Download Short peptides conferring resistance to macrolide antibiotics

Peptides 22 (2001) 1661–1668
Review
Short peptides conferring resistance to macrolide antibiotics
Tanel Tensona, Alexander S. Mankinb,*
a
b
Institute of Molecular and Cell Biology, Tartu University, Riia 23, Tartu 51010, Estonia
Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607, USA
Received 22 December 2000; accepted 17 April 2001
Abstract
Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis
upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with
different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the
peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the
ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved
in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and
antibiotics can occur in both cases. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction
1.1. Macrolide antibiotics: binding site and mechanism of
action
All proteins in the cell, large or small, are synthesized by
the ribosome. The ribosome consists of two subunits. The
small ribosomal subunit is engaged in decoding of genetic
information, while the large subunit assembles amino acids
into a polypeptide chain. Catalysis of peptide bond formation and amino acid polymerization occurs in the peptidyl
transferase center located on the large subunit. At a short
distance form the peptidyl transferase center is the entrance
to the nascent peptide channel through which a newly synthesized polypeptide leaves the ribosome.
Many natural and synthetic antibiotics inhibit bacterial
growth by interfering with protein synthesis. Most of these
drugs act upon the ribosome. Among the ribosome-targeted
antibiotics, one of the most important groups is the 14member ring macrolides [1]. Macrolides bind to the large
ribosomal subunit in the vicinity of the peptidyl transferase
center (see [37–39] for review). However, in contrast to
many other drugs that act upon the large subunit, macrolides
do not inhibit catalytic activity of ribosomal peptidyl trans* Corresponding author. Tel.: ⫹1-312-413-1406; fax: ⫹1-312-4139304.
E-mail addresses: [email protected] (A. Mankin), [email protected]
(T. Tenson).
ferase directly. Instead, they interfere with the growth of the
nascent peptide chain during early rounds of translation
[35,36]. Though the exact mechanism of such inhibition is
not clear, two (not necessarily exclusive) models have
emerged. In one commonly accepted scenario, macrolides
barricade the entrance to the nascent peptide channel, thus
preventing growth of the newly synthesized peptide [35].
On the other hand, macrolides were shown to compete with
the peptidyl tRNA for binding to the ribosome and therefore
can cause spontaneous dissociation of the peptidyl-tRNA
before the nascent peptide enters the exit channel [19]. In
either of these scenarios, the macrolide molecule is expected
to interact with the growing polypeptide chain a few amino
acids away from the peptidyl transferase center.
The central component of macrolide chemical structure
is a lactone ring carrying a number of substitutions (Fig. 1).
The first clinically important drug of this group of antibiotics, and also the best known, is erythromycin, which
represents the first generation of 14-member ring macrolides. Macrolides of the second generation, such as clarithromycin or roxithromycin, are characterized by better
stability and improved spectrum of activity. Subsequent
rapid spread of antibiotic-resistant strains has stimulated the
search for novel derivatives. Macrolides of the third generation, called ketolides, contain a keto group instead of the
cladinose residue at position 3 of the lactone ring and carry
alkyl-aryl side chains. Ketolides not only show an improved
activity profile compared to the drugs of the second gener-
0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 5 0 1 - 0
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1.3. rRNA-encoded E-peptide
Fig. 1. Chemical structures of macrolide antibiotics of the first (erythromycin), second (clarithromycin) and third (telithromycin) generation.
These and other macrolides were used to isolate and characterize peptides
conferring macrolide resistance.
ation, but are also more active against certain macrolideresistant strains [1,40].
