A Novel Negative Regulation Mechanism of Bacterial Outer Membrane Proteins in Response to Antibiotic Resistance Xiang-Min Lin, Jun-Ning Yang, Xuan-Xian Peng,* and Hui Li* Center for Proteomics, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, University City, Guangzhou 510006, People’s Republic of China Received July 17, 2010
Although some outer membrane (OM) proteins involved in antibiotic resistance have been previously reported, the OM proteins regulating chlortetracycline (CTC) resistance are largely unknown. In this study, we employed a subproteomics approach to identify altered OM proteins of Escherichia coli in response to CTC exposure. Upregulation of TolC and downregulation of LamB, FadL, OmpC, OmpT, and OmpW were found in E. coli strains exposed to CTC at a high concentration that was increased suddenly and at a half-minimum inhibitory concentration (MIC) that was kept constant in the culture medium. These changes in the level of protein expression were validated using Western blotting. In addition, the possible roles of these altered proteins and their regulation mechanisms in response to CTC exposure were investigated using genetically modified strains with gene deletion of these altered proteins. It was found that deletion of tolC, fadL, ompC, ompT, or ompW resulted in a decrease in the MICs and survival capabilities of the gene-deleted strains, whereas the absence of lamB led to an improvement of the two abilities. The downregulation of LamB expression in the CTC-resistant E. coli strain and the increased antibiotic resistance in its gene-deleted strain suggested a negative regulation mechanism in E. coli in response to CTC exposure. Meanwhile, the direction of the regulation pattern in response to CTC exposure was different from that in E. coli in response to exposure to other antibiotics. These findings uncover a novel antibiotic-resistant mechanism in which bacteria respond to exposure to antibiotics through alteration of the direction of regulation of OM proteins. Keywords: Regulation direction • negative regulation • antibiotic resistance • outer membrane proteins • LamB • E. coli • proteomics
1. Introduction Antibiotics have been widely used to treat infectious diseases in human and non-human subjects for approximately six decades. Inappropriate use or overuse of antibiotics results in the emergence of antibiotic-resistant strains that evolve under man-made selection.1-3 The number of antibiotic-resistant bacteria has rapidly increased, and almost all antibiotics are involved.3-5 In general, these antibiotic-resistant mechanisms include enzyme and target modifications, permeability decrease, and efflux system activation.3,6 The decrease in membrane permeability and the activation of the efflux pump are the major characteristic features of outer membrane (OM) proteins of Gram-negative bacteria in response to antibiotics.3,6 Several papers have reported the antibiotic-resistant OM proteomes of Escherichia coli in response to streptomycin, chloramphenicol, nalidixic acid, ampicillin, or tetracycline using a subproteomics approach, detection of the subcellular fraction.7-11 Among these subproteomes, alteration of TolC, OmpC, FadL, LamB, OmpT, and OmpW is always detected. Upregulation of TolC and OmpC and downregulation of FadL * To whom correspondence should be addressed: School of Life Sciences, Sun Yat-sen University, University City, Guangzhou 510006, People’s Republic of China. E-mail:
[email protected] or
[email protected].
5952 Journal of Proteome Research 2010, 9, 5952–5959 Published on Web 08/18/2010
are found to be unchangeable in these antibiotic-resistant bacteria, but changes in LamB, OmpT, and OmpW at the protein expression level are involved in antibiotic resistance. The level of expression of OmpT is elevated in streptomycinand nalidixic acid-resistant bacteria and decreased in chloramphenicol-resistant bacteria.7-9 The level of expression of LamB is decreased in chloramphenicol- and tetracycline-resistant bacteria but increased in streptomycin-resistant bacteria.7,8,10,11 Downregulation and upregulation of OmpW are detected in streptomycin-resistant bacteria and the other four antibioticresistant bacteria, respectively.7-11 These results indicate that the directions of regulation of these altered OM proteins are associated with antibiotic resistance and may be a key in understanding antibiotic-resistant mechanisms. The key OM proteins in these antibiotic-resistant proteomes have been characterized using functional characterization of altered OM proteins in genetically modified strains. TolC is always the most important OM protein in each of these antibiotic-resistant proteomes, but others may not always be, depending on the types of antibiotics used. It has been reported that TolC and OmpC, and OmpT and LamB, play more important roles in nalidixic acid and streptomysine resistance, respectively, than other OM proteins identified.7-9 In summary, the same altered OM proteins identified from different antibiotic-resistant bac10.1021/pr100740w
2010 American Chemical Society
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Negative Regulation of LamB in Response to CTC teria may have differential activities in response to antibiotics, and their alterations at the protein expression level and the importance of the changes are somewhat antibiotic-dependent. Thus, an improved understanding of these specific OM subproteomes, especially identification of key OM proteins and their regulation mechanism, including the regulation of direction, is critical for the elucidation of bacterial antibioticresistant mechanisms. Chlortetracycline (CTC), the first member of the tetracycline group, has been widely clinically used since the late 1940s. Bacteria resistant to this drug have been reported more frequently, especially in non-human applications.12-15 However, information regarding the CTC-resistant OM proteome is not available, which hampers our understanding of the key OM proteins and their role in CTC resistance. The aim of this study was to investigate the OM proteome that is responsible for CTC resistance, especially to identify key OM proteins and their possible roles in CTC resistance and the possible resistant mechanism.
