Phylogenetic sequence variations in bacterial rRNA affect species-specific susceptibility to drugs targeting protein synthesis

Abstract

Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket.

Fig. 1.

(a) Structures of erythromycin (blue) (PDB accession code 3OHJ) and telithromycin (pink) (PDB accession code 3OI3) bound to the Thermus thermophilus ribosome. Nucleotides investigated in this study are indicated in black. (b) Secondary structure of domain V of the 23S rRNA and sequence conservation in eubacteria. rRNA residues are numbered according to their homologous positions in E. coli 23S rRNA. Phylogenetic sequence variations analyzed in this study are highlighted in colors: base pair 2057 · 2611 is represented in blue and the adenine at position 2058 in red. (c) Detailed view of the hydrogen bond interaction between N-1 of A2058 and the 2′-OH moiety of erythromycin's deosamine sugar (the desosamine sugar is highlighted, and the hydrogen bond interaction is shown as a red dotted line). The hydrogen bond interactions between A2057 and U2611 are indicated by gray dotted lines.

Fig. 2.

(a) Secondary structure of 16S rRNA helix 44 decoding site and sequence conservation in eubacteria. rRNA residues are numbered according to their homologous positions in E. coli 16S rRNA. Phylogenetic sequence variations analyzed in this study are highlighted in colors: 1409, 1410, 1411, 1489, and 1490 in blue; the adenine at position 1408 in red; and the guanine at 1491 in green. (b) Structures of neomycin (blue) (PDB accession code 2QOY) and gentamicin (pink) (PDB accession code 2QB9) bound to the Escherichia coli A site. Ring 1 of the aminoglycosides is highlighted in yellow, nucleotides investigated in this study are numbered in black, and the hydrogen bond interactions between C1409 and G1491 are indicated by gray dotted lines. (c) Detailed view of the hydrogen bond and stacking interactions of neomycin's ring 1 with A1408 (shown as red dotted lines) and G1491, respectively. The gray dotted lines indicate hydrogen bonding between C1409 and G1491.