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.The bacterial ribosome is a target for many antibacterial agents that interfere with protein synthesis, such as aminoglycosides, macrolides, ketolides, oxazolidinones, and lincosamides (23). These compounds target different steps in translation, including decoding, peptide bond formation, and translocation (33,37,40). While different classes of antibiotics bind to different ribosomal regions and interfere with different steps in translation, they all interact directly with rRNA nucleotides at or near functionally important sites (38, 39). These rRNA residues typically show high phylogenetic sequence conservation within bacteria. It is largely unclear whether the minor sequence variations present in the bacterial drug binding sites affect antibiotic susceptibility and/or resistance development.Structures of antibiotics bound to the ribosome have been resolved primarily with extremophiles such as Thermus thermophilus, Deinococcus radiodurans, or Haloarcula morismortui (2, 9, 30). Most genetic data, however, have been generated with Escherichia coli and Mycobacterium smegmatis (4-6, 10, 14, 15, 20-22, 26, 28, 29, 32). It has still to be established whether the conclusions drawn from diverse model organisms hold true for other bacterial clades as well. To address this question, we investigated rRNA alterations corresponding to phylogenetic sequence variations that are found in bacteria and which are located in two major drug binding sites, the 23S rRNA peptidyltransferase region and the 16S rRNA-decoding region. These two regions are targeted by different classes of antibiotic compounds: macrolides/ketolides (23S rRNA) and aminoglycosides (16S rRNA) (23, 37).Macrolide/ketolide antibiotics are a diverse class of naturally occurring and synthetic compounds based on a polyketide macrolactone ring substituted with one or more nonnitrogenous and/or amino sugar moieties (27). These compounds exert their inhibitory effect on protein synthesis by binding to the opening of the ribosomal polypeptide exit tunnel to obstruct elongation of the nascent polypeptide chain (30, 34). Aminoglycos...

Species identification of Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii using rpoB and hsp65, and susceptibility testing to eight antibiotics

“…The first comprises mutations in the drug-binding pocket, in particular at nucleotide positions 2058 and 2059, of the bacterial 23S rRNA gene. [25][26][27][28] Corresponding mutations confer high-level resistance to clarithromycin (MIC >256 mg/l) in bacterial species with a limited number of chromosomal rRNA operons, including Mycobacterium chelonae and M. abscessus. [29][30][31][32] The second mechanism is conferred by a class of genes coding for inducible erythromycin ribosomal methylases (Erm), which monoor dimethylate the adenine at position 2058 of the 23S rRNA.…”

The macrolide antibiotic renaissance

“…This unusual behaviour is explained by the 2057-2611 base pair, which is part of the cradle housing the lactone ring of the macrolide (Pfister et al, 2005;Kannan and Mankin, 2011). The 2057-2611 base pair is conserved across bacteria (Akshay et al, 2011), and the polymorphism of this base pair (i.e. the presence of G-C vs. A-U) determines the ketolide susceptibility of A2058G mutants (Pfister et al, 2005).…”

New trends in the use of aminoglycosides

“…56 Other residues, such as the purine/pyrimidine switches 1411•1489 and 1410•1490, were reported to have minimal effect on MIC values while 1409•1491 was found to be critical for AG susceptibility. 69 …”