From the Center for Pharmaceutical Biotechnology, University of
Illinois, Chicago, Illinois 60607
Clones expressing pentapeptides conferring
resistance to a ketolide antibiotic, HMR3004, were selected from a
random pentapeptide mini-gene library. The pentapeptide MRFFV conferred
the highest level of resistance and was encoded in three different
mini-genes. Comparison of amino acid sequences of peptides conferring
resistance to a ketolide with those conferring resistance to
erythromycin reveals a correspondence between the peptide sequence and
the chemical structure of macrolide antibiotic, indicating possible interaction between the peptide and the drug on the ribosome. Based on
these observations, a "bottle brush" model of action of macrolide
resistance peptides is proposed, in which newly translated peptide
interacts with the macrolide molecule on the ribosome and actively
displaces it from its binding site. Temporal "cleaning" of the
ribosome from the bound antibiotic may be sufficient to allow
continuation of protein synthesis even despite the presence of the drug
in the medium.
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INTRODUCTION |
Erythromycin and other macrolides are important antibacterial
antibiotics. The primary mode of action of macrolides is inhibition of
protein synthesis, although they can also interfere with ribosome assembly (1-3). The best studied macrolide, erythromycin, binds to the
large ribosomal subunit in the vicinity of the peptidyl transferase
center, where it forms tight contacts with rRNA (4-6) and maybe
ribosomal proteins (7, 8). Although the molecular mechanisms of action
of erythromycin remain obscure, it is clear that the antibiotic blocks
the elongation step of translation during early rounds of protein
synthesis. The drug has a high affinity for ribosomes with nascent
peptides shorter than 2-5 amino acids, but does not bind to ribosomes
that carry long nascent peptide chains (9). It was suggested that
erythromycin sterically hinders growth of the nascent peptide chain (2)
and may promote dissociation of peptidyl-tRNA (10, 11).
The clinical use of macrolides is significantly hampered by the growing
number of resistant strains. Different mechanisms of resistance have
been described (reviewed in Ref. 6). We have recently described a novel
mechanism of erythromycin resistance, which is based on interaction of
specific short peptides with the ribosome. The discovery of this
resistance mechanism came from an observation that overexpression of a
short segment of Escherichia coli 23 S rRNA (positions
1235-1268) rendered cells resistant to erythromycin (12, 13).
Mutational and biochemical analyses demonstrated that resistance is
caused by translation of a pentapeptide open reading frame
(ORF)1 encoded in E. coli 23 S rRNA and is mediated by interaction of the newly
translated pentapeptide with the ribosome. The rRNA-encoded pentapeptide is not normally expressed because the Shine-Dalgarno region of the peptide ORF is sequestered in the 23 S rRNA secondary structure. However, the peptide expression can be activated by site-specific fragmentation of rRNA or by rRNA mutations that increase
accessibility of the Shine-Dalgarno region of E-peptide ORF (14). In
order to get insights into peptide structural features that are
essential for erythromycin resistance, other erythromycin resistance
peptides were selected from in vivo expressed random peptide
libraries (15). It was found that only short peptides, ranging in size
from 3 to 6 amino acids, with specific amino acid sequence can confer
erythromycin resistance. Analysis of more than 70 pentapeptides that
can confer resistance to erythromycin (E-peptides) revealed a consensus
sequence, MXLXV, which could be recognized in the
majority of E-peptides and was especially pronounced in the most active
E-peptides that could confer very high levels of erythromycin
resistance (15).
In vitro studies suggested that the ribosome is the most
plausible target of action of E-peptides (12). Interestingly, we observed that chemically synthesized peptides added exogenously to the
cell-free translation mixture did not render ribosomes resistant to
erythromycin. In contrast, ribosomes that could translate E-peptide
mRNA exhibited increased resistance to erythromycin in a cell-free
translation system (15). Based on this observation, a model of
cis-acting E-peptide was proposed where a newly synthesized E-peptide remains tightly bound to the ribosome and prevents binding of
erythromycin into its functional site. Although this model accounted
for most of the experimental data, it did not satisfactorily explain
why synthetic peptides were not functional in vitro.
Furthermore, in order to explain erythromycin resistance of cells
expressing E-peptide, we had to invoke a remote possibility that the
ribosome-bound E-peptide could be displaced by a newly initiated
growing nascent peptide chain.
According to this model, E-peptide did not have to interact directly
with the drug, and the only requirement for peptide activity was its
tight binding to the ribosome in the vicinity of the erythromycin binding site. To test whether there is an interaction between the drug
and peptide on the ribosome, we selected peptides conferring resistance
to a chemically different macrolide with a mode of action similar to
that of erythromycin. We report here isolation of pentapeptides
conferring resistance to a new class of macrolide antibiotics,
ketolides. The results of this study show a correspondence between
peptide structure and the chemical nature of macrolides to which
peptide confers resistance, suggesting direct interaction between the
resistance peptide and macrolide antibiotic on the ribosome. This
finding allowed us to propose a novel model of action of macrolide
resistance peptides.
