The rapid rise of antimicrobial resistance urges to develop new antimicrobial strategies that fit within a One Health perspective. Leaderless bacteriocins are attractive candidates, but because they are synthesized in their active form inside the cell, they raise a key question: how do producer bacteria protect themselves, and what does this mean for their safe use as antibiotic alternatives? To harness these molecules therapeutically, we need a clear picture of how they are produced and how immunity is ensured in the producing strain.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by Enterococcus faecalis 14 that shows antibacterial, antiviral and immunomodulatory activities. Yet the molecular basis of self-immunity during active EntDD14 production has remained unclear. Here, we focused on DdD, a protein encoded within the EntDD14 biosynthetic cluster. In silico analyses suggested that DdD is a membrane-associated protein, and our genetic and functional data show that it is not required for EntDD14 production or export, but is crucial for self-immunity. Deleting ddD dramatically reduced resistance to both intracellularly produced and exogenously added EntDD14, whereas complementation with ddD restored full resistance.

By identifying DdD as a new and highly effective immunity factor, our work updates current models of leaderless bacteriocin biology and underline the importance of accessory proteins as protective “safety locks”, providing new insights into the defense mechanisms associated with leaderless bacteriocin production. These findings can be exploited to engineer safer bacteriocin-producing probiotics, limit off-target toxicity, supporting the responsible use of bacteriocins as antibiotic alternatives in One Health strategies against antimicrobial resistance.