Chapter 35 : Macrolide Resistance Conferred by Alterations in the Ribosome Target Site

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This chapter concentrates on resistance to a subset of antibiotics, the 14-membered ring macrolides, and considers their fate as effective antibacterial agents now that resistance is widespread among bacterial pathogens. The ribosome target site for macrolides lies within the 23S rRNA at the peptidyltransferase center of the 50S subunit. Shortly after the introduction of erythromycin in therapy in the 1950s, resistance to the drug became widespread in numerous pathogens. The degree of resistance that is conferred seems to depend on the proportion of ribosomes that contain a modification (or mutation) at position 2058. Recent technical advances (not the least being reverse transcription-PCR methods) have made it possible to identify rRNA mutations that arise in pathogens during macrolide therapy. Pathogens that attain macrolide resistance by spontaneous rRNA mutations generally contain only one or two operons. The occurrence of MLS resistance mutations is not limited to erythromycin derivatives nor to human pathogens but can occur in any species with a low copy number that is exposed to macrolides. The details of the molecular contacts made between the Erm methyltransferases and the conserved rRNA motif await resolution by X-ray diffraction or NMR techniques. It can be hoped that determination of the three-dimensional structure of this motif will facilitate the design of compounds that will act as specific and effective competitive inhibitors of Erm methyltransferases.

Citation: Douthwaite S, Vester B. 2000. Macrolide Resistance Conferred by Alterations in the Ribosome Target Site, p 431-440. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch35

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Image of Figure 1.
Figure 1.

Three types of macrolide antibiotics. The first-generation drug erythromycin A gave rise to a second generation of macrolides, including clarithromycin (6--methylerythromycin A). The third generation of drugs, the ketolides, include HMR 3647, which is presently undergoing clinical trials. The ketolides are characterized by a 3-keto group in place of the cladinose sugar residue. HMR 3647 also has an alkyl-aryl chain extending from a cyclic hydrazono-carbamate substitution at the 11/12 position of the lactone ring.

Citation: Douthwaite S, Vester B. 2000. Macrolide Resistance Conferred by Alterations in the Ribosome Target Site, p 431-440. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch35
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Image of Figure 2.
Figure 2.

Hairpin 35 in domain II (a) and the peptidyltransferase center in domain V (b) of 23S rRNA. Nucleotides at which chemical footprint effects were observed upon erythromycin binding are circled ( ). A mutation at position 754 conferring mild drug resistance is indicated ( ). A mutation in ribosomal protein L22, conferring erythromycin resistance, affects the accessibilities of G748 (the N7 position becomes more reactive to dimethyl sulfate) and T747 (becomes more reactive to carbodiimide) ( ).

Citation: Douthwaite S, Vester B. 2000. Macrolide Resistance Conferred by Alterations in the Ribosome Target Site, p 431-440. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch35
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Image of Figure 3.
Figure 3.

(a) Structure of a 27-mer RNA, the smallest substrate that could be methylated (albeit poorly) by ErmE ( ). The structure corresponds to a portion of 23S rRNA helix 73 (Fig. 2b) and the single-stranded region adjacent to A2058 (boxed). (b) Part of the structure of the 72-mer RNA used in the negative in vitro selection study ( ). The RNA was doped at 34 positions (uppercase nucleotides) and passed through several rounds of methylation and selection (see the text). Of 187 selected subclones, 43 had single-base substitutions that were limited to the 12 positions in boxes; Δ represents a single-nucleotide deletion. The relative effects of mutations at these positions on lowering the rate by ErmE methylation are indicated (wild type = 1). The lowercase nucleotides and the 5' and 3' sequences (not shown) were not doped and were used for amplifying and subcloning the selected RNA sequences. The shaded nucleotide is equivalent to A2058. (c) Summary of the results from a site-directed mutagenesis study on a domain V transcript of 23S rRNA and its methylation by ErmE ( ). The bases investigated by mutagenesis are boxed, and those most important for the Erm interaction are shaded. The nucleotides in the lower stem (from position 2611) are collectively essential for the methylation reaction ( ), but the identities of individual bases here do not seem important provided their substitution does not unduly disrupt the stem structure ( ). (d) Consensus of the new sequences that are methylated by ErmE when its fidelity for A2058 is reduced by removal of magnesium ions ( ). ErmE methylation occurs exclusively at adenosines (boxed), and these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060, and there are slight preferences for the nucleotides shown in lowercase. H, any nucleotide except G; N, any nucleotide.

Citation: Douthwaite S, Vester B. 2000. Macrolide Resistance Conferred by Alterations in the Ribosome Target Site, p 431-440. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch35
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