Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance
Category
Published on
Type
journal-article
Author
Kaitlyn Tsai and Vanja Stojković and Lianet Noda-Garcia and Iris D Young and Alexander G Myasnikov and Jordan Kleinman and Ali Palla and Stephen N Floor and Adam Frost and James S Fraser and Dan S Tawfik and Danica Galonić Fujimori
Citation
Tsai, K., Stojković, V., Noda-Garcia, L., Young, I. D., Myasnikov, A. G., Kleinman, J., Palla, A., Floor, S. N., Frost, A., Fraser, J. S., Tawfik, D. S., & Fujimori, D. G. (2022). Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance. ELife, 11. CLOCKSS. https://doi.org/10.7554/elife.70017
Abstract
Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.
