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

Three types of macrolide antibiotics. The first-generation drug erythromycin A gave rise to a second generation of macrolides, including clarithromycin (6-O-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.

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 ( Moazed and Noller, 1987 ; Hansen et al., 1999a ; Xiong et al., 1999 ). A mutation at position 754 conferring mild drug resistance is indicated ( Xiong et al., 1999 ). 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) ( Gregory and Dahlberg, 1999 ).

(a) Structure of a 27-mer RNA, the smallest substrate that could be methylated (albeit poorly) by ErmE ( Vester et al., 1998 ). 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 ( Nielsen et al., 1999 ). 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 ( Villsen et al., 1999 ). 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 ( Vester et al., 1998 ), but the identities of individual bases here do not seem important provided their substitution does not unduly disrupt the stem structure ( Villsen et al., 1999 ). (d) Consensus of the new sequences that are methylated by ErmE when its fidelity for A2058 is reduced by removal of magnesium ions ( Hansen et al., 1999b ). 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.