Abstract
Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome.
Highlights
The ribosomal peptidyltransferase active site, the site of peptide bond formation during protein synthesis, is the target of a number of structurally unrelated classes of antibiotics, and mutations conferring resistance to many of these drugs have been identified in the genes encoding 23S rRNA or ribosomal proteins from a wide range of organisms
Based on chemical footprinting of 23S rRNA in Escherichia coli ribosomes, pleuromutilins bind to the PTC and compete for binding with the 16-atom macrolide and peptidyltransferase inhibitor carbomycin [6], and thereby inhibit peptide bond formation [7]
Tiamulin interacts exclusively with rRNA residues, ribosomal protein uL3 is in close proximity to the tiamulin binding site without making direct contact with the drug
Summary
The ribosomal peptidyltransferase active site ( referred to as the PTC), the site of peptide bond formation during protein synthesis, is the target of a number of structurally unrelated classes of antibiotics, and mutations conferring resistance to many of these drugs have been identified in the genes encoding 23S rRNA or ribosomal proteins from a wide range of organisms. Advances in structural biology have led to numerous high-resolution structures of ribosomes in complex with antibiotics, providing a wealth of information about the mechanisms of action of these important drugs and facilitating predictions about mechanisms of antibiotic resistance reviewed by [1,2,3]. Based on chemical footprinting of 23S rRNA in Escherichia coli ribosomes, pleuromutilins bind to the PTC and compete for binding with the 16-atom macrolide and peptidyltransferase inhibitor carbomycin (though not with the 14-atom macrolide erythromycin) [6], and thereby inhibit peptide bond formation [7]. Tiamulin interacts exclusively with rRNA residues, ribosomal protein uL3 is in close proximity to the tiamulin binding site without making direct contact with the drug
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