ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2005, p. 15531555 0066-4804/05/$08.00 0 doi:10.1128/AAC.49.4.15531555.2005 Copyright 2005, American Society for Microbiology.

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Vol. 49, No. 4

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Molecular Mechanisms by Which rRNA Mutations Confer Resistance to Clindamycin
Jacob Poehlsgaard,1* Peter Pster,2 Erik C. Bottger,2 and Stephen Douthwaite1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark,1 and Institut fur Medizinische Mikrobiologie, Universitat Zurich, Zurich, Switzerland2
Received 7 October 2004/Returned for modication 11 November 2004/Accepted 10 December 2004

The mechanisms by which rRNA mutations confer clindamycin resistance were examined in Mycobacterium smegmatis strains containing homogeneous populations of ribosomes with base substitutions at nucleotides A2058 and A2059. Computer graphic predictions based on structural studies correlate with the resistance phenotypes for six of seven strains with unique rRNA mutations. Resistance to antimicrobial agents in bacterial pathogens, including mycobacteria (4, 6), is a growing concern worldwide, and elucidation of the resistance mechanisms remains an important task. Many clinically relevant antibiotics, including the macrolide-lincosamide-streptogramin B (MLSB) drugs, target the bacterial ribosome (3, 7). Although the MLSB antibiotics are structurally diverse, they all bind to overlapping targets in the 50S subunit exit tunnel (1, 2, 8, 13), and their binding here is disturbed by mutations at key 23S rRNA nucleotides (14). Nucleotides A2058 and A2059, which are the main rRNA interaction sites for the lincosamide clindamycin (5), have been mutagenized in Mycobacterium smegmatis rRNA. Here, the resultant decreases in clindamycin susceptibility are compared with the molecular effects predicted from the crystal structure of clindamycin bound to the 50S subunit (13). Strains of M. smegmatis SMR5 rrnB::aph with homogeneous populations of mutant ribosomes were created by transformation with plasmid pMV361 (10) carrying mutagenized fragments of the 23S rRNA gene (12). RecA-mediated conversion of the single chromosomal rRNA gene (11) gives rise to cells that exclusively express mutant 23S rRNA. Drug-resistant recombinants with mutations at A2058G, A2058C, A2058U, A2059G, A2059C, A2058G/A2059G, and A2058C/A2059C were selected and sequenced (10), and their susceptibilities to clindamycin were determined in microtiter plates (3). In brief, freshly grown cultures of M. smegmatis were diluted to an optical density (A600) of 0.025 in Luria-Bertani medium and incubated in the presence of twofold serial dilutions of clindamycin (Sigma). The MICs (Table 1) are the lowest drug concentrations at which no growth was observed by 72 h of incubation. The ribosomal subunit was visualized and rendered from the crystallographic coordinates (13) by using PovRay and MolMol (9). van der Waals (VDW) overlaps were visualized by performing an intersection operation in PovRay. Mutant structures were adapted from the Deinococcus radiodurans 50S ribosomal subunit crystallographic coordinates (13) by superimposing the relevant bases over the native D. radiodurans residues, using the original orientation. The A-to-G transition at position 2058 results in high-level clindamycin resistance; in contrast, the corresponding transition at position 2059 confers only slight resistance (Table 1). At 23S rRNA positions 2058 and 2059, transversion of A to C surprisingly has only a marginal effect on drug susceptibility, whereas the 2058 A-to-U transversion results in signicant drug resistance. According to the crystal structure (13), clindamycin makes extensive electrostatic interactions with the backbone and bases of the rRNA region within its binding site. However, the main interaction occurs via a hydrogen bond between the N-1 of A2058 and the 2-OH of clindamycin (Fig. 1A). Other interactions, such as at A2059 (Fig. 1B), appear to be of a weaker electrostatic nature due to the relatively large distances between the donors/acceptors at the N-1 and N-6 of the base and the 2-OH and 3-OH of clindamycin, which are too great to support authentic hydrogen bonds. A single substitution at A2058G or A2059G seems to confer resistance mainly by the 2-amino group of the guanine base sterically hindering placement of the drug into its binding site. A larger overlap of the VDW radii is predicted to occur with the A2058G mutation than with A2059G (Fig. 1), correlating well with the MICs observed for these two mutant strains

