Mutations in 23S rRNA at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance

Abstract

The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid. Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C2055A-A2572U had remarkable synergistic effects on linezolid resistance relative to the effects of the corresponding single mutations. This study emphasizes that effects of rRNA mutations at the PTC are organism dependent. Moreover, the data show a nonpredictable cross-resistance pattern between linezolid, chloramphenicol, clindamycin, and valnemulin. The data underscore the significance of mutations at distal nucleotides, either alone or in combination with other mutated nucleotides, in contributing to linezolid resistance.

FIG. 1.

The positions of mutated 23S rRNA nucleotides relative to the linezolid binding site. (A) A cut view of the D. radiodurans 50S subunit showing linezolid (magenta) bound at the PTC and the peptide exit tunnel (PDB accession number 3DLL [49]). The image was made with the VMD software program (19) and contributed by Jacob Pøhlsgaard. (B) Secondary structure of the M. smegmatis 23S rRNA peptidyl transferase loop. The positions of mutated nucleotides are highlighted with colored circles, and the corresponding E. coli numbering is boxed in colored rectangles. The coloring of mutations indicates first-layer (blue), second-layer (green), third-layer (orange), and outer-layer (red) nucleotides with respect to linezolid. Wedges point to first-layer nucleotides that form the linezolid binding pocket. Nucleotides in bold type are those where a mutation(s) is identified to confer linezolid resistance in at least one organism (1, 14, 22, 30, 33, 43, 52, 53). (C to E) Close-up views of the linezolid binding site made with the PyMOL software program (7), where nucleotides are colored as described above. (C) The locations of mutated nucleotides with respect to bound linezolid. First-layer nucleotides are shown in surface representation. (D) The position of G2576 with respect to linezolid. (E) The positions of the nucleotides involved in the double mutations with respect to linezolid.