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Previous Article | Next Article 
Journal of Bacteriology, December 2001, p. 6898-6907, Vol. 183, No. 23
Center for Pharmaceutical Biotechnology,
University of Illinois, Chicago, Illinois
60607,1 and Infectious Disease Research,
Abbott Laboratories, Abbott Park, Illinois
600642
Received 31 July 2001/Accepted 19 September 2001
Macrolides represent a clinically important class of antibiotics
that block protein synthesis by interacting with the large ribosomal
subunit. The macrolide binding site is composed primarily of rRNA.
However, the mode of interaction of macrolides with rRNA and the exact
location of the drug binding site have yet to be described. A new class
of macrolide antibiotics, known as ketolides, show improved activity
against organisms that have developed resistance to previously used
macrolides. The biochemical reasons for increased potency of
ketolides remain unknown. Here we describe the first mutation that
confers resistance to ketolide antibiotics while leaving cells
sensitive to other types of macrolides. A transition of U to C at
position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for
ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA
was further confirmed by RNA footprinting. The newly isolated
ketolide-resistance mutation, as well as 23S rRNA positions shown
previously to be involved in interaction with macrolide antibiotics,
have been modeled in the crystallographic structure of the large
ribosomal subunit. The location of the macrolide binding site in the
nascent peptide exit tunnel at some distance from the peptidyl
transferase center agrees with the proposed model of macrolide
inhibitory action and explains the dominant nature of macrolide
resistance mutations. Spatial separation of the rRNA residues involved
in universal contacts with macrolides from those believed to
participate in structure-specific interactions with ketolides provides
the structural basis for the improved activity of the broader spectrum
group of macrolide antibiotics.
Macrolide antibiotics inhibit
protein synthesis in a wide range of pathogenic bacteria. The drugs
bind to a single site in the large ribosomal subunit located near the
entrance to the nascent peptide tunnel and are thought to sterically
hinder the growth of the polypeptide chain and cause dissociation of
peptidyl-tRNA from the ribosome (1, 10, 18, 33). Macrolide
antibiotics were also shown to affect ribosome assembly
(9).
The macrolide binding site is composed primarily of RNA. Two segments
of 23S rRNA, the central loop of domain V and the loop of hairpin 35 from domain II, are believed to be the major components of the drug
binding site on the ribosome. Mutations of A2058 (Escherichia coli numeration) and several neighboring positions in the central loop of domain V confer resistance to macrolides (27), as
reviewed in references 34 and 35. Methylation
of A2058 by Erm-type methyl-transferases drastically reduces the
affinity of macrolides for the ribosome, suggesting the direct
interaction between this RNA residue and the drug (11, 17,
28). The interaction of macrolides with this region of rRNA is
further supported by RNA footprinting: all of the macrolide antibiotics
investigated strongly protect A2058 and A2059 from chemical
modification (5, 16, 20, 37; L. Xiong and A. Mankin,
unpublished results). The accessibility of several other positions
within the central loop of domain V is also affected by macrolide
binding (11, 25). Genetic and biochemical evidence also
revealed interaction of macrolides with the helix 35 in domain II of
23S rRNA (16, 37). Though rRNA appears to be the major
component of the macrolide binding site, mutations in two ribosomal
proteins, L4 and L22 (1, 8, 23, 31, 36), were shown to
confer resistance. It remains unclear, however, whether these proteins
directly participate in the drug binding or the mutations
allosterically affect the conformation of rRNA in the drug binding site
(12, 13).
Erythromycin A, which was introduced in clinical practice in 1953, represented the first generation of the 14-member-ring macrolides (Fig.