1.2. Main mechanisms of macrolide resistance
The binding site of 14-member ring macrolides includes
two distinct segments of the 23S rRNA located in close
vicinity in the ribosome tertiary structure. The best characterized rRNA site involved in macrolide binding is a segment of the so called central loop of domain V, in which
positions 2058 and 2059 (E. coli numbering) are critical for
the drug binding [20]. The main mechanism of macrolide
resistance is based on a modification of the drug binding site
in the ribosome, specifically, methylation of A2058 by specific methyl transferase enzymes [16,25]. Methylation of
A2058 results in a dramatic reduction of drug binding [10].
rRNA mutations in the vicinity of A2058 and also in helix
35 of domain II, another rRNA segment involved in macrolide binding, can also render cells resistant to the macrolide action [14,42]. Not only mutations in rRNA, but also
mutations in ribosomal proteins L4 and L22 confer resistance to macrolides [2,8,29]. Analysis of the protein mutations suggests that they may cause allosteric changes in the
conformation of rRNA in the macrolide binding site [13].
The second most important mechanism of cell resistance
to macrolides is based on antibiotic efflux (27,28, see also
26,40 for review). Specialized or broad-spectrum drug-efflux transporters can efficiently reduce the intracellular drug
concentration and render cells drug resistant.
Resistance mechanisms based on enzymatic chemical modification of the drug which were described for several isolates
do not play a major role in clinical macrolide resistance [3].
A novel mechanism of macrolide resistance, based on the
expression of a specific short peptide in the cell, was discovered in our laboratories several years ago [30,31]. In this
paper, we review previous data and more recent findings,
which provide insights into this mechanism of drug resistance. We believe that understanding how short peptides can
render cells resistant to macrolides will not only explain this
particular resistance mechanism, but will also provide important clues to the mode of macrolide action and basic
mechanisms of protein synthesis.
1.3.1. The first encounter
The phenomenon of peptide-mediated macrolide resistance was first discovered coincidentally in an experiment
where fragments of rRNA were expressed in vivo in search
for fragments that can bind antibiotics [30]. E. coli cells
expressing random fragments of rrnB operon were screened
for the presence of erythromycin (Ery) resistant clones.
Several Eryr clones were identified. In all such clones,
resistance depended on expression of rRNA fragments all of
which encompassed the region 1233–1348 in 23S rRNA.
The rRNA fragments expressed in these clones were designated E-RNA for erythromycin resistance RNA. The smallest of the E-RNAs in the originally selected Eryr clones
contained a 116 nucleotide long segment of the 23S rRNA.
Subsequent deletion analysis showed that expression of an
rRNA fragment only 34 nucleotides long (E-RNA34), corresponding to the E. coli 23S rRNA sequence 1235–1268,
was sufficient to render cells resistant to erythromycin.
1.3.2. rRNA or mRNA?
Neither biochemical nor genetic data implicated the segment of 23S RNA corresponding to E-RNA34 in erythromycin binding, suggesting a completely new mode of drug
resistance conferred by its expression. Mutational analysis
of E-RNA34 revealed three regions important for the Ery
resistance: the 5⬘-terminal five nucleotides, GGAGG, a trinucleotide GUG and yet another tri-nucleotide UAA (Fig.
2A) [30]. Remarkably, these three segments correspond to
the Shine-Dalgarno sequence, initiator and terminator
codons of a short open reading frame (ORF) which codes
for a pentapeptide MRMLT. Subsequent biochemical and
genetic analysis showed that translation of this mini-gene is
required and sufficient to render cells resistant to low concentrations of erythromycin.
