Investigating the mechanism of action of ribosome-targeting antibiotics and resistance by RNA methylation

The ribosome is a major antibiotics target, and a large portion of transitional inhibitors bind to the peptidyl transferase center (PTC). A prominent form of resistance to PTC-targeting antibiotics, including the “last-resort” antibiotic linezolid, is through acquisition the Cfr enzyme which methylates a PTC nucleotide to form m8A2503. To understand how bacteria adapt Cfr resistance under antibiotic pressure, we performed directed evolution of Cfr to generate variants with increased resistance via improved Cfr expression and stability. We used an evolved variant with superior methylation activity to obtain a 2.2 Å cryo-EM structure of a Cfr-methylated ribosome, revealing that the Cfr modification directly interferes with antibiotic binding. Building upon this knowledge, we sought to understand how the linezolid derivative, radezolid, overcomes Cfr-mediated resistance. We discover that linezolid and radezolid have similar context-specificity, preferring to inhibit translation with a penultimate alanine in the nascent peptide. The obtained high resolution cryo-EM structures of the antibiotic-stalled ribosome complexes reveal that the alanine forms a favorable interaction with the antibiotic. Furthermore, the determined structure of radezolid in complex with a Cfr-modified ribosome indicates that radezolid overcomes resistance through interactions with its extra ring system, forcing m8A2503 to tilt away to accommodate the antibiotic. Together, these findings identify strategies that boost Cfr methylation and provide molecular rationale for how second-generation antibiotics can overcome Cfr-mediated resistance