1). The later developed drugs, such as
clarithromycin, roxithromycin, and the 15-member-ring azithromycin,
were characterized by better stability and a broader spectrum of
action. The subsequent spread of resistant strains prompted a
search for newer macrolide derivatives. The new class group of
macrolides
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6898-6907.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Binding Site of Macrolide Antibiotics on the
Ribosome: New Resistance Mutation Identifies a Specific Interaction of
Ketolides with rRNA
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
ketolides, which contain a keto function instead of the
3-cladinose residue and carry an 11,12 carbamate and an additional
alkyl-aryl side chain (Fig. 1) exhibit an improved activity profile
and, importantly, show a significant activity against some
macrolide-resistant isolates (21, 26). (Note that, in this
paper, we will refer to cladinose-containing macrolides as
cladinosolides, while drugs with the keto function at position 3 of the
lacton ring will be referred to as ketolides.) The enhanced
potency of ketolides correlates with their higher affinity for the
ribosome, which depends, at least in part, on a stronger interaction
with the helix 35 loop (11, 16, 37). Ketolides may
interact idiosyncratically with the other rRNA segments as well: some
rRNA mutations that render cells resistant to cladinosolides produce
little or no effect on cell sensitivity to ketolides (11).
View larger version (16K):
[in a new window]
FIG. 1.
Chemical structures of macrolide antibiotics used in
this study. Ketolides, containing the 3-keto group, are represented by
ABT-773, telithromycin, and A-192803. Erythromycin and RU69874 are
examples of cladinosolides that contain a 3-cladinose residue.
In this paper, we characterize the first ketolide-specific resistance mutation in rRNA. The mutation at position 2609 of rRNA confers resistance to ketolides, but not to cladinose-containing macrolides. The location of this and other RNA residues involved in interaction with macrolides in the ribosome tertiary structure positions the macrolide binding site in the nascent peptide exit tunnel. Structural information provides the rationale for the improved activity of ketolide antibiotics and illuminates a possible mode of macrolide action.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents, strains, and plasmids. (i) Antibiotics. ABT-773 and A-192803 were from Abbott Laboratories, telithromycin, and RU 69874 from Aventis Pharma. Erythromycin was purchased from Sigma.
(ii) Chemicals and enzymes. Dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT), used in RNA probing experiments, were from Aldrich. All the other chemicals were purchased from either Fisher or Sigma. Restriction enzymes were from MBI Fermentas.
(iii) Plasmids.
Plasmid pKK3535 (Table
1) contains a complete wild-type
rrnB operon of E. coli cloned in the
BamHI site of pBR322 vector (7). A closely
related pKK3535 derivative, pNK, lacking a 903-bp NaeI-BamHI fragment from the vector portion,
provided a more favorable restriction site configuration for the
fragment exchange (38). pKK754A is identical to pKK3535,
except for the presence of a T754A mutation in the 23S rRNA gene
(37). pSTL102 contains the rrnB operon with a
C1192T spectinomycin resistance mutation in the 16S rRNA gene and an
A2058G mutation in the 23S rRNA gene (32).
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(iv) E. coli strains.
Strain JM109
(39) [endA1 recA1 gyrA96
thi
hsdR17(rK
mK+) relA1
supE44 
(lac-proAB) (F'
traD36, proAB
lacIqZM15)] was from
Promega. Strain TA531 (EBHGzADcCcrecA/pTRNA66, pHK-rrnC+) (3) was a gift of C. Squires (Tufts University).
Selection of ketolide resistance mutations. The E. coli strain TA531 was used for selection of the resistant mutants (3). In this strain, all seven chromosomal rRNA operons are deleted and the only source of rRNA is a pHK-rrnC+ plasmid carrying the rrnC operon as well as a kanamycin resistance marker. The plasmid pKK3535 was randomly mutagenized by passing through the E. coli mutator strain XL1-Red as described previously (38). TA531 cells were transformed with the mutant pKK3535 library and grown overnight in liquid culture in the presence of ampicillin to promote loss of the pHK-rrnC+ plasmid. A total of 106 Ampr cells were plated onto agar plates containing 100 µg of ampicillin per ml and 10 µg of ABT-773 per ml. About 50 ketolide-resistant (Ketr) clones appeared after a 24-h incubation. They were replica plated onto Luria-Bertani agar containing ampicillin (100 µg/ml) or kanamycin (30 µg/ml). Two clones that exhibited an Ampr Ketr Kans phenotype, containing only mutant pKK3535 as a rRNA source, were chosen for further analyses.