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EXPERIMENTAL PROCEDURES |
Reagents, Strains, and Plasmids--
Ketolide (HMR3004) was from
Roussel Uclaf, France. Erythromycin was purchased from Sigma. E. coli JM109 strain (16) (endA1, recA1,
gyrA96, thi, hsdR17
(rK
,
mK+relA1, supE44,
D(lac-proAB), F', traD36,
proAB, lacIqZDM15) was used for
propagation of the random pentapeptide library. Construction of the
library in the pPOT1AE vector has been described previously (15), and
the physical map of the library plasmid is shown in Fig. 1.
Selection of Ketolide Resistance Peptides--
The mini-gene
library (15) was transformed into competent E. coli JM109
cells and plated onto agar plates containing 100 µg/ml ampicillin, 60 µM ketolide, and 2 mM IPTG. Plates were
incubated overnight at 37 °C. Twenty-five individual colonies that
appeared on the plate were grown in liquid cultures. Plasmids were
isolated from 3-ml cultures and used to transform fresh E. coli cells. Transformants were plated onto ampicillin plates, and
individual colonies of transformed cells were streaked onto plates
containing 100 µg/ml ampicillin and 60 µM ketolide with
or without addition of 2 mM IPTG. All the selected clones
exhibited IPTG-dependent ketolide-resistant phenotype
co-transferable with the plasmids. The level of ketolide resistance of
the isolated clones was tested in liquid cultures as described
previously (15).
Mini-genes in the selected plasmids were sequenced using CCW sequencing
primer d(GCCATCGGAAGCTGTGG) (Fig. 1).
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RESULTS |
Construction of a random five-codon mini-gene library was
described previously (15). In this library, mini-genes, containing the
ATG initiator codon, followed by four random codons and the TAA
terminator codon, were cloned into the pPOT1AE vector where they are
expressed from an IPTG-inducible Ptac promoter (Fig. 1). This library has been used previously
to select for clones that express peptides rendering cells resistant to
erythromycin (E-peptides) (15). In the present study, we asked the
question whether the amino acid sequence of the selected resistance
peptides depends on the chemical structure of macrolide antibiotic used in selection.

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Fig. 1.
A 5-codon mini-gene library in pPOT1AE vector
(15). Locations of Ptac promoter, lac operator
(Olac), and Ttrp terminator are shown by
shaded bars. The Shine-Dalgarno (21) sequence,
initiator codon and terminator codon of the peptide mini-gene are
underlined. The orientation of the sequencing primer, CCW,
is shown by an arrow.
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Ketolides represent a new generation of 14-member-ring macrolide
antibiotics exhibiting increased activity toward erythromycin-resistant ribosomes (17, 18). The L-cladinose sugar moiety at
position 3 of the erythromycin macrolide ring is replaced by a keto
group in ketolides (Fig. 2). The general
mechanism of action of ketolides is probably similar to that of
erythromycin, and both macrolides have overlapping binding sites on the
ribosome.2,3
We used one of the ketolide derivatives, HMR3004, to select clones expressing ketolide resistance peptides.
The minimal inhibitory concentration of HMR3004 for E. coli
JM109 was 10 µM. Clones expressing ketolide resistance
peptides (K-peptides) were selected by plating the library onto an agar plate containing 2 mM IPTG and 60 µM
ketolide. Several clones appeared on the plate after 24 h of
incubation, and 25 clones were analyzed. Resistance phenotypes were
co-transferable with plasmids isolated from the clones. Furthermore,
dependence of ketolide resistance on the presence of IPTG clearly
indicated that it was mediated by expression of plasmid-encoded peptide mini-genes (Fig. 3). Sequencing of
plasmids from 25 isolated ketolide-resistant clones revealed 6 different peptide mini-genes (Table I),
each found in several independent isolates. Remarkably, three
mini-genes (clones K3, K9, and K17), which had different nucleotide
sequences, encoded the same pentapeptide, MRFFV. The minimal inhibitory
concentration of HMR3004 for clones expressing different K-peptides
ranged from 60 to 100 µM and thus, significantly exceeded
the drug concentration required to inhibit growth of the control
E. coli cells (Table II).

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Fig. 3.
Ketolide resistance of cells
expressing different K-peptides. Cells containing plasmids were
streaked onto agar plates containing 2 mM IPTG, 100 µg/ml
ampicillin, and either none (A) or 40 µM
(B) ketolide HMR3004 and grown for 24 h at 37 °C.
