1 Institut für Mikrobiologie, Biochemie und Genetik, Universität Erlangen, Erlangen, Germany; 2 Wyeth Research, Cambridge, MA, USA
Received 10 December 2003; returned 17 December 2003; revised 19 December 2003; accepted 21 December 2003
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Abstract |
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Methods: Fe2+-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites.
Results: We identified three prominent Fe2+-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit.
Conclusions: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.
Keywords: glycylcyclines, mode of action, tetracycline resistance
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Introduction |
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The latest crystal structure data show up to six tetracycline binding sites on the 30S subunit of the ribosome,7,8 where site-1 is composed of a binding pocket formed by helix (h) 31 and h34 of 16S rRNA (helical numbering according to Mueller & Brimacombe9). Site-1 exhibits the highest degree of tetracycline occupancy.8 Various experiments provide additional biochemical evidence for tetracycline binding to site-1, e.g. enhanced dimethylsulphate (DMS) methylation at U1052 and C1054 in h3410 or inhibition of the RNARNA photocrosslink between h31 and h44 in the presence of tetracycline.11 The ribosomal protection proteins TetO and TetM may interfere with tetracycline binding to this high affinity site to confer resistance.12 In the presence of TetO, the enhanced DMS methylation induced by tetracycline at C1054 in h34 is abolished.13 If tigecycline shares this high-affinity binding site with tetracycline, the question arises of why TetM does not mediate tigecycline resistance.5 We compared rRNA interaction sites of tetracycline and tigecycline to analyse whether this discrepancy is the result of a different mechanism of action by tigecycline or a different binding mode. A detailed comparison of tetracycline and tigecycline binding sites on 16S rRNA required a new probing method, since only DMS modification in h34 correlates with biological activity of tetracycline derivatives.13,14 Other RNA probing methods like kethoxal or europium cleavage yielded no tetracycline-specific signals.10,15 Tetracycline-mediated Fe2+ cleavage has been used to identify the tetracycline binding site on Tet repressor and on the tetracycline efflux protein TetA.16,17 Since Fe2+ cleavage has also been employed to detect metal ion binding sites in group I intron RNA,18 we adapted this technique for the identification of tetracycline binding sites on rRNA in 70S ribosomes of Escherichia coli. We identified three cleavage sites induced by the presence of tetracycline or tigecycline. These sites are consistent with data obtained from DMS and mutational analysis of 16S rRNA.
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Materials and methods |
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E. coli DH519 was routinely used as plasmid host. E. coli TA527 [
rrnE F ara
lac thi
(rrsB-gltT-rrlB)101
(rrsH-ileV-alaV-rrlH) 103
(rrsG-gltW-rrlG)30::lacZ+
(rrsA-ileT-alaT-rrlA)34
(rrsD-ileU-alaU-rrlD) 25::cat+
(rrsC-gltU-rrlC)15::cat+ ilv+]20 was used to measure the MIC of tetracycline and tigecycline of mutated 16S rRNA in the absence of wild-type background. For viability, E. coli TA527 contains the pSC101 derivative pHK-rrnC+ bearing the entire rrnC operon from E. coli. pKK3535, a pBR322 derivative that carries the 7.5 kb BamHI fragment from
rifd, containing the entire rrnB operon from E. coli,21 was taken as a wild-type rRNA control. pKK1058C is a pKK3535 derivative bearing a point mutation of G
C at base 1058 of the 16S rRNA.22 The plasmid pKK966U was constructed by site-directed two-step PCR mutagenesis, whereby only part of the 16S rRNA was amplified using the primers 2220f_BglII (5'-AAG AAG ATC TGG AGG AAT ACC G-3'), 2916r_BsrGI (5'-GGT GTG TAC AAG GCC CGG G-3') and 2489r-966U (5'-CGC GTT GAA TCG AAT TAA ACC-3'), and plasmid pKK3535 as template. The PCR fragment obtained was digested with BglII and BsrGI, and cloned into the likewise digested pKK3535. Finally, the entire 16S rRNA was sequenced to ensure that only the desired and no additional mutations were present.
