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 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 antibacterial proteins (ABPs) enzymatically degrade essential cellular components including DNA, tRNA, and 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 bacterial phyla. Target cell entry by ABPs requires proteolytic activation by a cognate, co-exported 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. Significance StatementMany bacteria produce proteins with antibacterial properties. However, owing to their reliance on a specific surface receptor for target cell entry, all known antibacterial proteins are only active against a narrow range of organisms. Using biochemical and genetic approaches, this study reports the discovery of a new family of antibacterial proteins secreted by many Gram-positive bacteria that enter and kill a broad spectrum of bacteria. Entry of these proteins into susceptible bacteria does not require a receptor and instead relies on cleavage by a co-secreted protease and the proton motive force of the target cell. Overall, our findings reveal a new family of antibacterial proteins and provides insight into how these proteins enter and kill a broad range of bacteria.