The clone names correspond to those shown in Table I. Note that clones
K3, K9, and K17, which express the same pentapeptide MRFFV, contain
different mini-genes (see Table I). Cells transformed with an empty
vector pPOT1AE or with an unselected plasmid expressing the
pentapeptide MDVEQ were used as controls.
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Table I
The amino acid sequences of the selected K-peptides and nucleotide
sequences of corresponding mini-genes
The number of independent isolates containing the same mini-gene is
shown in the second column.
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Table II
Minimal inhibitory concentration of ketolide HMR3004 for E. coli JM109
cells transformed with an empty vector pPOT1AE or recombinant plasmids
expressing K-peptides
The antibiotic MIC was determined in liquid cultures as described in
Ref. 15.
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In previous studies, we found that the amino acid sequence of
E-peptides conferring erythromycin resistance conformed to the consensus MXLXV, where the second and the fourth
positions were significantly less conserved than the third and the
fifth (the first position was occupied by methionine by default) (15). Interestingly, the sequence of the K-peptide MRFFV (see Table I)
closely resembled that of the E-peptides MRLFV, which conferred resistance to a very high erythromycin concentration (up to 1 mg/ml)
(15). We tested whether peptides selected with one antibiotic could
confer resistance to the other (K-peptides to erythromycin and,
conversely, E-peptides to ketolide). The clone K3 expressing the MRFFV
K-peptide and the clone expressing the E-peptide MRLFV were streaked
onto agar plates containing ampicillin, IPTG, and either ketolide or
erythromycin (Fig. 4). Cells expressing
E-peptide were resistant to low concentrations of ketolide, while
K-peptide could confer resistance to low concentrations of erythromycin (data not shown). Remarkably, however, only E-peptide rendered cells
resistant to high erythromycin concentration and, conversely, only
K-peptide conferred high level of ketolide resistance. Thus, different
peptides exhibit a clear specificity toward different types of
macrolide antibiotics.

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Fig. 4.
Erythromycin and ketolide resistance of cells
expressing E-peptide and K-peptide. Cells expressing the E-peptide
MRLFV or the K-peptide MRFFV (clone K3) were streaked onto agar plates
containing 2 mM IPTG, 100 µg/m ampicillin, and no
macrolide (A), 1 mM erythromycin (B),
or 40 µM ketolide (C) and grown at 37 °C
for 24 h.
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DISCUSSION |
Ketolide Resistance Peptides--
Unraveling the mechanism of
peptide-mediated macrolide resistance may provide important clues for
understanding the mode of action of macrolide antibiotics. It may also
provide insights into interactions between the ribosome and the nascent
peptide. In the current study, we investigated whether resistance
peptides act simply by competing with the antibiotic for the ribosomal binding site, or whether macrolide resistance conferred by short peptides is mediated by direct interaction between the peptide and the
drug on the ribosome. To address this question, peptides conferring
resistance to a ketolide, a macrolide antibiotic different from
erythromycin, were selected and their amino acid sequences were
compared with those of E-peptides.
The previously selected erythromycin resistance peptides conformed to a
general sequence consensus, MXLXV, where the
third position was the most conserved and occupied by leucine in the majority of E-peptides (15). Among the ketolide resistance peptides selected in the current study, neither had leucine in the third position. This result indicates that different peptides confer resistance to different macrolides. This conclusion was corroborated by
comparing the resistance patterns conferred by a very similar E-peptide
and K-peptide (MRLFV and MRFFV, respectively). The E-peptide rendered
cells resistant to high concentrations of erythromycin, but not
ketolide; conversely, expression of K-peptide afforded high level of
ketolide resistance but did not protect the cell from high erythromycin
concentrations (Fig. 4). Thus, the nature of the expressed peptide
determines to which macrolide the cell will become resistant.
Out of 25 analyzed ketolide-resistant clones, 10 expressed K-peptide
MRFFV. This peptide is apparently one of the "best" peptides conferring resistance to the ketolide HMR3004 used in the selection. Cells expressing this peptide exhibited the highest level of ketolide resistance compared with other selected clones (Table II). Furthermore, this same peptide was encoded in three different mini-genes (Table I),
demonstrating strong selection for a specific amino acid sequence.
Since one of the best E-peptides had the sequence MRLFV, which differed
from the K-peptide MRFFV only in the third amino acid position, one can
suppose that, within this context, it is the third amino acid (leucine,
in the E-peptide or phenylalanine, in K-peptide) that allows the
peptide to discriminate between the two macrolides.