E-RNA34 with its pentapeptide mini-gene is derived
from 23S rRNA. Does it mean that wild-type E. coli cells
are constitutively resistant to erythromycin due to expression of the rRNA-encoded pentapeptide? Probably not. It is
unlikely that significant amounts of the rRNA-encoded pentapeptide can be expressed in the cell under “normal” conditions. Not only is 23S rRNA associated with ribosomal
proteins and can hardly be directly translated, but also the
Shine-Dalgarno region of the mini-gene is sequestered in
the 23S rRNA secondary structure (Fig. 2B) - thus making
its translation impossible. All E-RNAs expressed in the Eryr
clones selected from the random rRNA fragment library
lacked the 5⬘ half of the hairpin sequestering the ShineDalgarno region of the mini-gene and therefore, peptide
expression was not hampered. Expression of the rRNAencoded pentapeptide can be activated by specific mutations. A spontaneous deletion of 12 nucleotides (positions
1219 –1230) from the 23S rRNA gene was shown to cause
resistance to erythromycin [11,12]. Such deletion evidently
destabilized the hairpin, making the ribosome-binding site
T. Tenson, A.S. Mankin / Peptides 22 (2001) 1661–1668
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peptide was added to the cell-free translation system, inhibitory action of erythromycin was diminished. In this experiment, ribosomes were allowed to synthesize the MRMLT
peptide by translating E-RNA34 and then were “re-programmed” with MS2 RNA. The ribosomes that had a
chance to translate E-RNA34 prior to translation of MS2
RNA became tolerant to erythromycin. This result suggested that a ribosome becomes erythromycin resistant only
if the MRMLT peptide was synthesized on that very same
ribosome (in the other words, that the peptide acts in cis).
The important implication of this finding was that the ribosome is the primary site and possibly the target of action of
the rRNA-encoded MRMLT peptide.
Fig. 2. A. Nucleotide sequence of an E. coli 23S rRNA fragment encoding
the E-peptide mini-gene. Nucleotide sequences whose mutations eliminate
peptide-mediated resistance are underlined. Amino acid sequence of the
encoded E-peptide is shown beneath the corresponding codons of the
mini-gene. B. Secondary structure of the segment of E. coli 23S rRNA
encompassing the E-peptide mini-gene. The mini-gene and its ribosome
binding site are shown in bold and the Shine-Dalgarno sequence of the
mini-gene is boxed. E. coli 23S rRNA numeration is shown.
of the E-peptide mini-gene more accessible, thus activating
E-peptide expression. Other deletion and nucleotide substitutions in this hairpin can activate mini-gene expression
through a similar mechanism [9].
1.3.3. How does the peptide work?
How can a short peptide with a molecular weight comparable to that of erythromycin render the bacterial cell
resistant to the drug? One of the plausible models was that
the peptide could bind erythromycin and sequester it in an
inactive complex. To test this model, activity of a synthetic
pentapeptide was tested in a cell-free translation system. In
vitro translation of phage MS2 RNA is efficiently inhibited
by low concentrations of erythromycin. Contrary to expectations, addition of excess of the synthetic MRMLT peptide
(up to 1 mM) to the cell-free translation system did not
alleviate inhibitory action of erythromycin, therefore ruling
out direct sequestering of the drug by the peptide [30].
In contrast, when E-RNA34 instead of the synthetic
1.3.4. Peptide gene in rRNA: why is it there?
The idea that rRNA in addition to its structural and
functional role in the ribosome could code for proteins has
been around for a long time. A few relatively long open
reading frames have been identified in rRNA [5,6,23]. Nevertheless, the translation products of these ORFs were never
detected and functions of the putative proteins were never
recognized.
Finding of the E-peptide raises the question about translation of ribosomal RNA again. Because of the association
of 23S rRNA with ribosomal proteins and because of its
secondary structure, the MRMLT peptide mini-gene is not
expressed unless mutations occur in the 23S rRNA gene or
23S rRNA undergoes site-specific fragmentation. Such
fragmentation can potentially occur under specific physiological conditions. However, so far, we were unable to
identify the conditions that would lead to the expression of
the rRNA-encoded peptide in the cell.
In general, it remains unclear if the presence of the
peptide mini-gene in rRNA has a biological purpose (in the
other words, whether it is a result of evolutionary selection
or whether it is simply coincidental). The mini-gene is well
conserved among different branches of eubacteria, missing
only in the Rhodobacter-Flexibacter branch. This conservation argues in favor of functional importance of the rRNAencoded mini-gene. Nevertheless, its functions remain obscure. We can only speculate what the possible
physiological significance of an rRNA-encoded peptide
could be. The cis mode of peptide action in the macrolide
resistance mechanism suggests that the peptide can possibly
modulate properties of the ribosome. However, while expression of some other short peptides have profound
effects on protein synthesis and cell growth [15,32],
overexpression of the rRNA-encoded E-peptide did not
significantly change growth characteristics of the cell. If
the peptide mini-gene is present in rRNA for a reason, it
is certainly not to render cells resistant to erythromycin.