Plasmids isolated from these two clones were used to re-transform fresh TA531 cells and the plasmid-exchange procedure was repeated. The Ketr phenotype was cotransferable with the mutant pKK3535 plasmids, indicating that the resistance determinant was plasmid borne.Mapping the site of the Ketr mutation. DNA sequencing revealed the presence of the U2609C mutation in the 23S rRNA gene in plasmids isolated from both analyzed Ketr clones. Fragment exchange was used to test whether the presence of this only mutation was sufficient for the ketolide resistance. A Bpu1102I-PaeI 2.2-kbp restriction fragment from mutant pKK3535 plasmids containing the U2609C mutation was used to replace the corresponding fragment in the wild-type rrnB operon on plasmid pNK (38). The resulting plasmid, pNK2609C, conferred the same level of ketolide resistance as the originally selected mutant pKK3535. DNA sequencing confirmed the presence of only the U2609C mutation in the exchanged Bpu1102I-PaeI 2.2-kbp fragment, demonstrating that the presence of this mutation was sufficient to confer ketolide resistance.
Determining antibiotic MICs.
To determine the MICs of
various macrolide and ketolide antibiotics, cultures of
Ampr Kans TA531 cells
carrying one of the following plasmidswild-type or mutant pKK3535,
pNK, or pSTL102were grown overnight in the presence of 100 µg of
ampicillin per ml, diluted 1,000-fold into a fresh
ampicillin-containing medium supplemented with various drug
concentrations, and grown overnight with constant shaking. The optical
densities of the cultures were measured at 650 nm and plotted.
RNA footprinting. E. coli ribosomes (200 nM) were incubated for 10 min at 37°C, followed by 10 min at 20°C in 50 µl of the modification buffer (30) containing 5 µM macrolide antibiotics. DMS or CMCT modification was carried out at 37°C essentially as described previously (19), except that the CMCT concentration was increased twofold. Primer extension was performed according to the method of Stern et al. (30).
Antibiotic binding.
One
A260 unit (23 pmol) of ribosomes was
incubated with [14C]ABT-773 (27.2 mCi/mmol)
(prepared by Abbott Laboratories) or
[14C]erythromycin (55.1 mCi/mmol) (NEN Life
Science) at concentrations ranging from 2 nM up to 200 nM. The
reactions were carried out at room temperature for 4.5 h in
buffers containing 10 mM Tris-HCl (pH 7.2), 10 mM
NH4Cl, 4 mM MgCl2, and 100 mM KCl in a final volume of 0.5 ml in sterile microcentrifuge tubes. At
the end of the reactions, ribosomes were pelleted by centrifugation at
100,000 × g for 2 h in a Beckman TL-100
centrifuge (rotor TL-45). The amount of radioactive drug associated
with the ribosomes was determined by scintillation counting. Data were
analyzed by using KaleidaGraph software (Synergy Software).
Kd values were determined with the formula: Kd = D1/2 
1/2Rtotal, where
D1/2 = concentration of the drugs at
which 50% ribosomes bind antibiotic and
Rtotal = total concentration of active ribosomes.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Selection of the ketolide-resistant mutant.
To identify rRNA
mutations that confer resistance to ketolides, the plasmid pKK3535
containing a complete copy of E. coli rrnB operon and an
ampicillin resistance marker was mutagenized by passing through a
mutator E. coli strain. For mutant selection, we used an
E. coli strain, TA531, with seven chromosomal rRNA operons
deleted. rRNA in TA531 cells is transcribed from the wild-type rrn operon contained on a plasmid
pHK-rrnC+ carrying a kanamycin resistance marker
(2, 3). Growth of TA531 cells transformed with the
wild-type pKK3535 was significantly inhibited at 5 µg of ketolide
antibiotic ABT-773 per ml and completely abolished on agar plates
containing 10 µg of ABT-773 per ml. The mutagenized pKK3535 plasmid
library was introduced into the TA531cells and
Ketr clones were selected on agar plates
containing 10 µg of ABT-773 per ml. Replica plating allowed
identification of two clones that exhibited the
Kans phenotype, indicating that they had lost the
pHK-rrnC+ plasmid. rRNA in these clones was
expressed exclusively from the mutant pKK3535, and the ribosomes were
expected to contain only mutant rRNA. The ABT-773 MIC for both clones
was 75 µg/ml, 15 times higher than the ABT-773 MIC for the
Ampr Kans TA531 cells
transformed with the wild-type pKK3535 (Table
2). The ketolide resistance was
cotransferable with the mutant pKK3535 plasmids from these clones,
demonstrating the presence of the ketolide resistance marker on the
plasmid-borne genes.
