Mechanism of Action of Spiramycin and Other Macrolides

Journal of Antimicrobial Chemotherapy (1988) 22, Suppl. B, 13-23

Mechanism of action of spiramycin and other macrolides Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 a a Anne Brisson-Noel , Patrick Trieu-Cuot" and Patrice Courvalin • b

aUnite des Agents Antibacteriens, CNRS UA271, Institut Pasteur, 75724 Paris Cedex 15; b National Reference Center for Antibiotics, Institut Pasteur, 75724 Paris Cedex 15, France

Macrolide antibiotics constitute a group of 12 to 16-membered lactone rings substituted with one or more sugar residues, some of which may be amino sugars. They inhibit bacterial protein synthesis both in vivo and in vitro with varying potencies. Macrolides are generally bacteriostatic, although some of these drugs may be bactericidal at very high concentrations. The mechanism of action of macrolides has been a matter of controversy for some time. Spiramycin, a 16- membered macrolide, inhibits translocation by binding to bacterial 50S ribosomal subunits with an apparent I : I stoichiometry. This antibiotic is a potent inhibitor of the binding to the ribosome of both donor and acceptor substrates. Spiramycin induces rapid breakdown of polyribosomes, an effect which has formerly been interpreted as occurring by normal ribosomal run-off followed by an antibiotic induced block at or shortly after initiation of a new peptide. However, there is now convincing evidence that spiramycin, and probably all macrolides, act primarily by stimulating the dissociation of peptidyl-tRNA from ribosomes during translocation. Although the ribosomes of both Gram-positive and Gram-negative organisms are susceptible to macro Ii des, these antibiotics are mainly used against Gram-positive bacteria since they are unable to enter the porins of Gram-negative bacteria. Resistance to macrolides in clinical isolates is most frequently due to post transcriptional methylation of an adenine residue of 23S ribosomal RNA, which leads to co-resistance to macrolides, lincosamides and streptogramins type B (the

so-called MLSB phenotype). Other mechanisms of resistance involving cell impermeability or drug inactivation have been detected in Staphylococcus spp. and Escherichia coli. These strains are resistant to 14-membered macrolides (erythromycin and oleandomycin) but remain susceptible to spiramycin.

Introduction Spiramycin belongs to the group of macrolide antibiotics which contain a large lactone ring (12-16 atoms) with few double bonds and no nitrogen atoms. In addition, the ring is substituted with one or more sugar residues, some of which may be aminosugars. Closer examination of the structure of macrolides suggest that they fall into several groups, the most important of which are the erythromycin group (erythromycin and oleandomycin), the spiramycin group (tylosin, relomycin, angolamycin and others) and the carbomycin group (including niddamycin and leucomycin). Structures of erythromycin, spiramycin and carbomycin are given in Figure 1. These antibiotics, with different potency, inhibit bacterial protein synthesis

Correspondence to: Dr P. Courvalin. 13 0305-7453/88/22B013 + 11 $02.00/0 © 1988 The British Society for Antimicrobial Chemotherapy 14 A. Brisson-Noel et al. Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021

Erythromycin A

Spiramycin R = H

II R=COCH3 III R = COCH 2CH)

Carbomycin A

Figure 1. Chemical structures of the major macrolide antibiotics. Mode of action of spiramycin 15 both in vivo and in vitro (Vazquez, 1967). Like many other inhibitors of protein synthesis, they are generally bacteriostatic, although at very high concentrations some macrolides may be bactericidal. Spiramycin and other macrolides display selective toxicity, since they are active against bacteria but not against eukaryotes. Although it has been now clearly established that macrolides block the functions of the bacterial ribosome, the mechanism by which they accomplish this inhibition is still not clear. Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 Mechanism of protein synthesis Proteins are constituted by one or more polypeptide chains which are polymers of L-aminoacids. Before integration into polypeptides, amino acids are attached to specific transfer RNA (tRNA) by enzymes specific for each aminoacid (aminoacyl-tRNA synthetases). The resulting complexes are carried to the ribosome where the specificity of protein synthesis is achieved by pairing between the anticodon in the incoming aminoacyl-tRNA and the complementary codon in messenger RNA (mRNA). Protein synthesis occurs in Escherichia coli through three steps (initiation, elongation and termination) requiring different cofactors (Figure 2).

