Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates

Abstract

SummaryThe pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA ( Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 ( Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

“…** Pleuromutilin Resistance. The slow stepwise manner of the appearance of pleuromutilin resistance is consistent with the finding that several pleuromutilin resistant strains include more than one mutation (31). Furthermore, the majority of the nucleotides that are mutated for acquiring resistance do not directly interact with the bound compound, consistent with the essentiality of the interacting nucleotides for ribosomal function.…”

“…Furthermore, the addition of hydroxyl group at C2 in SB-571519 did not result in remarkably different binding orientation. Hence, it is not surprising that many pleuromutilin resistance mutations, identified in animal pathogens, involve nucleotides residing in the vicinity of the mutilin core, namely G2032, C2055, G2447, C2499, A2572, and U2504 (31). A similar effect has been previously observed for several mutations in the nearby protein L3.…”

“…It was previously suggested that mutations in ribosomal protein L3 reduce bacterial susceptibility to pleuromutilins by indirect influence of the binding pocket conformation (22,30,31). Unambiguous tracing of protein L3 Arg-144 could be performed owing to the high quality of the electron density map of SB-571519 complex, as it was constructed from a complete dataset collected form a single crystal with significant redundancy ( Table 1), showed that it extends toward the PTC, and interacts electrostatically with U2506 phosphate, but does not make any contacts with the pleuromutilin compound (Fig.…”

“…These interactions may account for the protection of N3 in U2506 and U2585 on chemical footprinting by 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate in Escherichia coli and B. hyodysenteriae in the presence of tiamulin, valnemulin, pleuromutilin, and the carbamate derivative SB-264128 ( Fig. 1) (20,(30)(31)(32). It is worth noting that U2506 and U2585 motions are not conditional for pleuromutilins binding because slightly different location of the compound can partially compensate for it, as observed for tiamulin, an additional C14-sulfanyl acetate derivative (22).…”

“…Although different in sequence, in all known structures of the large ribosomal subunit (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) protein L3 penetrates deeply toward the PTC (Fig. 4 a-c).…”