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hence proving that a single U2609C
mutation in the 23S rRNA gene can confer resistance to ABT-773.
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Peculiar spectrum of resistance of the U2609C mutant. The spectrum of macrolide resistance conferred by the newly selected U2609C mutation was compared with that of the previously characterized mutation, A2058G (29). TA531 cells were transformed with pNK2609C or with pSTL102 carrying the A2058G mutations. Both of these plasmids contained an ampicillin resistance marker. The results of antibiotic sensitivity testing of the Ampr Kans transformants expressing exclusively mutant 23S rRNA are shown in Table 2. The A2058G mutation rendered cells resistant to all of the macrolides tested. In contrast, the U2609C mutation conferred considerable resistance to two ketolides, telithromycin and ABT-773, but made cells slightly more sensitive to the cladinosolides, erythromycin and azithromycin. Comparison of the effect of the U2609C mutation on cell sensitivity to telithromycin or its cladinose derivative, HMR69874, showed no resistance to the latter compound. Therefore, the U2609C mutation represents the first example of a nucleotide change in 23S rRNA that confers resistance specifically to ketolides.
Footprinting of ketolides and cladinosolides on the ribosome reveal
structure-specific interactions.
Ketolide resistance of the U2609C
mutant suggested that U2609, as well as the neighboring rRNA sites,
might be involved in direct interaction with the drug. To explore such
possible interaction more directly, we used RNA footprinting
(19). All of the macrolides tested strongly protected
A2058 and A2059 in domain V from DMS modification (Fig.
3A), confirming the previous conclusion
that these nucleotides may directly participate in macrolide binding (16, 20, 37). The ketolides ABT-773 and telithromycin
enhance somewhat the accessibility of A2062 to chemical modification; similar effects were reported previously for telithromycin
(11). Cladinosolides only marginally affected modification
of A2062 (a minor protection of this position by erythromycin seen in
Fig. 3A could not be reproduced in other experiments). It has been noted, however, that mutations of A2062 do not affect cell sensitivity to 14-member-ring macrolides (both cladinosolides and ketolides). Therefore, changes in the accessibility of A2062 to DMS modification in
the presence of ketolides are probably the result of allosteric effects
rather than an indication of a direct drug-RNA interaction.
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The U2609C mutation reduces the affinity of ketolides for the
ribosome.
rRNA mutations can confer resistance by reducing drug
binding to the ribosome or by interfering with the drug action without perturbing its affinity (24). In order to verify the mode
of resistance rendered by the U2609C mutation, binding studies were performed with radioactive erythromycin or ABT-773 and either wild-type
or mutant (U2609C) ribosomes. Antibiotic binding was performed under
equilibrium conditions, and the ribosome-drug complex was separated
from the unbound antibiotic by high-speed centrifugation. The data
showed that the U2609C mutation reduced binding of the ketolide,
whereas the affinity of erythromycin, a cladinosolide, was slightly
increased (Table 3), thus indicating that
the U2609C mutation renders cells ketolide resistant by interfering with antibiotic binding. The results of the binding experiments were in
excellent agreement with the resistance data (Table 2), in which the
U2609C mutation rendered cells resistant to ABT-773 but slightly
increased sensitivity to erythromycin.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dominant nature of macrolide resistance mutations. In this paper, we described the selection of a ketolide-resistant mutant of E. coli. The presence of a single mutation, U2609C, in 23S rRNA was shown to be sufficient to confer resistance to ketolide antibiotics, but not to cladinose-containing macrolides.