Initiation step The first event of this step is the binding of the 30S subunit to the correct site on mRNA, which involves complementary base pairing between the 3' terminus of 16S rRNA and a sequence of nucleotides upstream from the initiator codon (Shine & Dalgarno, 1974). Polypeptide chain synthesis is initiated by particular tRNA specific for methionine, tRNAF , which allows formylation of the aminogroup of bound methionine. The anticodon of tRNAF recognizes the AUG or GUG initiation co dons in mRNA. Another species of tRNA, tRNAM , does not allow formylation of bound methionine and is used to insert methionine at internal positions in polypeptide chains. Specific initiation factors (IF-I, IF-2, and IF-3) and GTP direct the binding of formylmethionine-tRNAF and mRNA to the smaller ribosomal subunit to form the 'initiation complex'. Then the 50S ribosomal subunit is added to the initiation complex while GTP is hydrolysed and the initiation factors are released.

Elongation step Addition of the large ribosomal subunit to the initiation complex places the fMet tRNAF in a particular site of this subunit, the peptidyl donor or P site. The aminoacyl tRNA bearing the next amino acid binds to an adjacent site, the aminoacyl acceptor or

A site. Peptide bond formation involves transfer of the fMet-tRNA from the tRNAF bound in the P site to the aminoacyl-tRNA bound in the A site. This reaction, which does not require exogenous energy or supernatant factors, is catalysed by an enzyme, the peptidyl transferase, which is an integral part of the 50S subunit. Then the new peptidyl-tRNA (tRNA bearing the nascent peptide) shifts from the A site to the P site and the old deacylated tRNA (which previously bore the nascent peptide) is released from the P site, while the ribosome moves towards the 3' end of the mRNA by one codon shift. This reaction, designated translocation, involves the elongation factor EF-G and hydrolysis of one GTP molecule. The amino acyl-tRNA binding reaction requires the elongation factor EF-Tu and hydrolysis of one GTP molecule. -0\ • INITIATION • IF 1,IF-2 5 m'''' Q ~ "/f ~ /// ~..~: /-t> f IF-3 IF 1,IF-2 T T GTP '.",""z 1t ra n sfer 0 ~ ~ - .... reaction t- :3. G « =fI) fI) /(

amino acid; EF, elongation factor; !Met, forrnylmethionine; IF, initiation factor; P, peptidyl donor site; RF, releasing factor. Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 October 02 on guest by https://academic.oup.com/jac/article/22/Supplement_B/13/714054 from Downloaded Mode of action of spiramycin 17 Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021

Puromycin Figure 3. Structure of puromycin.

Termination step Termination of polypeptide chains is signalled by three termination codons (UAA, UAG, and UGA) in mRNA. When a termination codon is reached, the release of the peptidyl-tRNA from the ribosome is promoted by three releasing factors (RF-l, RF-2, and RF-3). The hydrolysis of the ester linkage between the polypeptide and the tRNA is achieved by a peptidyl-tRNA hydrolase. Detachment of the deacylated-tRNA and mRNA from the ribosome is promoted by factor EF-G and another protein designated RRF (ribosome release factor). Clarification of the mechanism of protein synthesis was associated with the elucidation of the mode of action of puromycin. This antibiotic, which is a structural analogue of the 3'-terminus of aminoacyl-tRNA (Figure 3), takes part in the ribosomal peptide-bond forming reaction and accepts the nascent peptide chain. Since puromycin binds only weakly to ribosomes, the resultant peptidyl-puromycin molecule usually falls off the ribosome almost at once.