2. Materials and Methods 2.1. Bacterial Strains. The bacterial strains used in this study were E. coli K12 BW25113 and its mutants. E. coli K12 BW25113, ∆lamB, ∆ompT, ∆ompW, and ∆fadL were kindly provided by NBRP (NIG). E. coli16 and ∆tolC, ∆ompC, and +lamB (gene complementation strain) were the same strains that were used in our previous report from the collections in our laboratory.17 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., Solon, OO). Drug stocks were prepared in sterile water and stored at -70 °C until they were used. A CTC-resistant strain (CTC-R) was selected from E. coli K12 BW25113 (CTC-R-O) with the use of 10 sequential subcultures at the half-minimum inhibition concentration (MIC) of CTC as described previously.10 2.2. Bacterial Culture and OM Protein Extraction. Bacterial cultures and OM protein extraction were performed as described previously with some modifications.8 Four bacterial cultures, CTC-R, CTC-R-O, and CTC-S (the strain was stressed by a large dose of CTC suddenly) and its control (CTC-S-C), were used here for extraction of OM proteins. For the samples of CTC-R and CTC-R-O, they were separately cultured in LB medium overnight and then diluted 1:100 using fresh LB medium. When the sample reached an OD of 1.0, these bacteria were harvested. For the samples of CTC-S and CTC-S-O, the overnight culture was diluted 100-fold in fresh LB medium. A CTC solution was added to an OD ) 1.0 culture at a final concentration 8-fold greater than the MIC (50 µg/mL); the cultures were kept for 2 h as CTC-S, and no drug was added as CTC-S-C. These resulting CTC-R, CTC-R-O, CTC-S, and CTCS-O cells were used for OM protein extraction via the lauryl sarcosinate method.8 Briefly, these harvested bacterial cells were washed in sterile saline, resuspended in sonication buffer [50 mM Tris-HCl (pH 7.4) and 1 mM PMSF], and disrupted by intermittent sonic oscillation for a total of 15 min in 9 s intervals on ice. Unbroken cells and cellular debris were removed by centrifugation at 5000g for 20 min. The supernatant was further ultracentrifuged for pellet at 100000g for 40 min at 4 °C. The pellet was resuspended in 10 mL of 2% (w/v) sodium lauryl sarcosinate (Sigma) and incubated at room temperature for 1 h, followed by ultracentrifugation at 100000g for 40 min at 4 °C. The resulting pellet was resuspended in sterile pure water and
stored at -20 °C. The concentration of the OM proteins in the final preparation was determined using the Bradford method. 2.3. Two-Dimensional Electrophoresis (2-DE) and Mass Spectrometric (MS) Analysis. 2-DE was performed according to a procedure described previously.18 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) was conducted by mixing pH 3-10 and pH 4-6 carrier ampholyte (ratio 1:1) for 10000 V h. After being equilibrated for 15 min, the gels were subjected to twodimensional electrophoresis using a 12% acrylamide gel. The preparative gels were stained with Coomassie Blue-R250. Subsequently, gels were scanned with ImageScan (Amersham Bioscineces, Uppsala, Sweden), and the raw images were analyzed using the 2-DE software ImageMaster version 5.0. Altered spots were standardized and compared on the basis of their volume percentages in total spot volume over the whole gel image. Significantly changed spots were selected by a rate increased or decreased >2-fold or complete appearance or disappearance. Mass spectrometric analysis was conducted using a procedure described previously.9 Briefly, the differential proteins were digested with trypsin as a routine procedure. The sample solution (30-100 ppm) with an equivalent matrix solution was applied to the MALDI TOF-Target and prepared for MALDI-TOF MS analysis. HCCA was used as the matrix. MALDI-TOF spectra were calibrated using trypsin autodigestion peptide signals and matrix ion signals. All MALDI analysis was performed with a fuzzy logic feedback control system (Reflex III MALDI-TOF system, Bruker) equipped with delayed ion extraction. Peptide masses were searched against the NCBI database using Mascot (http://www.matrixscience.com); the E. coli protein database was defined as a matching species, and the mass tolerance was 150 ppm. The protein subcellular locations were determined with PSORTb version 2.0 (http:// www.psort.org/psortb/). 2.4. 