Rather, resistance to the drug is a consequence of some
special mode of interaction between the peptide and the
ribosome (see below).
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onstrated that any mutations eliminating the stop codon of
the pentapeptide mini-gene abolished erythromycin resistance [30]. Therefore, the mere presence of an E-peptide
sequence at the N-terminus of a longer polypeptide could
not apparently render ribosomes resistant to erythromycin.
The result of screening a 21-codon library confirmed this
observation and also showed that presence of the E-peptide
sequence at the C-terminus of a polypeptide could not
confer erythromycin resistance. Thus, an erythromycin resistance peptide cannot be part of a longer protein and the
small size of the peptide is critical for its activity. The small
size of the peptide can be important if termination of translation plays a role in the mechanism of the peptide action.
Fig. 3. Plasmid library in pPOT1AE vector [33] used for selection of
peptides conferring resistance to macrolides. Promoter (Ptac) and terminator (Ttrp) of the mini-gene are shown by black boxes. Shine-Dalgarno
sequence, initiator and terminator codons of the mini-gene are underlined.
The pentapeptide library contained 4 random codons (12 random nucleotide positions) while the 21-codon library contained 20 random codons (60
random nucleotide positions).
1.4. Mini-gene libraries as a tool for selection of
functional peptides
Mutational analysis of the rRNA-encoded mini-gene
suggested that peptides with sequences other than MRMLT
can render cells resistant to erythromycin. In order to clarify
which properties allow a peptide to confer resistance, a
variety of erythromycin resistance peptides (E-peptides)
were selected from random mini-gene libraries (Fig. 3) [33].
In such libraries, each individual bacterial clone expresses a
peptide encoded in a specific mini-gene. Two libraries were
used to select peptides conferring resistance to erythromycin. One contained 5-codon-long mini-genes (4 of them
randomized). The other contained 21-codon-long minigenes
(20 of them randomized). Comparison of peptides expressed in erythromycin resistant clones revealed sequence
and size requirements for the peptide activity.
1.4.1. Size is critical for activity of E-peptides
There are three termination codon in the genetic code.
Therefore, about 2/3 of the clones in the 21-codon library
are expected to have in-frame stop codons. Because of this,
the library provides an excellent tool for investigating the
size requirement for functional peptides. As expected, a
broad distribution of sizes of the encoded peptides were
found in unselected, randomly picked clones. In contrast,
the majority of the peptides expressed in Eryr clones fell
within an amazingly narrow size range. Eleven out of
twelve peptides were four, five or six amino acids long,
suggesting that the optimal length of erythromycin resistance peptides should be within this range [33]. In agreement with this conclusion, the originally described rRNAencoded peptide (MRMLT) was five amino acids long.
Genetic analysis of the rRNA-encoded mini-gene dem-
1.4.2. E-peptide consensus sequence
If analysis of clones isolated from the 21-codon library
revealed peptide size requirements, then screening the
5-codon library provided information regarding the sequence of the active E-peptides. Comparison of sequences
of 52 pentapeptide mini-genes found in Eryr clones showed
a strong tendency for having Leu or Ile in the third position
and a hydrophobic amino acid (most commonly Val) in the
C-terminal position. Of sixteen peptides that conferred resistance to high concentrations of erythromycin (1 mg/ml),
only one lacked Leu or Ile in the third position, and only one
peptide did not have a hydrophobic amino acid in the 5th
position [33] (Fig. 4A). Though all the resistance peptides
were selected in E. coli system, their activity is not limited
to this organism because at least some of the selected
peptides could confer erythromycin resistance in evolutionary distant Bacillus subtilis and Proteus mirabilis [22].