Binding of spiramycin to ribosomes Spiramycin, like other macrolides, binds to 50S ribosomal subunits (Vazquez, 1967) apparently with 1: I stoichiometry (Pestka, Nakagawa & Omura, 1974). This antibiotic and various macrolides compete with each other (Fernandez-Munoz et aI., 1971) for ribosomal binding sites to which they attach with dissociation constants in the range 10 - 8 -10 - 7. This competition suggested that all macrolides may bind at overlapping or identical sites on the ribosome. From experiments based on competitive binding of antibiotics and leucyl-pentanucleotide fragment (CACCA-Leu-Ac) from leucyl-tRNA on 50S subunit, it was reported that spiramycin and carbomycin blocked the P site of peptidyl transferase and also exerted an inhibitory action on substrate binding at the A site (Celma, Monro & Vazquez, 1970). On the contrary, erythromycin was shown to stimulate substrate-binding at the P site. Several studies intending to identify the ribosomal proteins that bound the antibiotics gave conflicting results. Using reconstitution experiments of the 50S subunit, Teraoka & Nierhaus (1978) demonstrated that proteins LI5 and LI6 were 18 A. Brisson-Noel et al. required for both erythromycin binding and peptidyl transferase activity. Siegrist, Moreau & Le Goffic (1984) used the technique of protein labelling with photoreactive dihydrorosaramycin to show that rosaramycin could bind to proteins L 1, L5, L6 and S 1. With the same techhique, Tejedor & Ballesta (1985) found that carbomycin and niddamycin boundto proteins L27, L28, L2, S12, and to a lesser extent to L6/11, L8 and L32/33. The authors explain their diverging results by differences either in the nature of the antibiotic or in their experimental conditions. However, the latter data seem to be interesting since proteins L2, L27 and L32/33 have been reported to be Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 associated to the P site 6f the peptidyl transferase (Saltzman & Apirion, 1976). An alternative hypothesis is that the actual target of macrolide binding is the 23S ribosomal RNA molecule of the 50S subunit (Cundliffe, 1987). It has been shown that modification, either by methylation or base substitution, of the adenine residue at position 2058 of E. coli 23S rRNA prevents the binding of macro Ii des to ribosomes and yields to cross-resistance to erythromycin and other macrolides (spiramycin, carbomycin), to lincomycin and to streptogramins type B (Saito, Hashimoto & Mitsuhasi, 1969; Lai & Weisblum, 1971; Weisblum et aI., 1971; Sigmund, Ettayebi & Morgan, 1984). Analysis of the 23S rRNA of other erythromycin resistant prokaryotic or eukaryotic organisms revealed the involvement of an analogous adenine residue corresponding to the same position 2058 (Cundliffe, 1987). Direct binding of macrolides to 23S rRNA was demonstrated by Moazed & Noller (1987), who showed that erythromycin, carbomycin and vernamycin B (a streptogramin type B antibiotic) protected overlapping sites in the 2058-2062 region of the rRNA from E. coli ribosomes. Given the results obtained both on 23S rRNA and ribosomal proteins, it is likely that macrolides would actually bind to 23S rRNA and that the affinity of binding would be enhanced by the presence of one or more ribosomal proteins. This has been demonstrated in the case of the antibiotic thiostrepton, which can bind directly to 23S rRNA, with increased affinity in the presence of protein LII (Cundliffe, 1987). Effect of spiramycin on protein synthesis Macrolide antibiotics do not prevent the binding of aminoacyl-tRNA to ribosomes nor do they inhibit ribosome-dependent hydrolysis of GTP in the presence of factors EF-G or EF-Tu and aminoacyl-tRNA (Mao & Robishaw, 1971). Previously it was thought that the inhibitory action of macrolides on protein synthesis reflected impairment of the peptidyl transferase reaction and/or of translocation. Monro & Vazquez (1967) showed that spiramycin and other macrolides inhibit the 'fragment' reaction, which consists of a ribosome-catalysed reaction between puromycin and the fMet