2-DE Western Blotting. Western blotting on 2-DE was performed as previously described.7 Briefly, the 2-DE gels of OM proteins were transferred to a NC membrane for 1 h at 100 mA in transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol) at 4 °C. The rabbit antiserum to TolC, LamB, FadL, OmpC, OmpT, or OmpW was separately used as the primary antibody, and the horseradish peroxidase (HRP)conjugated goat anti-rabbit antibody was used as the secondary one (Guangzhou Chengxue Corp.). Western blotting results were displayed by DAB. 2.5. Testing of Antimicrobial Susceptibility by Minimal Inhibitory Concentration (MIC) and Survival Capability Assays. Susceptibility to antibiotic was determined by MIC and survival capability assays as described previously.8 The MIC assay used a 2-fold standard broth microdilution method. For the survival capability assay, E. coli strains were first cultured at 37 °C for 16 h to saturation. The cultured medium was then diluted 1:1000 using 5 mL of LB medium containing CTC at different concentrations. They were shaken at 200 rpm for 6 h, and the OD values were then measured at 600 nm. The survival capability was determined by dividing the OD values of the cultures with CTC by the values of those without CTC. The experiments were performed in triplicate.
3. Results 3.1. Thirteen Altered Spots Representing Seven Proteins Were Identified in CTC-R by 2-DE. E. coli K12 BW25113 was subinoculated in LB medium with a half-MIC of CTC for 10 Journal of Proteome Research • Vol. 9, No. 11, 2010 5953
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Figure 1. 2-DE subproteomics for investigation of altered OM proteins in response to CTC. (A) Growth curve of CTC-R-O and CTC-R. (B) Representative 2-DE map of the sarcosine-insoluble fraction of CTC-R-O grown in LB medium. (C) Representative 2-DE map of the sarcosine-insoluble fraction of CTC-R grown in LB medium. (D) Enlarged partial 2-DE gels showing altered expression spots. (E) Histogram displaying the changes in spot intensity of altered proteins between CTC-R-O (gray) and CTC-R (white). Bars represent spot intensity, with the relative volume divided by the total volume over the whole image, according to ImageMaster version 5.0.
serial passages, and the MIC of the resulting strain (CTC-R) increased from 6.25 to 50 µg/mL, which was 8-fold higher than that for CTC-R-O that underwent 10 serial passages without the antibiotic. The result suggested that CTC-R was selected. 5954
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Compared to CTC-R-O, CTC-R exhibited a bit slower growth in the postlog phase (Figure 1A). To identify altered OM proteins from CTC-R, we extracted OM proteins of CTC-R-O and CTC-R by lauryl sarcosinate
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Negative Regulation of LamB in Response to CTC Table 1. Identification of Altered Spots by PMF Searching in a Chlortetracycline-Resistant Strain spot
accession number
character description
subcellular location
MW/pI
no. of peptides matched
Moscow scorea
volume % difference (CTC-R-O/CTC-R)
1 2 3
TOLC_ECOLI TOLC_ECOLI TOLC_ECOLI
multidrug efflux and protein export multidrug efflux and protein export multidrug efflux and protein export
Upregulated OM 53967/5.46 OM 53967/5.46 OM 53967/5.46
8 13 7
86 138 60
0.405 ( 0.148/1.290 ( 0.202 0.212 ( 0.133/0.473 ( 0.084 0.137 ( 0.034/0.590 ( 0.182
4 5 6 7 8 9 10 11 12 13
ACEA_ECOLI FADL_ECOLI LamB_ECOLI OmpT_ECOLI OmpT_ECOLI OmpT_ECOLI OmpT_ECOLI OmpC_ECOLI OMPW_ECOLI OMPW_ECOLI
isocitrate lyase long-chain fatty acid transport protein maltose-inducible porin protease protease protease protease Omp 1B a receptor for colicin S4 a receptor for colicin S4
Downregulated cytoplasm 47200/5.44 OM 48742/5.09 OM 49941/4.85 OM 35540/5.76 OM 35540/5.76 OM 35540/5.76 OM 35540/5.76 OM 40343/4.58 OM 22928/6.03 OM 22928/6.03
10 8 7 13 10 12 12 8 6 6
109 62 70 176 121 159 151 125 65 65
0.852 3.145 0.238 0.694 0.753 0.709 0.309 0.223 2.826 0.891
( ( ( ( ( ( ( ( ( (
0.103/0.061 0.307/1.491 0.075/0.120 0.091/0.322 0.146/0.373 0.184/0.376 0.060/0.088 0.057/0.107 0.294/0.420 0.095/0
( ( ( ( ( ( ( ( (
0.061 0.219 0.066 0.153 0.215 0.184 0.001 0.071 0.121
a Peptide masses were searched against the NCBI database using Mascot (http://www.matrixscience.com); the E. coli protein database was defined as a matching species, and the mass tolerance was 150 ppm.