1.5. Different peptides confer resistance to different
macrolides
E-peptide did not affect cell sensitivity to chloramphenicol and clindamycin, structurally different antibiotics
which compete with erythromycin for binding to the ribosome [7]. However, E-peptide rendered cells resistant to
low concentrations of oleandomycin and spiramycin, which,
together with erythromycin, belong to the family of macrolide antibiotics. This observation raised the possibility that
E-peptides could render cells resistant to all types of macrolides. Alternatively, different peptides could be required
to confer resistance to different macrolide antibiotics. Selection of resistance peptides using a variety of macrolide
antibiotics confirmed the latter model [34] (Fig. 4). Two
general groups of resistance peptides emerged from these
experiments. The first group consisted of peptides that render cells resistant to macrolide antibiotics containing a
cladinose residue at position 3 of the lactone ring (C-peptides). C-peptides show a very strong bias in amino acid
composition with a prevalence of the hydrophobic amino
acid residues Leu, Ile, Val, Ala, Phe and Trp. The preponderance of hydrophobic amino acid residues is especially
notable in the third and fifth position of the pentapeptide,
T. Tenson, A.S. Mankin / Peptides 22 (2001) 1661–1668
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Fig. 4. Nucleotide sequences of mini-genes and amino acid sequences of the encoded peptides conferring resistance to different macrolide antibiotics. A.
C-peptides conferring resistance to cladinose-containing compounds, erythromycin, clarythromycin, roxithromycin and RU69874. B. K-peptides conferring
resistance to ketolides, telithromycin, RU64399 and HMR3004. All the compounds used in selection experiments were obtained from Aventis-Pharma.
while the second and especially the fourth position exhibit
higher amino acid diversity. The most conserved position in
C-peptides is the third, which is most commonly occupied
by leucine. Peptides of the second group (K-peptides) confer resistance to ketolides - antibiotics which possess keto
function in place of the cladinose residue. K-peptides are
less hydrophobic than C-peptides. They contain positively
charged Arg or Lys in the second or sometimes fourth
position, but lack Leu in the third position. The relationship
between the drug structure and sequence of the resistance
peptides is especially evident when compounds differing
from each other only by the presence or absence of cladinose moiety are compared (for example, RU69874 and
telithromycin). This example clearly shows that structurally
distinct peptides are required to confer resistance to the
drugs with only a small difference in their molecular structures. Other variations in the drug structure also call for
different consensus sequences of the resistance peptides
(Tenson and Mankin, unpublished results).
1.6. Brushing the ribosome
The most straightforward explanation of the correlation
between the sequence of the resistance peptides and the
structure of a macrolide antibiotic is that at some point, a
peptide will interact directly with the drug molecule. The
cell-free experiments with the rRNA-encoded MRMLT
peptide (discussed above) showed that E-peptides do not
bind the drug “outside” the ribosome. Thus, we arrive at the
conclusion that peptide-drug interaction must take place in
the ribosome.
Crystallographic structure of the large ribosomal subunit
showed that extended segments of ribosomal proteins L4
and L22 form a narrow “gate” located at the entrance of the
nascent peptide channel [4,21]. Biochemical and genetic
data indicate that macrolides bind very close to this gate.
This is the most probable site where the interaction between
the drug and the resistance peptide occurs. The distance
between the peptidyl transferase center and macrolide binding site is approximately 14Å. Therefore, when the C- or
K-peptides synthesized by the ribosome reach the size of
4 –5 amino acids, they can come in direct contact with the
macrolide molecule.
