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

Abstract

Many clinically useful antibiotics inhibit the bacterial ribosome. The ribosomal RNA-modifying enzyme Cfr methylates an adenosine (m8A2503) in the peptidyl transferase center and causes cross-resistance to several classes of antibiotics. Despite the prevalence of this mode of resistance, mechanisms of adaptation to antibiotic pressure that exploit ribosome modification by Cfr are poorly understood. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. To address these questions, we evolved Cfr under antibiotic selection to generate variants that confer increased resistance and methylation of rRNA, provided by enhanced Cfr expression and stability. Using a variant which achieves near-stoichiometric methylation, we determined a 2.2Å cryo-EM structure of the Cfr-modified ribosome, revealing the molecular basis for resistance and informing design of antibiotics that overcome Cfr resistance.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

“…A prevalent resistance mechanism to LZD identified in multiple clinical isolates worldwide [22][23][24][25][26][27][28] involves methylation of rRNA by the Cfr enzyme, which adds a methyl group at the C8 atom of A2503 (m 8 A2503) in 23S rRNA [29][30][31][32][33] . The Cfr modification disrupts LZD binding to the ribosome by introducing a steric clash between the installed methyl mark and the C5 group of the antibiotic.…”

“…To perform in vitro assays, we expressed Cfr in E. coli and isolated ribosomes with near-complete methylation of m 8 A2503 as described previously 33 . As expected, RZD retains activity against the m 8 A2503 ribosomes.…”

“…Cfr-modified 70S ribosomes were prepared using previously published protocol with modification 33 . In short, E. coli BW25113 expressing the evolved variant CfrV7 were grown to an OD 600 of ~0.7 in LB media containing ampicillin (100 µg/mL) and AHT inducer (30 ng/mL) at 37°C.…”