Figure 2. Confirmation of these altered proteins in response to CTC exposure by 2-DE Western blotting. (A) 2-DE Western blotting maps for comparison of these altered OM proteins between CTC-R-O and CTC-R. (B) Histogram displaying the 2-DE changes in the spot volume of these altered protein spots between CTC-R-O (gray) and CTC-R (white). Bars represent the spot intensity with relative volume, using ImageMaster version 5.0. The detection was performed in the same two NC papers, one from CTC-R and the other from CTC-R-O. Comparison between these altered proteins, some of which showed upregulation and the others downregulation in one NC paper, could produce more reliable results.
extraction and analyzed them by 2-DE. Approximately 50 spots were determined from each of these gels (Figure 1B,C). Thirteen differentially expressed spots, named 1-13 as marked in Figure 1B-D, were characterized in CTC-R as compared to CTC-R-O. Of these 13 spots, three were upregulated and 10 were downregulated. The enlarged partial 2-DE gels showing altered protein spots and a detailed comparative histogram view are shown in panels D and E of Figure 1. These 13 altered spots were excised from gels and digested with trypsin after being destained. The resulting digestion mixture was analyzed by MALDI-TOF MS to assign putative functions to these proteins. Table 1 showed the identities of these spots by MALDI-TOF MS. They were identified as seven unique proteins, TolC (up spots 1-3), AceA (down spot 4), FadL (down spot 5), LamB (down spot 6), OmpT (down spots 7-10), OmpC (down spot 11), and OmpW (down spots 12 and 13). All these proteins except AceA were OM proteins on the basis of protein subcellular location analysis. 3.2. The Altered Protein Expression Was Confirmed by 2-DE Western Blotting. To confirm the altered OM protein expression detected by 2-DE gels, 2-DE Western blotting rather
than 1-DE was used. The Western blotting result indicated a good correlation between 2-DE and its Western blotting in the determination of the expression levels of TolC, LamB, OmpC, and OmpW. On the other hand, 2-DE Western blotting results seemed to be more sensitive and reliable than 2-DE. A decrease for one FadL spot was detected in 2-DE gels, while downregulation of three FadL spots was revealed by Western blotting. Similarly, four and five detectable OmpT spots were detected by 2-DE gels and by 2-DE Western blotting, respectively. In summary, upregulation of TolC and downregulation of LamB, FadL, OmpC, OmpT, and OmpW were detected, which was consistent with the result of 2-DE gel analysis (Figure 2A). The detailed comparative histogram views are shown in Figure 2B. These results demonstrated that TolC, LamB, FadL, OmpC, OmpT, and OmpW were CTC-responsive OM proteins. 3.3. Ten Altered Spots Representing Six OM Proteins in Response to a Suddenly Increased High Concentration of CTC Were Identified by 2-DE. To investigate whether these altered OM proteins in CTC-R were detected in CTC-S, bacterial OM proteins were extracted from both CTC-S and CTC-S-C and Journal of Proteome Research • Vol. 9, No. 11, 2010 5955
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Figure 3. Differential OM protein expression profiles of E. coli 2 h after a suddenly strong CTC treatment detected by 2-DE and confirmed by Western blotting. (A) Enlarged partial 2-DE gels showing altered expression spots. (B) Enlarged 2-DE Western blotting results showing these target spots detected. The detection was performed in the same two NC papers, one from CTC-S and the other from CTC-S-O. Comparison between these altered proteins, some of which showed upregulation and others downregulation in one NC paper, could produce more reliable results.