MIC determination
The activities of the antibiotics were determined by the agar dilution method following the recommendations of the NCCLS.23 The inocula of E. coli TA527 cells, which were adjusted to 107 cfu/mL, were applied to the surfaces of MuellerHinton agar plates to give 104 cfu/spot. This was confirmed by plating the corresponding number of cells on MuellerHinton agar plates containing no antibiotics. The plates were incubated at 36°C for 20 h in ambient air. MIC tests were performed three times.
Preparation of 70S ribosomes
Cultures from E. coli MRE600 were grown at 37°C to an OD600 of 0.5 in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl), harvested and resuspended in ribosomal buffer 1 (20 mM TrisHCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 10 mM ß-mercaptoethanol). The isolation of 70S ribosomes via ultracentrifugation was performed as described previously.24
DMS modifications
Chemical modification of the 70S ribosome with DMS was carried out as described previously.25 For the DMS modification assays, 10 pmol of ribosomes in ribosomal buffer 2 [20 mM TrisHCl, pH 7.5, 100 mM NH4Cl, 6 mM MgCl2, 3 mM dithiothreitol (DTT)] were incubated at 37°C for 30 min, followed by incubation at room temperature for 10 min in the absence or presence of antibiotics (final volume 49 µL). The DMS reaction was started by addition of 1 µL DMS (1:50 dilution in 96% ethanol) and incubation at room temperature for 5 min. The reaction was stopped with 1 µL of ß-mercaptoethanol (1:5 dilution in water).
Fe2+-mediated hydroxyl radical cleavage reactions
Fe2+-mediated hydroxyl radical cleavage of the 70S ribosome was carried out as described previously.18 Four microlitres of ribosomes (5 pmol) and 1 µL of a 10x antibiotic stock solution in TAKA7 (50 mM TrisHCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2)26 were incubated with 2 µL of 5x native cleavage buffer (25 mM MOPSKOH, pH 7.0, 3 mM MgCl2, 400 µM spermidine) and incubated for 30 min at 37°C, followed by 10 min incubation at room temperature. One microlitre of 1.25 mM FeCl2 was added to the reaction tube, mixed by centrifugation and incubated for 1 min before adding 1 µL of 6.25 mM sodium ascorbate. After 1 min, 1 µL of 6.25 mM H2O2 was added to initiate the reaction, and rapidly mixed. The final concentrations were 125 µM for Fe2+ and 625 µM for both sodium ascorbate and H2O2. The cleavage reaction was stopped after 1 min by the addition of thiourea to a final concentration of 100 mM.
Mg2+ competition of Fe2+ cleavage
In the Mg2+ competition experiments, MgCl2 was added as a 10x stock solution of the final Mg2+ concentration to the 1.25 mM FeCl2 solution. The Fe2+/Mg2+ mixture was then pipetted into the reaction tube and the cleavage reaction continued as above.
Extraction of rRNA
The RNA was extracted by treatment of the ribosomes with 200 µL of ribosomal extraction buffer (0.3 M sodium acetate, 0.5% SDS, 5 mM EDTA) at room temperature followed by phenolization (3x) and isoamylalcohol/chloroform (1:24) treatment. Thereafter, the RNA was ethanol precipitated, resuspended and residual phenol was eliminated by diethylether treatment. After a final ethanol precipitation, the RNA was resuspended in 10 µL of Millipore water.
Primer extension and gel electrophoresis
RNA (2.5 µL) was mixed with 1 µL of 4.5x hybridization buffer (225 mM HEPESKOH, pH 7.0, 450 mM KCl) and 1 µL of 5' 32P-labelled primer (0.25 pmol; 100 000 cpm). The hybridization was carried out by heating the mixture for 1 min at 96°C followed by 15 min incubation at room temperature. The extension reaction was started by the addition of 5x first-strand buffer (50 mM TrisHCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 mM DTT, 167 µM dNTP mix, 3 U RNAguard RNase inhibitor and 10 U SuperscriptII RnaseH Reverse Transcriptase (Invitrogen), and carried out in a final volume of 15 µL at 42°C for 50 min. To stop the reaction, the RNA was degraded by addition of 2 µL of 1 M NaOH, followed by a 15 min incubation at 42°C. After neutralization by adding 2 µL of 1 M HCl, the DNA was precipitated with ethanol. The DNA was resuspended in 10 µL of loading buffer (0.3% each of Bromophenol Blue and xylene cyanol, 10 mM EDTA, pH 7.5, and 97.5% deionized formamide) and electrophoresed in denaturating 815% polyacrylamide (PAA) sequencing gels. Modifications were identified and quantified by phosphoimager analysis (FUJIFILM Bas-1500; TINA200, Raytest, Straubenhardt, Germany). The positions of the modifications were mapped by dideoxynucleotide sequencing using pKK3535 or pKK1058C as a template.
