Proteolytically activated antibacterial toxins inhibit the growth of diverse gram-positive bacteria

Many species of bacteria produce small-molecule antibiotics that enter and kill a wide range of competitor microbes. However, diffusible antibacterial proteins (ABPs) that share this broad-spectrum activity are not known to exist. Here, we report a family of proteins widespread in gram-positive bacteria that display potent antibacterial activity against a diverse range of target organisms. Upon entering susceptible cells, these ABPs enzymatically degrade essential cellular components including DNA, transfer ribonucleic acid (tRNA), and ribosomal ribonucleic acid (rRNA). Unlike previously characterized bactericidal proteins, which require a specific cell surface receptor and therefore display a narrow spectrum of activity, we find that ABPs act in a receptor-independent manner and consequently kill bacteria spanning multiple phyla. Target cell entry by ABPs requires proteolytic activation by a cognate, coexported serine protease, and the liberated toxin component of the cleaved ABP is driven across the target cell membrane by the proton motive force. By examining representative ABPs from diverse pathogenic, commensal, and environmental bacteria, we show that broad-spectrum antibacterial activity is a conserved property of this protein family. Collectively, our work demonstrates that secreted proteins can act as broad-spectrum antibiotics, suggesting that ABPs represent one of potentially many such families produced in nature.