then analyzed by 2-DE. Upregulation of two TolC spots (TolC1, 2), one OmpW spot (OmpW1), and one OmpT (OmpT1) and downregulation of a FadL spot, a LamB spot, two OmpT spots (OmpT2-3), one OmpW spot (OmpW2), and an AceA were detected in these gels (Figure 3A). These results indicate that CTC-S exhibited almost the same changes as CTC-R did. Furthermore, Western blotting was used to confirm the 2-DE results (Figure 3B). These altered CTC-S OM proteins identified by 2-DE were confirmed by Western blotting. These results suggested the diversity of OmpT and OmpW spots in CTC-S with respect to CTC-R, but the change trends of six OM proteins were similar for CTC-R and CTC-S. In summary, TolC, LamB, FadL, OmpC, OmpT, and OmpW are commonly changed proteins from the two CTC strains. 3.4. Functional Characterization of Altered OM Proteins Using Genetically Modified Strains. To further investigate the capability of these altered OM proteins in CTC resistance, MIC and survival capability assays were performed using their gene deletion mutants. Deletion of tolC or lamB resulted in a 4-fold decrease or increase in the MIC, respectively. However, MICs in the mutants with deletion of ompT, ompC, fadL, or ompW remained unchanged (Figure 4A). These results may indicate the importance of TolC and LamB in response to CTC. After this study, the survival capability assay was also performed to investigate the ability of CTC-R and these mutants in response to CTC. A dose-dependent experiment indicated that the ability of CTC-R in resistance to CTC was improved with CTC concentration in comparison with that of CTC-R-O (Figure 4B). Importantly, compared with CTC-R-O, these mutants exhibited significantly increased or decreased levels of growth when they survived in the medium with a 1/16 MIC (Figure 4C). The absence of TolC, OmpC, OmpT, OmpW, or FadL led to the elevation of bacterial susceptibility to CTC. The order of susceptibility was as follows: TolC > OmpW or FadL > OmpC or OmpT. The deletion of LamB resulted in an increase in the level of bacterial growth. These results further indicated the importance of TolC and LamB in the response to CTC exposure. 3.5. Negative Regulation of LamB Was Validated Using a Dose-Dependent and Gene Complementation Assay. One result that attracted our attention was the negative regulation 5956
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of LamB that was in great contrast with the positive regulation of TolC in the CTC resistance. We were interested in validating the LamB strong negative regulation mechanism in comparison to a well-known positive regulation of TolC in resistance to antibiotics. Thus, a dose-dependent experiment was used to investigate the role of LamB using E. coli K12 BW25113 as a control. ∆lamB grew significantly faster than E. coli K12 BW25113 in a CTC dose-dependent manner (Figure 5A). Furthermore, we investigated effect of LamB on the survival capability in ∆lamB. As a result, +lamB (∆lamB-pACYC184lamB) grew just like negative control E. coli K12 BW25113 in a dose-dependent manner, whereas positive controls ∆lamBpACYC184 and ∆lamB exhibited significantly faster growth (Figure 5B). These results validated the mechanism of negative regulation of LamB in response to CTC.
4. Discussion In this study, the OM proteome in response to CTC was identified. Given that pathogenic bacteria may face exposure to a suddenly strong antibiotic treatment and persistent exposure to the drug,8 we designed this study to identify OM proteins that were commonly altered in E. coli in response to these two different exposure situations. OM proteins, TolC, LamB, FadL, OmpC, OmpT, and OmpW, were found to be the commonly altered OM proteins in the two exposure cases. They belong to different functional classes of porins (LamB and OmpC), and others (FadL, by long-chain fatty acids, OmpT, a serine protease; TolC, a transport protein; OmpW, thought to be a porin19,20) according to the SWISS-PROT database search results.21 TolC is a well-known antibiotic-resistant OM protein and a key component in the multidrug efflux pump system to efflux many drugs. Its role in CTC resistance has been reported.22 The other five proteins, LamB, FadL, OmpC, OmpT, and OmpW, were first identified here to be CTC-resistant proteins in E. coli, although they were reported to be involved in other antibiotic resistance.7-11 In addition, AceA was identified as a CTC-responsive and downregulated protein.8 Similar expression patterns have been found in chloramphenicolresistant bacteria.
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Figure 4. Testing for antimicrobial susceptibility. (A) MICs of CTC-R, CTC-R-O, and gene deletion mutants of these altered proteins by the broth microdilution method. (B) Dose-dependent experiment for the growth percentage of CTC-R and CTC-R-O. (C) Survival capability of CTC-R-O and these mutants in medium with a 1/16 MIC CTC of CTC-R-O. The asterisks denote the datum points that differed significantly (P < 0.05) between the wild-type strain and the mutant strains by the paired Student’s t test.
