Sequence diversity of apidaecin-like peptides arresting the terminating ribosome

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit bacterial translation.
• Apidaecin (Api), an 18-amino acid PrAMP, functions by binding to the ribosomal nascent peptide exit tunnel.
• Api traps release factors (RF1/RF2) and arrests ribosomes at stop codons, halting protein synthesis.

Purpose of the Study:

• To investigate the sequence variability of apidaecin that maintains its ribosomal translation inhibition activity.
• To identify novel PrAMP variants with potential therapeutic applications.

Main Methods:

• Screening of a large library (~350,000 variants) of synthetic mutant apidaecin genes expressed in bacterial cells.
• Application of orthogonal negative and positive selection strategies to identify functional variants.
• Assessment of antibacterial activity of selected multi-substituted apidaecin variants.

Main Results:

• Identification of multiple apidaecin variants with multiple substitutions that retain the ability to arrest ribosomes at stop codons.
• Demonstration of the crucial role of specific amino acid residues in apidaecin's function.
• Discovery of several synthesized multi-substituted apidaecin variants exhibiting enhanced antibacterial activity compared to wild-type apidaecin.

Conclusions:

• Specific amino acid residues are critical for apidaecin's ribosomal arrest function.
• A broad range of PrAMP sequences can target the terminating ribosome.
• Engineered apidaecin variants show promise as a starting point for developing new clinical antimicrobials.