The Mechanism of Peptide-mediated Macrolide Resistance--
We
have previously demonstrated the cis-mode of E-peptide
action so that only the ribosome that translated an E-peptide became resistant to erythromycin, while exogenously added E-peptide did not
affect inhibition of cell-free translation by erythromycin (12). Based
on this observation, a model of action of E-peptides was proposed in
which the newly translated E-peptide remained bound to the ribosome in
the vicinity of the peptidyl transferase center thus blocking the
erythromycin-binding site (15). Within this model, the peptide did not
interact directly with the erythromycin molecule and resistance
depended only on the relative affinities of the E-peptide and the drug
to the ribosome. Our new results, however, show that the macrolide and
newly synthesized peptide do interact with each other; such interaction
is reflected in correlation between the chemical structure of macrolide
and the amino acid sequence of resistance peptides. To reconcile the
model with the new experimental data, we suggest that the newly
synthesized peptide does not just passively occupy the drug binding
site, but instead, actively displaces the macrolide from the ribosome (Fig. 5). During this process, the
peptide directly interacts with the drug. In the previous model, which
was based on an idea of tight association of E-peptides with the
ribosome, we had to explain why E-peptide bound in the nascent peptide
channel does not inhibit protein synthesis. Therefore, we had to
speculate that after initiation of a new polypeptide synthesis, the
growing nascent peptide chain has either to displace the E-peptide from its binding site or go around it. This problem is easily solved within
the new model which does not require association of the resistance
peptide with the ribosome. Instead, the peptide acts as a "bottle
brush" that "cleans" the ribosome from the bound antibiotic.
After antibiotic is removed, the ribosome can either initiate synthesis
of a new polypeptide or bind another molecule of the drug. In the
former case, when the nascent peptide becomes longer than 5 amino
acids, the ribosome will become "resistant" to macrolides until
completion of polypeptide translation because macrolides cannot bind to
ribosomes with long nascent peptide chains (19, 20). Thus, translation
of macrolide resistance peptides opens for ribosomes a window of
opportunity to translated cellular proteins. Notably, the new model is
fully compatible with the cis-mode of the peptide action,
because only the ribosome that translated the resistance peptide
mini-gene would become transiently competent for translation of other
genes, which, in an experiment, would be observed as antibiotic
resistance.

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Fig. 5.
A model of action of macrolide resistance
peptides. I, binding of a macrolide
(hatched) to the large ribosomal subunit hinders growth of
the nascent peptide chain in the early rounds of protein synthesis.
II, translation of a macrolide resistance peptide
"cleans" the ribosome from antibiotic. If an antibiotic-free
ribosome initiates synthesis of a cellular protein and polymerizes the
first 2-5 amino acids, it will become "immune" to an antibiotic
until the completion of polypeptide synthesis because macrolides cannot
bind to ribosomes containing long nascent peptides.
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Macrolide Resistance Peptides and Translation--
The
correspondence between the peptide amino acid sequence and chemical
nature of the macrolide strongly suggests a possibility of interaction
between the resistance peptide and the antibiotic. However, free
peptide apparently does not interact with the drug, since even
1000-fold excess of the synthetic E-peptide added to the cell-free
translation system did not reduce inhibitory action of erythromycin
(12). Therefore, the peptide-drug interaction occurs most probably only
on the ribosome. Previously, we have shown that only short peptides
(3-6 amino acids long) could confer resistance to erythromycin (15).
The size of active peptides is compatible with direct interaction
between resistance peptide and the drug on the ribosome. Erythromycin
hinders growth of nascent polypeptide when it reaches the size of 2-5
amino acids. Therefore, when the ribosome decodes the stop codon of an
E- or K-peptide mini-gene, the newly synthesized pentapeptide should be
in contact with the ribosome-bound antibiotic. The ribosome may
constrain the newly synthesized peptide in a conformation competent for specific interaction with the antibiotic. Such interaction may reduce
affinity of the drug to its binding site on the ribosome, and it can be
ejected together with the completed peptide.
The known mechanisms of antibiotic resistance are usually divided into
three main groups: 1) mechanisms affecting accumulation of the drug in
the cell, 2) mechanisms involving modification of the drug target, and,
finally, 3) mechanisms based on modification of the drug. The proposed
mechanism of action of macrolide resistance peptides does not fall
within any of the three categories, but rather represents a hybrid of
the latter two groups, since "modification of the drug" (its
ability to bind to the ribosome) occurs directly within the target (the
ribosome). Multiple examples of peptide sequences capable of conferring
resistance to different macrolide antibiotics suggest that similar
mechanisms of antibiotic resistance may potentially operate in nature
and may account for macrolide resistance of some clinical
pathogens.
We thank Dr. P. Mauvais (Roussel Uclaf,
Romainville, France) for encouragement and Dr. S. Douthwaite
(University of Odense, Odense, Denmark) for communicating unpublished
results.