Resistance to linezolid caused by modifications at its binding site on the ribosome

Abstract

Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

Fig 1

(A) The chemical structure of linezolid. (B) The E. coli 70S X-ray structure with 16S rRNA in orange, 30S ribosomal proteins in yellow, 23S rRNA in gray, and 50S ribosomal proteins in blue (PDB file from reference 75). (C) A cutaway view of the large ribosomal subunit with a red circle at the PTC (PDB file from reference 77). A P-site-bound tRNA is shown in magenta, and the PTC and peptide exit tunnel are indicated. (D and E) Close-up views of the linezolid binding site made with the PyMOL software program. The coloring of nucleotides indicates first-layer (blue), second-layer (green), third-layer (orange), and outer-layer (red) nucleotides with respect to linezolid. (D) The locations of mutated nucleotides with respect to bound linezolid. First-layer nucleotides are shown in surface representation. (E) The position of G2576 with respect to linezolid. (F) Illustration of how parts of the L3 (in purple) and L4 (in green) ribosomal proteins extend toward the PTC where linezolid (in red) is bound. Selected amino acids are marked with the corresponding S. aureus numbering. The coordinates are from PDB file 3DLL (95).

Fig 2

Secondary structure of the peptidyl transferase loop of domain V of 23S rRNA (M. smegmatis sequence in E. coli numbering). The nucleotides that form the linezolid binding pocket are indicated with black triangles. Nucleotide positions where mutations confer linezolid resistance are marked with yellow circles. The nucleotides where mutations have a significant effect on linezolid MIC (>4-fold MIC increase) are in bold type, whereas those where mutations have a small to moderate effect on the linezolid MIC (4-fold or less MIC increase) are in regular type. The mutations and corresponding organisms are indicated with two-letter abbreviations: Ec (E. coli), Sa (S. aureus), Se (S. epidermidis), Sh (S. haemolyticus), Sp (S. pneumoniae), Es (E. faecalis), Em (E. faecium), Ms (M. smegmatis), Mt (M. tuberculosis), and Hh (H. halobium). Asterisks indicate mutations found in clinical isolates. Only mutations where some evidence or strong indication of the mutation-resistance relationship has been published are marked on the figure (8, 19, 30, 41, 47, 50, 56, 59, 60, 62, 66, 69, 73, 97, 99, 100).

Fig 3

The sequences of 50S ribosomal proteins L3 and L4 from Staphylococcus aureus MRSA252. The amino acids highlighted in purple and green correspond to those shown as balls in Fig. 1F. For the L3 sequence, A157 is the equivalent of E. coli L3 N149.