Ribosomal and non‐ribosomal resistance to oxazolidinones: species‐specific idiosyncrasy of ribosomal alterations

Abstract

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.

Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

“…6C) and prevent drug binding (56). The G2447U mutation, which renders M. smegmatis, M. tuberculosis (57), and E. coli (58) linezolid-resistant, has been suggested to be involved in functional differences between Gram-positive and Gram-negative bacteria (59). Of interest, significant structural similarity was observed in this region of ribosomes from T70S, D50S, E70S, and SA50S, which represent both Gram-positive and Gram-negative species.…”

Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis

“…However, subsequent crystallographic studies of the first clinically approved ketolide telithromycin bound to the bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) ribosome showed the placement of the 11,12 side chain in a position that was hardly compatible with rRNA protections and mutations observed in the E. coli ribosome (3,43). Furthermore, the orientation of the alkyl-aryl side chain differed significantly between the reported D. radiodurans and H. marismortui structures, conforming to the general notion of idiosyncratic interactions of antibiotics with ribosomes of various bacterial species (36,47) and therefore leaving open the question of how the drug may bind to the ribosomes of pathogenic bacteria targeted by ketolide antibiotics.…”

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning

“…5D). Linezolid can bind to the free A site of an empty 50S subunit (31) or Pi state ribosome (15,20) and stabilize U2585 in a nonproductive conformation (Fig. 5E).…”

“…4F). Thus, it is unclear why in some systems oxazolidinones have little inhibitory effect on fMetpuromycin formation (23,31,32), whereas in others an obvious effect is observed (15,20,22). Because the drug does not encroach on the P site (Fig.…”

“…1A and supporting information (SI) Fig. S1] (14)(15)(16)(17)(18)(19). Although translation is recognized as the main target of oxazolidinones, there still remains some controversy as to the step of inhibition.…”