Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance.

Kaitlyn Tsai,Danica Galonić Fujimori,Ali Palla,Jordan Kleinman,James S Fraser,Alexander G Myasnikov,Dan S Tawfik,Stephen N Floor,Iris D Young,Vanja Stojković,Adam Frost,Lianet Noda-Garcia

doi: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.

Highlights

A large portion of clinically relevant antibiotics halt bacterial growth by binding to the ribosome and inhibiting protein synthesis (Tenson and Mankin, 2006; Wilson, 2009; Arenz and Wilson, 2016)
The ribosomal RNA (rRNA)-methylating enzyme Cfr modifies an adenosine nucleotide located within the peptidyl transferase center (PTC), a region of the ribosome essential for catalyzing peptide bond formation and a common target for antibiotics (Schwarz et al, 2000; Kehrenberg et al, 2005)
Over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic

Summary

Introduction

A large portion of clinically relevant antibiotics halt bacterial growth by binding to the ribosome and inhibiting protein synthesis (Tenson and Mankin, 2006; Wilson, 2009; Arenz and Wilson, 2016). Since antibiotic binding sites are primarily composed of ribosomal RNA (rRNA), rRNA-modifying enzymes that alter antibiotic binding pockets are central to evolved resistance (Vester and Long, 2013; Wilson, 2014). Due to the proximal location of A2503 to many antibiotic binding sites, introduction of a single methyl group is sufficient to cause resistance to eight classes of antibiotics simultaneously: phenicols, lincosamides, oxazolidinones, pleuromutilins, streptogramin A (PhLOPSA), in addition to nucleoside analog A201A, hygromycin A, and 16-membered macrolides (Long et al, 2006; Smith and Mankin, 2008; Polikanov et al, 2015). Direct evidence for how m8A2053 alters antibiotic binding sites to inform the design of next-generation molecules that can overcome Cfr resistance is lacking. The obtained structural insights provide a rationale for how m8A2503 causes resistance to ribosome antibiotics

Results

Discussion

Materials and methods

Funding Funder