Genetic and biochemical studies revealed several important facts pertaining to the mode of action of the resistance
peptides. 1) Resistance peptides are small, 4-to-6 amino
acids long. 2) There is a correlation between the sequence of
the resistance peptides and the structure of the drug to which
the peptide confers resistance. 3) Peptides act in cis: only
the ribosome on which the peptide was synthesized becomes resistant to the drug. 4) Free peptides do not bind to
the antibiotic but may interact with the drug on the ribosome. All these data can be accounted for by a “bottle
brush” model of the peptide action [34] (Fig. 5). Macrolides
do not block initiation of translation or the first several
rounds of amino acid polymerization. Therefore, the ribosome with the bound macrolide antibiotic can still translate
the resistance peptide mini-gene. When synthesis of the
peptide is almost finished (when the nascent peptide is 4 –5
amino acid residues long), the N-terminal segment of the
peptide reaches the site where the antibiotic is bound and
the peptide forms specific contacts with the drug molecule.
During termination of peptide synthesis when the peptide is
Fig. 5. “Bottle brush” model of the resistance peptide action. Translation of
a resistance peptide (one of the C-peptides is shown) may remove antibiotic from its binding site on the ribosome. This will free the ribosome for
translation of a cellular protein. The cumulative effect of translating the
mini-genes encoding resistance peptides will be an overall increase in the
population of drug-free ribosomes in the cell.
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T. Tenson, A.S. Mankin / Peptides 22 (2001) 1661–1668
released from the peptidyl-tRNA, or during the last translocation event, the peptide “kicks” the drug out of its binding site thus removing antibiotic from the ribosome. Once
the antibiotic is removed, the ribosome can engage in translation of a cellular protein. If it has enough time to polymerize
the first 4 – 6 amino acids before a macrolide molecule binds
again to the ribosome, then the drug binding site will be
occupied by the nascent polypeptide and the ribosome can
successfully finish translation of the protein. After the release
of the completed protein, the ribosome can either initiate translation of a new protein or it can be invaded by an antibiotic and
remain inactive until it is again “cleaned” by translation of a
resistance peptide. Frequent translation of resistance peptide
mini-genes essentially increases the population of drug-free
ribosomes in the cell. At a relatively low drug concentration
this is sufficient for the cell to continue its growth.
1.7. Resistance peptides and inducible resistance
The bottle brush model suggests direct interaction of the
growing polypeptide chain with the macrolide molecule on
the ribosome. Such interaction is reminiscent of that implicated in the induction of Erm genes (see [38] for review).
Erm genes code for methyltransferase enzymes that render
cells resistant to macrolides (as well as streptogramins and
lincosamides) by methylating a specific adenine residue
(A2058) located in the drug binding site in 23S rRNA.
Induction of erm expression involves conformational
isomerization of its mRNA into a translationally active
form. The leader region of erm mRNA that precedes the erm
cistron contains a short open reading frame. In the absence
of antibiotic, mRNA assumes a conformation where the
translation initiation region of erm is sequestered in a hairpin structure, which precludes erm expression (Fig. 6). In
the presence of low concentrations of an inducing macrolide
antibiotic (for example, erythromycin), the ribosome stalls
on the leader ORF. This switches the mRNA secondary
structure so that erm translation becomes possible.
Stalling of the ribosome in the leader ORF occurs when
the eighth codon (Val) of the leader ORF is positioned in the
ribosomal P site [17]. The identity of Val8 is essential for
stalling. Similarly, alterations of the two preceding amino
acids, Ile6 and Phe7, strongly diminishes ribosome stalling
and erm induction. The amino acid sequence of this leader
peptide segment that is critical for ribosome stalling (Ile6 Phe7 - Val8) is remarkably similar to the consensus sequence of E-peptides. When synthesis of the E-peptide is
completed, right before its translation is terminated, the last
(most commonly Val) codon of E-peptide ORF is positioned in the ribosomal P-site. The semi-ultimate position of
the E-peptide is frequently a hydrophobic amino acid and
the preceding position is either leucine or isoleucine (Fig.
