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

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• Antibiotic resistance is a growing threat, often mediated by modifications to the bacterial ribosome.
• The Cfr enzyme confers resistance to multiple antibiotic classes by methylating ribosomal RNA (rRNA) at position A2503 (m ⁸ A2503).
• Mechanisms by which bacteria modulate Cfr activity and the structural basis of m ⁸ A2503-mediated resistance remain poorly understood.

Purpose of the Study:

• To investigate how bacteria adapt Cfr-mediated rRNA methylation under antibiotic pressure.
• To elucidate the structural consequences of m ⁸ A2503 modification on antibiotic binding sites.
• To inform the development of novel antibiotics effective against Cfr-mediated resistance.

Main Methods:

• Directed evolution of the Cfr enzyme under antibiotic selection.
• Analysis of Cfr variants for enhanced rRNA methylation and antibiotic resistance.
• Cryo-electron microscopy (cryo-EM) to determine the structure of the modified ribosome.

Main Results:

• Directed evolution yielded Cfr variants with increased methylation efficiency through improved expression and stability.
• Mechanisms for enhanced Cfr activity involve both transcriptional and post-transcriptional regulation.
• A high-resolution cryo-EM structure revealed the molecular details of how m ⁸ A2503 alters the antibiotic binding pocket.

Conclusions:

• Bacteria can enhance Cfr methylation levels to adapt to antibiotic stress, suggesting potential clinical relevance.
• The determined structure provides a molecular explanation for broad-spectrum antibiotic resistance conferred by Cfr.
• This structural insight is crucial for designing next-generation antibiotics capable of overcoming Cfr-mediated resistance.