hexanucleotide fragment from fMet-tRNAF (Monro & Marcker, 1967). Cerna et al. (1971) studied the effects of macrolides on the reaction of puromycin with various donor substrates (polyLysyl-RNA, AcPhe-tRNA, AcPhe-pentanucleotide) in the presence of 70S E. coli ribosomes, and found that spiramycin and carbomycin were able to inhibit the transfer reaction for all substrates, whereas erythromycin gave varying results. In a more detailed analysis, synthesis of dilysine was shown to be inhibited by carbomycin, unaffected by spiramycin and stimulated by erythromycin, whereas synthesis of trilysine was inhibited by carbomycin and spiramycin, the effects of erythromycin varying with experimental conditions (Mao & Robishaw, 1971). Similar results were obtained with polyphenylalanine synthesis. From these data, Mao & Robishaw (1971) suggested that carbomycin inhibited peptide bond formation, Mode of action of spiramycin 19 spiramycin inhibited translocation and erythromycin inhibited neither of these two reactions. These differential effects on protein synthesis may reflect the different chemical structures of these antibiotics. Effects of macro Ii des on polyribosomes have been studied in various in-vivo or in vitro systems. Action of spiramycin on protoplasts of Bacillus megaterium resulted in rapid breakdown of at least 70% of the polyribosomes (Cundliffe, 1969). Since polyribosome breakdown occurred by normal ribosomal run-off, it was suggested that the residual polysomes might be blocked multi-initiation complexes and that Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 spiramycin might be an inhibitor of polypeptide chain initiation (Cundliffe, 1969). However, Pestka et al. (1974) showed that spiramycin inhibited the binding of chloramphenicol to polyribosomes only after removal of the nascent peptides. Gale et al. (1981) advanced the hypothesis that the ability of spiramycin to inhibit protein synthesis might depend critically upon the length of the nascent peptide on a given ribosome. According to this hypothesis, spiramycin should be regarded primarily as an inhibitor of the elongation of short peptides, which could be unable to affect ribosomes bearing nascent chains longer than a certain critical length. Similar considerations may apply to macrolides of the carbomycin group (Ennis, 1972). Again, erythromycin behaved differently since, when added to B. megaterium protoplasts, it stabilized polyribosomes bearing their nascent pep tides (Cundliffe, 1969). Preservation of polyribosome in the presence of macrolides of the erythromycin group was also observed by Ennis (1972) in intact cells of E. coli. More recently, a new experimental approach led to a different hypothesis to explain the mechanism of protein synthesis inhibition by macrolides (Menninger & Otto, 1982). The authors showed that erythromycin, spiramycin and carbomycin stimulate dissociation of the peptidyl-tRNA from ribosomes of E. coli during elongation. In normal cells of E. coli, hydrolysis of intact peptides from peptidyl-tRNA is catalysed by the peptidyl-tRNA hydrolase. When a strain bearing a temperature-sensitive mutation in the peptidyl-tRNA hydrolase gene is grown at non-permissive temperature (40°C), non-hydrolysed peptidyl-tRNAs accumulate, leading to inhibition of protein synthesis and cell death (Atherly & Menninger, 1972; Menninger, 1978, 1979). This type of mutant allows the measurement of the rate of dissociation of peptidyl-tRNA from ribosomes. The accumulation of peptidyl-tRNAs in cells has been shown to be markedly enhanced by erythromycin, spiramycin and carbomycin, owing to stimulation of dissociation of peptidyl-tRNA from ribosome in the presence of these drugs (Menninger & Otto, 1982). Although gross loss of peptidyl-tRNA from ribosomes exposed to erythromycin was not observed in some in-vitro assays (Cannon & Burns, 1971; Cundliffe & McQuillen, 1967), similar effects of erythromycin on peptidyl-tRNA dissociation from ribosomes in cell-free extracts have been reported by Otaka & Kaji, 1975). Menninger (1985) later confirmed the fact that erythromycin weakens the interactions between ribosome and peptidyl-tRNA. According to him, peptidyl-tRNAs are bound to ribosomes through non-specific and decoding-specific interactions; if macrolides preferentially weaken the non-specific interactions, a greater fraction of the binding energy will be due to decoding-specific interactions and better discrimination between erroneous and correct peptidyl-tRNAs should result. This idea has been tested with low doses of erythromycin, which were observed to counteract the error-inducing effects of streptomycin and ethanol on the synthesis of {3-galactosidase by E. coli. However, no evidence was given that this effect was not due to simple competition between these antibiotics. 20 A. Brisson-Noel et al.

Menninger's hypothesis conflicts with the idea that macrolides block protein synthesis by inhibiting peptide bond formation or translocation. Since the number of ribosomes in the ceIl is far lower than the number of tRNAs, peptidyl-tRNA accumulation can only occur if ribosomes are synthesizing several peptidyl-tRNAs successively, which would need fully active ribosomes. If ribosomes were blocked by macrolides during elongation, either by peptide transfer or translocation inhibition, one would expect a slowing of synthesis, of dissociation and of accumulation of peptidyl-tRNA. Accordingly, antibiotics that are known to interfere with protein Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 elongation, such as chloramphenicol and fusidic acid, have been shown to have a decreasing effect on peptidyl-tRNA accumulation (Menninger, 1976). The mechanism proposed by Menninger can explain several apparently conflicting data on macrolides. The observations of Mao & Robishaw (1971) on the stimulation of synthesis of shorter oligolysines and the inhibition of synthesis of longer oligolysines can be explained by the fact that dissociation of short peptidyl-tRNA prevents their utilization as substrates for further chain growth. The breakdown of polysomes upon treatment with spiramycin or carbomycin could result from stimulation of the dissociation of peptidyl-tRNA, folIowed by dissociation of the deacylated-tRNA and the messenger RNA from the ribosome. The absence of polysome breakdown after action of erythromycin could reflect the fact that this molecule, owing to its smalIer size, would not provoke dissociation of the deacylated-tRNA which could therefore allow the conservation of the mRNA-ribosome complex. Menninger's proposal has recently been restricted by the results of Andersson & Kurland (1987), who showed that elongating ribosomes were refractory to erythromycin. This suggests that peptidyl tRNA dissociation induced by erythromycin should occur only at an early stage after initiation.

