Stepwise Binding of Tylosin and Erythromycin to Escherichia coli Ribosomes, Characterized by Kinetic and Footprinting Analysis

Πετρόπουλος, Αλέξανδρος Δ.; Kouvela, Ekaterini C.; Dinos, George P.; Kalpaxis, Dimitrios L.

doi:10.1074/jbc.m708371200

Cited by 27 publications

(43 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…5B), illustrating that the U2609C mutation does not perturb the binding of erythromycin. This is in excellent agreement with resistance and antibiotic binding data published previously (11), as well as a more recent study showing that the U2609C mutation does not significantly change the kinetics of erythromycin binding to ribosomes (25). Collectively, these results suggest that although K-1325 interacts with domain II of the 23S rRNA like other ketolides, such as telithromycin, the differences in footprinting patterns indicate that the mode of interaction differs.…”

Section: Figsupporting

confidence: 81%

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

Kouvela

Kalpaxis

Wilson

et al. 2009

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin-resistant strains, while maintaining their activity against erythromycin-susceptible ones. In this study, we present a new ketolide, K-1325, that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies, K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild-type Escherichia coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the alkyl-aryl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increased affinity of U2609C mutant ribosomes for the drug and rationalizing the enhanced effectiveness of this new ketolide.

show abstract

Section: Figsupporting

confidence: 81%

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

Kouvela

Kalpaxis

Wilson

et al. 2009

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…1D). The K i values were within the range of K D values (Ϸ10-100 nM) measured for erythromycin binding to ribosomes containing wild-type L22 or ⌬MKR-L22 (21)(22)(23)(24). At the highest concentrations of erythromycin tested, no translation by ⌬MKR ribosomes was detected (Fig.…”

Section: ⌬Mkr Ribosomes Are Inhibited By Erythromycin In Vitromentioning

confidence: 76%

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (⌬MKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ⌬MKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ⌬MKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.ermC ͉ erythromycin ͉ ribosome ͉ TolC M any antibiotics inhibit bacterial protein synthesis. Understanding how microbes become antibiotic resistant is important both for developing effective treatment regimens and designing new therapeutics. Macrolides consist of a 14-to 16-member lactone ring with different appended sugars and comprise a key group of inhibitors of bacterial translation (1, 2). The inhibitory activity of macrolides, including erythromycin, depends on binding to a site near the polypeptide exit tunnel of the large ribosomal subunit (3, 4). Because macrolides do not bind to ribosomes with an occupied exit tunnel and cause the synthesis of 2-10 residue peptides in translation assays in vitro, it has been proposed that drug binding physically blocks elongation of nascent proteins beyond this size (5-7).Some macrolide-resistance mutations alter the ribosomal target site and prevent binding (4,8). Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8-10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11-13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2). When binding has been measured, ribosomes with macrolideresistant alterations in L22 bind erythromycin with near wild-type affinity (8,10,12,15).In a crystal structure of the E. coli ribosome, the MKR sequence is part of an extended L22 loop, which together with a similar loop in protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A) (16, 17). Cryo-EM studies initially revealed a widened exit tunnel in E. coli ⌬MKR ribosomes (18). This loop is also displaced to create an expanded tunnel in structures of ⌬MKR ribosomes from Thermus thermophilus and Haloarcula marismortui (4, 19). These results explain the altered chemical reactivity in E. coli ⌬MKR ribosomes of 23S-RNA bases (13). Together,...

show abstract

“…The similarly structured azithromycin binds in a two-step process (17) that could thus involve transient interactions with nucleotides other than those contacted in the crystal structure (1). Tylosin also binds to its ribosomal site in a two-step process (16), and the methylations at G748 and A2058 probably interfere with this process prior to impeding the final orientation of the drug in its binding site (9). In the case of tildipirosin, a 23-piperidine replaces the 23-mycinose of tylosin and tilmicosin (Fig.…”

mentioning

confidence: 99%

Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

Andersen

Poehlsgaard

Warrass

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC 50 ], 0.23 ؎ 0.01 M) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site. Recent approval has been given in Europe and the United States for the use of tildipirosin (20,23-dipiperidinyl-mycaminosyltylonolide; Zuprevo) in combating bovine and swine respiratory tract infections. Tildipirosin is a derivative of the natural compound tylosin with two piperidine rings and no mycarose sugar (Fig. 1). Despite its wide use as a veterinary macrolide, tylosin is not particularly effective at penetrating the outer membrane of Gram-negative pathogens (Table 1), and the substitutions in tildipirosin were made to improve efficacy against Mannheimia haemolytica and Pasteurella multocida, which are the two main etiological agents of bovine respiratory disease (8,22). Tildipirosin has additionally proven effective against Histophilus somni, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, and Haemophilus parasuis (6), which can also be associated with animal respiratory diseases. Different arrangements of hydrophobic and basic substituents are present in an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyltylonolide; Micotil) and the 15-membered triamilide tulathromycin (Draxxin) (Fig. 1), which are used for similar indications.Here we quantified the inhibitory activity of tildipirosin (MSD Animal Health) on protein synthesis and compare it with tylosin, tilmicosin (Sigma), and tulathromycin (extracted from Draxxin [Pfizer]). The concentration of each macrolide that inhibits 50% of protein synthesis (IC 50 ) was determined in an in vitro transcription/translation assay, and the effects of methylations and mutations at rRNA nucleotides within the macrolide binding site were evaluated in cell cultures. The data have been visualized using computationally calculated models of the binding site and reveal subtle differences in macrolide contacts, indicating how changes in the rRNA target have distinct effects on drug efficacy.An in vitro transcription/translation system based on cell extracts containing susceptible, wild-type Escherichia coli ribosomes (Promega) was adapted to translate the 27-kDa green fluorescent protein (GFP). GFP mRNA was transcribed from 3 g of plasmid pIVEX (Roche) in 50 l of 100 mM piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES)-KOH (pH 7.9), 5 mM MgCl 2 , 0.5 mM CaCl 2 , 100 mM KCl, 5 mM NH 4 Cl, 1 mM dithiothreitol, and 1 mM spermidine and translated to produce [35 S]methioninelabeled protein. Synthesis was followed over 155 min with macrolide an...

show abstract

Stepwise Binding of Tylosin and Erythromycin to Escherichia coli Ribosomes, Characterized by Kinetic and Footprinting Analysis

Cited by 27 publications

References 46 publications

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

Contact Info

Product

Resources

About