During mutant selection, the resistance phenotype conferred by the U2609C mutation was initially manifested in a mixed-ribosome environment, in which a portion of the ribosome population contained mutant rRNA encoded in mutagenized pKK3535 plasmid, while the rest of the ribosomes contained wild-type rRNA transcribed from the pHK-rrnC+ plasmid. Hence, the U2609C-mediated resistance must be dominant or codominant. This conclusion is further supported by the fact that the pKK2609C plasmid rendered JM109 cells ketolide resistant in spite of the presence of the chromosome-encoded wild-type ribosomes (data not shown). The previously studied macrolide resistance mutations in rRNA were also shown to be dominant (see reference 34 for references). The dominant nature of these mutations is in agreement with the proposed mode of macrolide action according to which the drugs block nascent peptide growth and cause dissociation of peptidyl-tRNA (18). The peptidyl-tRNA loss should facilitate disengagement of sensitive ribosomes from mRNA, allowing resistant ribosomes to continue translation. The dominant nature of the U2609C mutation, as well as other rRNA mutations conferring resistance to macrolides, has an unfortunate consequence: the appearance of the mutation in just one out of several chromosomal rRNA operons may already provide a selective advantage for the mutant during antibiotic treatment. This may result in the subsequent spread of the mutation between other rRNA loci, rendering the pathogen resistant to even higher concentrations of the drug (31).Macrolide binding site and specificity of ketolide interaction with the ribosome. Several mutations conferring resistance to cladinose-containing macrolides have been found previously in the central loop of domain V of 23S rRNA (34, 35). The U2609C mutation is unique because it is the first mutation that confers resistance exclusively to ketolides without increasing cell resistance to cladinosolides. It is likely that the interaction of the nucleotide residue 2609 with the ketolide molecule differs from its interaction with the cladinose-containing macrolides. Such interaction can contribute to a tighter binding of ketolides to the ribosome and should be important for their activity.
The recently solved crystal structure of the 50S ribosomal subunit of the archaeon Halobacterium marismortui (4) provides a useful framework for modeling antibiotic binding sites in the ribosome (6). It should be kept in mind, however, that archaeal ribosomes differ from their bacterial counterparts both in protein composition and in rRNA structure. In particular, a number of nucleotide residues that are involved in macrolide binding are different in Bacteria and Archaea (15). Nevertheless, even though the nature and precise orientation of the nucleotide residues in Archaea and Bacteria can be idiosyncratic, their general ribosomal localization should be the same, allowing the use of the available crystallographic data for characterization of the macrolide binding site on the ribosome. We used coordinates of nucleotides, according to genetic or biochemical criteria involved in macrolide binding, to map macrolide binding site in the ribosome. Residues 2057 to 2059, 2609, and 2611 in domain V and the residue at position 752 in domain II were included in our reconstructions (11, 16, 20, 25, 34, 37). All of the nucleotides implicated in interactions with macrolides form a relatively tight cluster located at approximately one-third the distance from the interface side of the 50S subunit (Fig. 4 A to C). The drug binding site is located near the bottom of a cavity containing the peptidyl transferase center, at the entrance into the nascent peptide exit tunnel. The easiest way for the drug molecule to access this site in the isolated 50S subunit is from the interface side. However, given a relatively wide size of the exit tunnel and its hypothetical "nonstick properties" (22), it is also possible for the drug to diffuse to its binding site through the tunnel. This may be a preferred or even the only way for the drug to reach its site of action in 70S ribosome during early rounds of translation when the peptidyl transferase cavity is occupied by peptidyl- and aminoacyl-tRNAs.
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ACKNOWLEDGMENTS |
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We thank S. Douthwaite, F. Francheschi, J. Sutcliffe, and P. Mauvais for stimulating discussion and advice; N. Polacek for advice regarding the manuscript, H. F. Noller, C. Squires, and S. Douthwaite for making their plasmid constructs available to us, and M. Gomez for help in preparing the manuscript. We also thank Aventis Pharma for providing telithromycin and RU 69847.
This work was supported by research grants from Abbott Laboratories and the National Institutes of Health (GM53762).
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FOOTNOTES |
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*
Corresponding author. Mailing address: Center for
Pharmaceutical BiotechnologyM/C 870, University of Illinois, 900 S. Ashland Ave., Rm. 3056, Chicago, IL 60607. Phone: (312) 413-1406. Fax: (312) 413-9303. E-mail: shura{at}uic.edu.
Present address: Departamento de Bioquimica, Facultad de Medicina,
Universidad Nacional, Autonoma de Mexico, Mexico City, D.F. 04510, Mexico
Present address: Givaudan Flavors Corporation, Cincinnati, OH 45216.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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