Resistance to spiramycin and other macrolides Resistance to macrolide antibiotics can be due to different mechanisms classified in two groups: resistance due to mutation of a character of the ceIl, or resistance due to the presence of a 'foreign' gene not implicated in the normal metabolism of the ceIl. Three different mutations affecting ribosomal proteins have been shown to confer macrolide resistance. eryA mutants display an alteration in protein L4 which leads to loss of the ability of ribosomes to bind erythromycin and to react with puromycin (Wittmann et at., 1973). Since the gene encoding the altered protein L4 is contransduced with resistance to erythromycin, eryA may be identical to rpID, the structural gene for protein L4. The second type of mutant, designated eryB, contains altered L22 protein. However, the eryB ribosomes bind erythromycin normalIy and are able to react with puromycin. Moreover, transduction experiments of altered protein L22 can result in erythromycin susceptibility (Wittmann et at., 1973). It seems therefore that the eryB locus is not the structural gene for protein L22. The mechanism of resistance conferred by eryB is stilI unclear. A third resistance mutation in a gene designated eryC may affect a protein of the 30S ribosomal subunit and may implicate both subunits in the mode of action of erythromycin (Pardo & Rosset, 1977). It is noticeable that none of the proteins which seem to be involved in macrolide resistance have been reported to interact with these antibiotics (see 'Binding of spiramycin to ribosomes'). Other types of erythromycin resistant E. coli mutants were obtained by in-vitro mutagenesis. One of the mutations confers cross resistance to all macrolides, lincosamide and streptogramins type B, and consists in an A to U transversion Mode of action of spirarnycin 21 involving an adenine residue located at position 2058 of the 23S ribosomal RNA (Sigmund, Ettayebi & Morgan, 1984) Mutations affecting other positions of the 23S rRNA genes confer various resistance phenotypes, e.g. cross resistance to erythromycin and chloramphenicol (Ettayebi, Prasad & Morgan, 1985). Resistance to macrolides by ribosomal mutations is very rare in clinical isolates and the most frequent mechanism of resistance consists in post-transcriptional modification of 23S ribosomal RNA by mono or dimethylation of an adenine residue. This mechanism leads to coresistance to all macrolides, to lincosamides and to Downloaded from https://academic.oup.com/jac/article/22/Supplement_B/13/714054 by guest on 02 October 2021 streptogramins type B (for a review see Weisblum, 1985). Determination of the site of methylation indicated that an analogous adenine residue of the 23S rRNA, corresponding to position 2058 of E. coli ribosome, is modified by different rRNA methylases both in Gram-positive bacteria and in E. coli (Skinner, Cundliffe & Schmidt, 1983; Thakker-Varia, Ranzini & Dubin, 1985). Studies of in-vitro methylation of 23S rRNA and of in-vivo expression of the MLSB phenotype after cloning of various erm (erythromycin resistance methylase) genes in B. subtilis or E. coli has demonstrated that ribosomal rRNA of various origins can be substrate for the same methylase (Malke & Holm, 1981; Carlier & Courvalin, 1982; Hardy & Haefeli, 1982; Skinner et al., 1983; Thakker-Varia et al., 1985; Brisson-Noel, Arthur & Courvalin, 1988). The synthesis of rRNA methylases may be constitutive or inducible by different subsets of MLS antibiotics, most frequently erythromycin and oleandomycin. The high incidence of strains exhibiting an inducible phenotype has led to an increase of the clinical use of non-inducer macrolides, especially spiramycin. However this practice has provoked the emergence of strains displaying a constitutive phenotype, which has raised from 30% to 60% of the total MLS resistant strains of Staphylococcus. Other mechanisms of resistance to macrolides have also been described. Inactivation of erythromycin has been detected in strains of E. coli highly resistant to this drug, which synthesize erythromycin esterases (Ounissi & Courvalin, 1985; Arthur, Autissier & Courvalin, 1986), and in strains of Staphylococcus hominis which have not been studied (Leclercq. Duval & Courvalin, 1987). Strains of S. epidermidis have been found to resist erythromycin by impermeability (Lampson, von David & Parisi, 1986). It is interesting to notice that strains displaying these phenotypes remain susceptible to spiramycin, which therefore constitutes a useful agent in the treatment of infections due to these types of microorganisms.

Conclusion "-!though it has been extensively studied since the early sixties, the mechanism of lction of spiramycin and other macrolides is still not clear. The most attractive lypothesis is that macrolides stimulate dissociation of peptidyl-tRNA from the 'ibosome during the elongation reaction, leading to the inhibition of protein synthesis. Resistance to spiramycin involves the synthesis of resistant ribosomes due to the )resence of methylases which modify the 23S ribosomal RNA.

References

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