamB deletion resulted in its decrease. Finally, OmpT and LamB were respectively related to other four or five proteins, and both might locate at the center of the network (Figure 6B), suggesting the important roles of LamB and OmpT in the regulation of other proteins in this network.
4. Discussion Several research groups including us have showed that bacterial OM subproteome rather than a single OM protein plays a role in intrinsic antibiotic resistance.6,9–13 However, little is known about the key proteins and the interaction network of the altered OM proteins. The questions we wanted to answer in the present study were (1) which OM protein may play a key role in SM resistance and (2) how altered OM proteins interacted with each other for the regulation to SM resistance. Our strategy included three steps: (1) Identification of differential OM subproteome in response to SM using 2-D gel electrophoresis and Western blotting; (2) Determination of key OM proteins in response to SM by functional characterization of the altered OM proteins using ∆SM-R-O and ∆SM-R; (3) Construction of a primary network among the altered OM proteins based on the effects of the absence of each of the altered OM proteins on the expression of other proteins in ∆SM-R. The up-regulation of TolC, OmpT, and LamB, and down-regulation of FadL, OmpW and Dps were observed in SM-R. Of the six altered proteins, TolC controlling effluxmediated multidrug resistance including SM has been reported in Salmonella enterica serovar Typhimurium DT104.19,20 However, functional characterization of the other five proteins in response to SM has not been performed, though a line of evidence has indicated that they are involved in other antibiotic resistance in E. coli.6,9–13 Our results indicate that TolC, OmpT, Dps and FadL are functionally linked to the SM resistance. They are key OM proteins directly contributing to positive or negative regulation of SM resistance in E. coli. It is worthy to mention
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Identification and Network of Outer Membrane Proteins that Dps was found to be a negative regulator in the network. This novel mechanism of negative regulation may open new revenue to the control of antibiotics-resistant bacteria. The present study also demonstrated that a regulating network of OM proteins for the regulation of SM resistance may exist. In this network, TolC may play a key role in the regulation of other protein expression, and OmpT, LamB and Dps may locate in the center of the network. Through the present study, we developed a method for the investigation of the effect of the altered proteins on the antibiotic-resistant ability using ∆SM-R-O strains which were propagated in the medium with SM. This method was based on the hypothesis that absence of an antibiotic-resistant gene may result in change in antimicrobial susceptibility of the mutant strain. The change may be appreciated by sequential propagation of the strain. The greater of the change is, the more important this deleted gene may be in the contribution of the resistance. Thus, the gene-deleted stains could be applied to the evaluation of importance of the OM proteins in the selection of antibiotic-resistant strains. Using this approach, we demonstrate that FadL, TolC and OmpT are more important than the others in the selection of SM-resistant strains. This is consistent with a previous report demonstrating the importance of TolC of S. enterica serovar Typhimurium in the selection of ciprofloxacin-resistant mutants.21 Equally important, our approach provides a novel way for the investigation of an interaction network of the altered OM proteins involved in the SM resistance. The network identified clearly specifies and strengthens the roles and importance of the six altered OM proteins in the regulation of SM resistance. Of the OM proteins in the network, TolC, OmpT, and Dps, may play more important roles in the positive or negative regulation of SM resistance than others. FadL may be a positive regulator in the selection of SM-resistant strains. These findings may provide novel insights into mechanisms of SM resistance in E. coli.
Acknowledgment. This work was sponsored by grants from “973” project (2006AA09Z432), NSFC project (30530610), Guangzhou Key Project (2006Z3-E0251) and Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals (2007A002). References (1) Kim, I. S.; Ki, C. S.; Kim, S.; Oh, W. S.; Peck, K. R.; Song, J. H.; Lee, K.; Lee, N. Y. Diversity of ampicillin resistance genes and antimicrobial susceptibility patterns in Haemophilus influenzae strains isolated in Korea. Antimicrob. Agents Chemother. 2007, 51, 453– 460. (2) Gherardi, G.; Fallico, L.; Del, G. M.; Bonanni, F.; D’Ambrosio, F.; Manganelli, R.; Palu ` , G.; Dicuonzo, G.; Pantosti, A. Antibioticresistant invasive pneumococcal clones in Italy. J. Clin. Microbiol. 2007, 45, 306–312. (3) Singer, R. S.; Ward, M. P.; Maldonado, G. OpinionsCan landscape ecology untangle the complexity of antibiotic resistance. Nat. Rev. Microbiol. 2006, 4, 943–952. (4) Davis, I. J.; Richards, H.; Mullany, P. Isolation of silver- and antibiotic-resistant Enterobacter cloacae from teeth. Oral Microbiol. Immunol. 2005, 20, 191–194.
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