Superposition figure
Superposition of tetracycline with DMG-DMDOT was carried out with VMD 1.8.1 structure viewer.27 VMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign (http://www.ks.uiuc.edu/Research/vmd/). Coordinates were taken from the RCSB protein data bank files 1I97 and 1ORK.
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Results |
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DMS modification experiments were carried out in the presence of tetracycline and tigecycline to test whether they show the same binding sites. 70S ribosomes from E. coli MRE600 were treated with DMS in the presence of tetracycline or tigecycline at concentrations ranging from 0.1 to 300 µM. The ribosomal RNA was isolated and used as a template for primer extension analysis. Protection from DMS methylation at A892 in h27 (see Figure 1) was only detected for tetracycline, but not for tigecycline (Figure 2a). Quantification showed that the intensity of the signal corresponding to A892 steadily decreased at tetracycline concentrations from 10 up to 300 µM, yielding a two-fold reduction compared with the control, while tigecycline did not affect signal intensity (Figure 2b).
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Tetracycline-directed Fe2+-mediated hydroxyl radical cleavage patterns
Fe2+ can replace Mg2+ chelated to tetracyclines forming a tetracyclineFe2+ complex, which is an inducer of TetR.28 We thus assume that tetracyclineFe2+ is isostructural to tetracyclineMg2+. We used the Fe2+-chelated complexes of tetracycline or tigecycline to map their binding sites on 16S rRNA by Fe2+ cleavage. After addition of H2O2, the bases and the RNA backbone are cleaved in the close proximity of bound tetracycline derivatives. We probed 70S ribosomes isolated from E. coli MRE600 with 125 µM Fe2+, 625 µM sodium ascorbate and 625 µM H2O2 in the presence of 1100 µM tetracycline or tigecycline. The rRNA was isolated and used as template for primer extension analysis. The primers chosen were spaced approximately every 250 nucleotides on the 16S rRNA. Three prominent regions of cleavage, seen as strong stops of reverse transcription, were observed. Sequence reactions run in parallel revealed one cleavage site in the internal loop of h31, one in h34 and one in h29 (compare Figure 1; helical numbering according to Mueller & Brimacombe9). Figure 3 shows sections of 10% denaturating polyacrylamide gels with cleavage sites.
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Cleavage products in h34 were mapped to nucleotides C1195A1197 (Figure 3c). Cleavage product quantification yielded an increase in signal intensity with 130 µM tetracycline. Saturation was reached with 30 µM tetracycline. No concentration-dependent change in signal intensity was detected for tigecycline. In addition, the overall effect for tigecycline was smaller than that for tetracycline (Figure 3d).
Cleavage products in h29 were mapped to region A1339U1341 (Figure 3e). Quantification showed that the signal strength increases from 1 to 100 µM tetracycline and from 1 to 10 µM tigecycline (Figure 3f). The signal intensity for 10 or 100 µM tetracycline resembled that for 1 or 10 µM tigecycline, respectively.
Mg2+ competition of Fe2+ cleavage
To show that Fe2+ and Mg2+ interact with the same sites, we performed a Mg2+ competition experiment. The cleavage reaction was carried out in the presence of increasing amounts of MgCl2 (1.520 mM) leading to Fe2+:Mg2+ ratios ranging from 1:12 to 1:160, since Fe2+ shows a 100-fold higher affinity to tetracycline than Mg2+.28 The results of a representative Mg2+ competition are shown for the internal loop of h31 (A964A969) in Figure 4a. Quantification shows that the extent of cleavage by Fe2+ was reduced for tetracycline- and tigecycline-directed reactions, reaching intensities close to the background at 20 mM Mg2+ (Figure 4b).
