Sequence diversity and antimicrobial activity of natural homologs of the translation terminator inhibitor Apidaecin.

Apiadecin 1b (Api1b), produced by the honeybee Apis mellifera, is a Proline-rich Anti-microbial Peptide (PrAMP), that enters Gram-negative bacteria via inner membrane transporters and arrests ribosomes at translation termination, causing cell death. Homologs of Api1b are found in genomes of several members of the bee and wasp families, often, with multiple isoforms found within the same insect. These homologs differ both in their lengths and amino acid composition, especially in the N-terminal part of the peptide. Apidaecins are synthesized by the insect as prepropeptides, which are proteolytically cleaved to yield biologically active peptides, making it challenging to identify the sequence of the active PrAMP from the gene sequence. Our bioinformatics analyses identified several potential homologs of Api1b from genomes of multiple species of bees and wasps. Peptide sequences that differed significantly from Api1b were chemically synthesized and tested against several Gram negative bacteria. Strikingly, a few of the selected peptides showed increased antimicrobial activity compared to Api1b as well as the more potent synthetic variant, Api137. Our in vitro experiments demonstrated that, despite differences in sequence, these active peptides maintain the ability to arrest ribosomes at stop codons, as we described for Api1b and Api137. Interestingly, some of the identified peptides also arrest the terminating ribosomes during in vitro translation but were unable to kill the tested bacteria, suggesting they are unable to penetrate these cells. Future studies may help uncover the sequence requirements for transporter recognition and elucidate whether these peptides have evolved to target specific bacterial species.