TABLE 1. MICs for the M. smegmatis strains examined in this study

Genotype MIC ( g/ml)

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Phone: (45) 6550 2370. Fax: (45) 6550 2467. E-mail: jacobp@bmb.sdu.dk. 1553

Wild-type.......................................................................................... 16 2058G ............................................................................................... 1,024 2058C................................................................................................ 64 2058U ............................................................................................... 256 2059G ............................................................................................... 64 2059C................................................................................................ 32 2058C/2059C .................................................................................... 256 2058G/2059G ................................................................................... 1,024

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FIG. 1. (A) Interaction of 23S rRNA nucleotides A2058 and A2059 (Escherichia coli numbering) with clindamycin (magenta). The rRNA backbone is in grey, riboses are highlighted with yellow windows, and adenines are highlighted with blue windows. The smallest drug-rRNA distances are indicated. (B and C) Clindamycin interactions with A2058U (B, blue window) and with A2058C (C, green window) were predicted to have the same geometry. (D) Steric VDW overlap (red) predicted to occur after substitution of A2058G and A2059G (green windows). This structure is viewed from above relative to the rst three panels. Hydrogens are not shown but were digitally added for VDW overlap calculations.

(Table 1). Remodeling the crystal structure to include the structurally smaller pyrimidine bases at 2058 or 2059 creates new hydrogen acceptors and donors, although the drug-target distance appears to be too large for strong interaction. Accordingly, the A2058U mutation (Fig. 1B) confers appreciable resistance to clindamycin, while A2059C gives very little resistance. Surprisingly, the A2058C mutation (Fig. 1C) has only a marginal effect on the MIC, and this is hard to reconcile with the molecular model. Ad hoc rationalizations, such as a local structural perturbation or the intervention of a water molecule or metal ion to bridge the drug and the binding site, can be invoked to explain this disparity, but none of these is wholly satisfactory. The double mutations confer substantial resistance to clindamycin and t well with the predictions from the molecular model. The double purine mutation A2058G/A2059G confers high-level resistance, and the mechanism behind this can be seen from the dual steric obstructions illustrated in Fig. 1D. The double pyrimidine mutation A2058C/A2059C also confers substantial resistance, although the MIC (Table 1) suggests that some residual clindamycin binding takes place. Thus, it appears that clindamycin is accommodated better by a binding site that is too loose (2058C/2059C) than one that is too tight (2058G/2059G). The double pyrimidine mutant presumably presents a smooth, albeit somewhat sunken, interaction surface where weak interactions with the drug are possible. In conclusion, the empirical resistance data presented here for rRNA mutants are by and large consistent with predictions that can be made from crystallographic data on the wild-type ribosome-drug complex. However, results can sometimes be counterintuitive (as in the case of the single A2058C mutant). This study highlights the need for gathering resistance data in organisms with similar genetic backgrounds and a homoge-

neous population of mutant ribosomes. Strains with homogeneous ribosomes facilitate a focused interpretation of molecular data and are without the unassigned variations that occur in random isolates with unknown genetic backgrounds and heterogeneous ribosome populations. It is predicted that as the sum of clear-cut data increases, better interpretations of the cause-and-effect relationship in mutational resistance will be possible and will lead to model systems that correctly predict the antibiotic resistance phenotypes conferred by rRNA mutations.
This study was supported in part by grants from the Swiss National Science Foundation (to E.C.B.), the European Commissions Fifth Framework Program (grant QLK2-CT2000-00935), and the Nucleic Acid Centre of the Danish Grundforskningsfond (to S.D.).
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