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As well as comparing cleavage patterns, we also examined the influence of mutations in 16S rRNA for both derivatives. E. coli TA527 carries deletions spanning the 16S and 23S coding regions in all seven chromosomal rRNA operons. A single E. coli rRNA operon (rrnC) carried on the plasmid pHK-rrnC+ (kanamycin resistant) supplies 16S and 23S rRNA to the cell.20 This strain allowed the analysis of plasmid-encoded 16S rRNA mutations on tetracycline susceptibility without wild-type background. Plasmid pKK3535, a pBR322 derivative (ampicillin resistant) that carries the ribosomal E. coli operon rrnB,21 was used as a wild-type control. pKK1058C is a derivative of pKK3535 containing the 16S rRNA mutation G1058C.22 Plasmid pKK966U was constructed in this study by site-directed mutagenesis of pKK3535 to introduce a G
U transversion at base 966 in h31. E. coli TA527 pHK-rrnC+ was transformed with pKK3535, pKK1058C or pKK966U, and plated on selective medium containing ampicillin to replace pHK-rrnC+. The loss of pHK-rrnC+ was confirmed by counter-selection for kanamycin susceptibility. We determined MICs for the strains TA527 pKK3535, TA527 pKK1058C and TA527 pKK966U of tetracycline and tigecycline (see the Materials and methods section for details). The results are summarized in Table 1. E. coli TA527 containing pKK1058C was eight-fold more resistant to tetracycline and to tigecycline compared with the same strain carrying pKK3535. The mutation encoded by pKK966U confers a fourfold increase in MIC of tetracycline and tigecycline as compared with TA527 pKK3535.
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Discussion |
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The third Fe2+ cleavage region is located in h29, close to tetracycline binding site-4. Oehler et al.30 observed tetracycline photocrosslinking to this region. 30S subunits containing crosslinked tetracycline showed a decreased translational activity when reconstituted with 50S subunits. Evidence for the physiological relevance of this site was also provided by the observation that a 942 mutation in h29 led to an increased MIC of tetracycline in H. pylori.29 We establish here that this site is also important for tigecycline binding.
Other tetracycline binding sites identified by crystallography have not been related to antibiotic activity. Since the three regions undergoing tetracycline- and tigecycline-dependent Fe2+ cleavage cover physiologically relevant sites, and since they are modified identically by both drugs, we conclude that tetracycline and tigecycline share the same binding sites on 16S rRNA. Tigecycline seems to bind more effectively to these binding sites, since maximal signal intensities for Fe2+ cleavage and DMS methylation are reached at lower concentrations compared with tetracycline. This is consistent with data from Bergeron et al.,6 who detected a five times higher binding affinity of the glycylcyclines DMG-DMDOT and DMG-DOX to the ribosome compared with tetracycline.
The qualitative differences in Fe2+ cleavage intensities indicate that tigecycline might bind these sites in another orientation, which is probably due to the large substituent at position 9. Such different orientations of tetracycline and another glycylcycline derivative, DMG-DMDOT, in complex with TetR has already been reported.31 We superimposed the co-crystal structures of DMG-DMDOT as seen in the TetR complex31 and tetracycline in the ribosomal tetracycline binding site-1,8 as shown in Figure 5. The bulky substituent at position 9 of DMG-DMDOT clashes with the phosphate backbone of G1053. It is therefore impossible for this molecule to bind site-1 identically to tetracycline. This difference must be even more pronounced for tigecycline, since it carries a t-butylglycylamido group, which is bulkier than the dimethylglycylamido substituent present in DMG-DMDOT. Since we demonstrated that tigecycline does bind to site-1, it must either be in a different orientation or the rRNA conformation must be altered to accommodate the bulkier drug.
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Acknowledgements |
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Footnotes |
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References |
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