Activity of the Antimicrobial Peptide Apidaecin Against Gram-Positive Bacteria

Apidaecin (Api) is a small antibacterial peptide produced by honeybees able to inhibit growth of Gram-negative bacteria via a unique mechanism of action. Once Api enters the cell through specialized transporters, it sequesters release factors (RF1 or RF2) on the terminating ribosome. Because RFs are outnumbered by ribosomes in E. coli cells, Api action leads to depletion of available RFs necessary for the end of translation and causes inhibition of protein synthesis. Gram-positive bacteria are impervious to Api because they lack the specialized transporters to internalize the antimicrobial. Furthermore, it is unknown whether Api can interact with the ribosomes and sequester the RFs of Gram-positive bacteria. To test whether Api is able to target the translation machinery of Gram-positive bacteria, we used a toeprinting assay which reveals the site of inhibitor-induced ribosome stalling on mRNA. Toeprintng analysis showed that Api arrests the Bacillus subtilis ribosome at stop codons, demonstrating for the first time that it can inhibit translation driven by ribosomes from Gram-positive bacteria. Having established Api’s ability to act on the Gram-positive ribosome in vitro, we used in-vivo assays to test if Api can inhibit growth of B. subtilis. To overcome the uptake issue, we endogenously expressed Api in B. subtilis cells by introducing an Api-encoding gene either into the chromosome or on a plasmid under the control of xylose-inducible promoter. Nevertheless, the addition of xylose to the cells carrying the api gene caused no toxicity. To test whether Api is expressed in B. subtilis, we appended it at the C-terminus of GFP, a chimeric construct that inhibits growth of E. coli. In spite of a high level of expression, verified by the fluorescence of the B. subtilis cells carrying Finding that Api has no inhibition of growth of G(+) bacteria we decided to test the inhibition of global translation in the presence of Api. Utilizing metabolic labeling of proteins using radioactive methionine demonstrated that global translation of B. subtilis is unaffected by Api. Knowing that Api can target the ribosome of Gram-positive bacteria but is unable to inhibit growth despite of strong expression, we decided to complete toeprinting assays utilizing B. subtilis release factors rather than the E. coli factors. We saw no inhibition or stalling of the ribosome when using the B. subtilis release factors. This demonstrated that even though Api can act on the Gram-positive ribosome it cannot sequester the Gram-positive release factors, losing all activity. Our findings that an antimicrobial peptide targeting the highly conserved ribosome has markedly contrasting effects on the growth of Gram-negative and Gram-positive bacteria may reveal important differences in the makeup of translation machinery factors among bacterial species. Understanding these differences may be critical for expanding the spectrum of activity of ribosome-targeting antibacterial peptides.