Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

Abstract

The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5-and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit.

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…Meanwhile, it has been reported that positions G748, A751 and A752 located within the loop of helix 35 of domain II are involved in tylosin-23S rRNA interactions in Gram-positive microorganisms (Hansen et al, 2002;Liu & Douthwaite, 2002a;Poehlsgaard & Douthwaite, 2005). Similarly, the role of position G745 in the tylosin-ribosome interactions in E. coli has been described (Liu & Douthwaite, 2002b), while positions G748 and A752 are reportedly involved in the interactions between ketolides and ribosomes (Novotny et al, 2004).…”

“…In the case of tylosin, the methylation at position A2058 does not by itself result in the development of resistance, but rather the concomitant methylation of other 23S rRNA positions, such as G745 or G748 in Gram-negative and Gram-positive microorganisms, respectively is needed (Liu & Douthwaite, 2002a, b). Thus, among others, Erm(32) (formerly TlrB or RlmA II ) encoded in the chromosome of members of the Streptomyces genus has been described to be able to methylate position G748 (Liu & Douthwaite, 2002a). In E. coli the presence of a related methylase, called RlmA I (formerly RrmA) has been described which acts by methylating position G745 (Liu & Douthwaite, 2002b).…”

“…In E. coli the presence of a related methylase, called RlmA I (formerly RrmA) has been described which acts by methylating position G745 (Liu & Douthwaite, 2002b). Nonetheless, when Erm(32) or RmlA I has been cloned in E. coli no effect was observed on the macrolide MIC, including tylosin (Liu & Douthwaite, 2002a). However, in both cases, the concomitant monomethylation of A2058, which by itself is unable to affect tylosin activity, leads to the development of tylosin resistance (Liu & Douthwaite, 2002a).…”

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

“…4B) nor disrupt base pairing with U2528, suggesting that methylation at this position indirectly confers resistance by inducing local conformational changes, possibly during ribosome assembly. We note that both AviRa and AviRb are required to obtain full protection against the AVI (31), suggesting that they function in a synergistic manner, similar to the methyltransferases that cause resistance to tylosin (32). The EVN methyltransferase EmtA, which was identified on a plasmid-borne insertion element in EVN-resistant E. faecium strains (isolated from animals given AVI as a growth promotant), was shown to methylate G2470 of H89 (13).…”

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

“…Most of these compounds inhibit cell growth by interfering with peptide bond formation (15). The second major antibiotic binding site in the large ribosomal subunit is located at the upper segment of the nascent peptide exit tunnel (NPET), adjacent to the PTC, and is used by macrolides and type B streptogramins (3,7,(16)(17)(18)(19)(20)(21). Binding to this site impedes progression of the nascent proteins toward the tunnel exit.…”