However, the direction of regulation of these altered OM proteins in response to CTC was significantly different from that responsible for other antibiotic resistance as described previously.7-11 Only TolC was upregulated in CTC resistance, whereas elevation of at least three altered OM proteins was detected in the other antibiotic resistance types.7-11 Interestingly, these six identical OM proteins and cytoplasmic protein AceA were also identified as altered proteins in the E. coli strains that encountered the same two exposures to chloramphenical in our previous report,8 but the direction of regulation of these shared OM proteins was not the same as that in CTCresistant bacteria. On the basis of the results from a comparison of these differential regulation patterns of the two antibiotic resistance types, it is clear that one key strategy for bacterial resistance to antibiotics included alteration of the direction of regulation of the altered OM proteins. All these altered OM proteins except TolC were downregulated in CTC-resistant bacteria, whereas upregulation of TolC, OmpC, and OmpW and downregulation of AceA, LamB, FadL, and OmpT (three downregulated and an elevated spot) were detected in chloramphenicol-resistant bacteria. These results indicate that the direction of OmpC and OmpW was inversely varied between the two antibiotic-resistant strains, and the change contributes to resistance to the two antibiotics. Furthermore, the difference in the direction of regulation also existed in the diversity of
OmpT and OmpW spots in CTC-S with respect to CTC-R. Thus, changes in the direction of regulation may be a simple and effective countermeasure against a variety of antibiotics and different exposures in bacteria. This finding provides novel insight into the mechanisms of antibiotic resistance. Our previous report has showed that an interaction network of these altered OM proteins, including TolC, LamB, FadL, OmpT, and OmpW, is involved in streptomycin resistance.7 Thus, bacteria may modify the network against other antibiotics by alteration of the direction of regulation of the OM proteins only. The results obtained from this study also further suggest the importance of OM proteomes in the regulation of antibiotic resistance. More importantly, distinctly negative regulation of LamB was found in this study. LamB is a trimeric λ receptor protein that forms permeability pores involved in the transport of maltose and maltodextrins. It is regulated by the mal regulon, which is repressed by a medium glucose concentration.23,24 Alteration of LamB was detected in streptomycin-, chloramphenicol-, and tetracycline-resistant E. coli.7,8,10,11 In the study presented here, 4-fold increased and decreased MICs were determined when lamB and tolC were absent, respectively, indicating that LamB played a role in CTC resistance almost equal in importance to that of TolC. It is also worth mentioning here that the absence of the other four downregulated OM proteins, FadL, OmpC, Journal of Proteome Research • Vol. 9, No. 11, 2010 5957
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Acknowledgment. This work was sponsored by grants from the National New Drug Research Foundation (2009ZX09103-640), NSFC projects (40876076), the Foundation of Guangdong for Natural Sciences (7117645), and the Special Fund of Central Universities for Basic Scientific Research Operating Expenses (Sun Yat-sen University, 10lgzd03) and SKLBC09B04. References
Figure 5. Effect of CTC concentration and gene complementation on survival of the lamB-deleted strain. (A) Dose-dependent experiment for the survival capability of the lamB-deleted strain, with E. coli K12 BW25113 used as a control. (B) Survival capability of the lamB complementation strain and control stains. The asterisks denote the datum points that differed significantly (P < 0.05) between the lamB-deleted strain and E. coli K12 BW25113 (A) and the lamB complementation stain and its three controls (B) by the paired Student’s t test.
OmpT, and OmpW, resulted in an elevated susceptibility to CTC, whereas deletion of lamB elevated the resistance to the drug. Thus, the level of LamB was inversely proportional to CTC resistance, which we call a negative regulation mechanism. Investigation of the negative regulation mechanism highlights the way to an improved understanding of bacterial countermeasures to antibiotic exposure through the alteration of the direction of regulation of OM proteins. In summary, we have identified a CTC-responsive OM proteome in this study. These altered proteins have been identified previously for their roles in antibiotic resistance, but the direction of the regulation of these proteins in response to CTC exposure is significantly different from previous reports of the resistance of bacteria to other antibiotics. In addition, TolC and LamB are also defined as the two key OM proteins in response to CTC and show two different regulation mechanisms. One is positive and the other negative. This novel insight is important for the understanding of antibiotic-resistant mechanisms. 5958
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Journal of Proteome Research • Vol. 9, No. 11, 2010 5959
