Radical SAM-dependent methylation in antibiotic resistance

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PROJECT SUMMARY More than 40% of clinically used antibiotics act by binding to the ribosome and inhibiting protein synthesis. One of the major mechanisms of resistance to these antibiotics results from the modification of the ribosome catalyzed by Cfr, an enzyme encoded by chloramphenicol-florfenicol resistance gene. By methylating the C8 position of a conserved adenosine nucleotide in the peptidyl transferase center of the bacterial ribosome, this enzyme confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutillins, streptogramin A, hygromycin A, nucleoside analog A201A and 16-member macrolides. This broad cross-resistance is unique to Cfr, and represents a major clinical challenge, one that is further exacerbated by the presence of cfr on mobile genetic elements, low fitness cost of its acquisition and its broad geographic distribution, as well as the ability to cause resistance in both gram-positive (eg, methicillin-resistant S. aureus) and gram-negative bacteria (eg, pathogenic E. coli). Cfr family is represented by over 600 unique sequences, with some member of the family sharing only ~50% sequence identity with the commonly investigated Cfr(A) enzyme from a clinical MRSA isolate. To date, only a handful of Cfr enzymes have been functionally characterized. Recent work on the structural basis of inhibition of translation by chloramphenicol and linezolid, an oxazolidinone antibiotic, shows that both antibiotics inhibit protein synthesis by binding to the ribosome-nascent peptide complexes containing specific nascent peptide residues. Sequence-specific stalling mechanisms have been exploited in nature to regulate inducibility of antibiotic resistance genes. Since cfr is often accompanied by upstream elements that may regulate its expression, we will investigate if antibiotic-induced ribosome stalling mechanisms may be involved in regulation of the expression of cfr resistance genes. Using directed evolution under antibiotic selection, we have generated variants of Cfr with improved antibiotic resistance properties. By improving enzyme expression and stability, these enzyme variants increase ribosomal RNA methylation, leading to an increase in the proportion of the ribosomes that carry the protective modification. Improved methylation of the ribosome has enabled structural determination of the Cfr-modified ribosome, which we achieved using cryo-electron microscopy. The directed evolution mutants also provide a roadmap for our future efforts to functionally annotate additional putative members of the vast and sequence-diverse Cfr enzyme family. This will be achieved through in vitro reconstitution and in vivo validation of methylation of the conserved adenosine nucleotide. Additionally, we will deploy an innovative strategy that relies on mechanism-based crosslinking of Cfr with its substrates and next-generation sequencing of crosslinked RNAs to identify, with nucleotide resolution, the sites of RNA methylation. Together, these studies have a potential to de-orphan additional Cfr enzymes and lead to identification of new substrates and biological functions of this protein family.