Preserved antibacterial activity of ribosomal protein S15 during evolution

•Amphioxus RPS15 was characterized as a new antimicrobial protein.•Amphioxus RPS15 interacted with bacterial membrane, inducing depolarization and intracellular ROS production.•Amphioxus RPS15 was not toxic towards mammalian cells.•Extracellular RPS15 was present across widely separated taxa.•Emergence of antibacterial activity of RPS15 was traced to its prokaryotic homolog RPS19. Conventional role of ribosomal proteins is ribosome assembly and protein translation, but some ribosomal proteins also show antimicrobial peptide (AMP) activity, though their mode of action remains ill-defined. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45–67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933−55, Aquificae RPS1933−55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial surface via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545–67 were toxic to mammalian cells, making them promising lead molecules for the design of novel AMPs against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.