Trifolitoxin represents a prototype of tiarins, a widespread family of ribosomally synthesized and post-translationally modified peptide antibiotics that selectively inhibit aminoacyl-tRNA synthetases through a Trojan-horse mechanism in which an unmodified N-terminal peptide mediates cellular uptake via oligopeptide transporters, followed by aminopeptidase-mediated proteolytic release of a post-translationally modified C-terminal warhead that targets specific aminoacyl-tRNA synthetases based on a predictable sequence-structure relationship.

Trifolitoxin (TFX) is a ribosomally synthesized and post-translationally modified peptide antibiotic produced by Rhizobium anhuiense T24. Although discovered more than half a century ago, its mechanism of action has remained elusive. Here we demonstrate that TFX inhibits arginyl-tRNA synthetase (ArgRS), an essential translation enzyme. TFX acts as a Trojan-horse antibiotic: it enters cells via the inner membrane oligopeptide transporter YejABEF and then undergoes partial proteolysis by the aminopeptidase PepN to release a “warhead” that binds ArgRS and arrests translation. A systematic analysis of prokaryotic genomes revealed that TFX belongs to a widespread family of modified peptides that we designate the tiarins. In addition to ArgRS inhibitors, the tiarin family includes compounds that specifically target at least seven other aminoacyl-tRNA synthetases. These findings define a previously unrecognized family of natural antibiotics and provide a framework for developing a new class of inhibitors targeting various tRNA synthetases.