Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes

• Published in: Biochemistry (Easton) Vol. 59; no. 36; pp. 3380 - 3391
• Main Authors: Muthunayake, Nisansala S, Islam, Rabiul, Inutan, Ellen D, Colangelo, Wesley, Trimpin, Sarah, Cunningham, Philip R, Chow, Christine S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.09.2020
• Subjects: Anti-Bacterial Agents - pharmacology; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - genetics; Antimicrobial Cationic Peptides - metabolism; Base Sequence; Escherichia coli - drug effects; Escherichia coli - growth & development; Escherichia coli - metabolism; Microbial Sensitivity Tests; Mutation; Pore Forming Cytotoxic Proteins - pharmacology; Ribosomes - chemistry; Ribosomes - metabolism; Sequence Homology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.0c00600

Peptides have important biomedical applications, but poor correlation between in vitro and in vivo activities can limit their development for clinical use. The ability to generate peptides and monitor their expression with new mass spectrometric methods and biological activities in vivo would be an advantage for the discovery and improvement of peptide-based drugs. In this study, a plasmid-based system was used to express the ribosome-targeting peptide oncocin (19 amino acids, VDKPPY­LPRPRP­PRRI­YNR) and to determine its direct antibacterial effects on Escherichia coli. Previous biochemical and structure studies showed that oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results that are consistent with those of in vitro studies but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides reveals which amino acids are important for antimicrobial activity. Overall, the in vivo peptide expression system can be used to investigate biological activities and interactions of peptides with their targets within the cellular environment and to separate contributions of the sequence to cellular transport. This strategy has future applications for improving the effectiveness of existing peptides and developing new peptide-based drugs.