4). Analogy between erm leader peptides and E-peptides
extends even further. While alteration of the E-peptide sequence changes the spectrum of antibiotics to which the
peptide confers resistance, alterations in the erm leader
Fig. 6. Similarity between erm leader peptides and macrolide resistance
peptides. A. “Inactive” conformations of the leader region of erm mRNA.
Shine-Dalgarno regions and initiator codons of the leader peptide and Erm
ORFs are underlined. Amino acid sequence of the leader peptide is shown
above the corresponding codons of the leader ORF. B. “Active” conformation of erm mRNA. Stalling of the ribosome occurs when the eighth
(Val) codon of the leader ORF is positioned in the ribosomal P-site.
Switching of mRNA conformation activates translation of the erm cistron.
C. Pre-termination complex of the ribosome with mRNA of the C-peptide
MVLFV that confers resistance to erythromycin.
peptide affects the spectrum of antibiotics which cause
ribosome stalling and erm induction [18]. This indicates that
not only resistance peptides but also erm leader peptides
interact with the drug on the ribosome. The effect of such
interaction is however, strikingly different: possible removal of antibiotic in case of resistance peptide and ribosome stalling in case of erm leader peptides. Such disparity
in the mode of peptide action can be related to a difference
in the length of the nascent peptides. Termination of the
translation of the five amino acid long nascent E-peptide
may lead to antibiotic expulsion, while a longer nascent
peptide of the erm leader ORF may get “stuck” in the
ribosome in the presence of the drug. Alternatively, the
difference in ribosome response can depend on the different
N-terminal sequences of the E- and leader ORF peptides.
2. Conclusions and future directions
Expression of C- or K- peptides renders cells resistant to
relatively low concentrations of macrolide antibiotics.
Therefore, this mechanism can not play a major role in the
T. Tenson, A.S. Mankin / Peptides 22 (2001) 1661–1668
drug resistance of clinical bacterial strains. Nevertheless, it
can contribute to the initial survival rate of the pathogenic
bacteria during antibiotic treatment and therefore increase
the chances of acquiring one of the major resistance mechanisms. A spontaneous 12 bp deletion in 23S rRNA gene
results in the expression of the E-peptide mini-gene encoded
in bacterial rRNA [12]. There are many other potential
mini-ORFs present in the bacterial genomes which can code
for resistance peptides. A single mutational event can generate an appropriate promoter or translation initiation site
for such an ORF and activate peptide expression.
Apart from its potential clinical significance, the phenomenon of peptide-mediated macrolide resistance is important because it reveals a new aspect of interaction of
nascent peptide with the ribosome and macrolide antibiotics. The correlation between the peptide structure and chemical structure of the drug suggests direct peptide-drug contact on the ribosome. However, the detailed mechanism of
such interaction remains obscure. It is possible that the
peptide structure needs to be constrained by the ribosome to
allow specific contacts with the drug, but it has never been
proven. Other questions closely related to the activity of Cand K-peptides and the mode of macrolide action also await
exploration. What is the exact size of the nascent peptide
when macrolides start to exert their inhibitory action? Does
this length depend on the nascent peptide sequence? Is
translation of all different polypeptides in the cell affected
by macrolides to the same extent?
The recently unveiled high resolution crystal structures
of the ribosomal subunits are expected to bring our understanding of how the ribosome works to a new level [4,24,
41]. The next critical and long-awaited leap in our knowledge of the macrolide binding site will be brought about
when crystal structures of ribosome-macrolide complexes
become public. There is a hope that these structures, in
combination with biochemical data, will help us to understand the intimate relations among the ribosome, nascent
peptide and macrolide antibiotics.
Acknowledgments
We thank Liqun Xiong and Marne Gaynor for their
expert technical assistance and Maria Gomez for help in
preparing the manuscript. This work was supported by National Institutes of Health grants GM53762 and TW00870
and Aventis Pharma Research Grant (to A.S.M.) and Estonian Science Foundation grant N 4443 (to T.T.).
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