The DdD protein confers intracellular and extracellular immunity to the leaderless bacteriocin enterocin DD14

Leaderless bacteriocins (LBBs) are ribosomally synthesized antimicrobial peptides that differ from classical bacteriocins because they are produced in their active form without leader peptides. This raises the key biological question of how producing strains protect themselves from their own bacteriocins during synthesis and prior to secretion. Enterocin DD14 (EntDD14) is a two-peptide LBB produced by Enterococcus faecalis 14. It has antibacterial and antiviral activities, along with immunomodulatory properties. The EntDD14 gene cluster contains 10 open-reading frames, most of which are involved in production, transport, or immunity. However, the function of the products of the ddC and ddD genes remains unknown. Here, we present the first functional characterization of the DdD protein. Although in silico analyses predicted that DdD is a membrane protein with multiple transmembrane domains and a prominent cytoplasmic helix, no homologs with defined functions were identified. Genetic analyses revealed that deletion of the ddD gene was only feasible in a Δbac background lacking the ddA and ddB bacteriocin structural genes. This indicates that lethality provoked by ddD deletion is bacteriocin dependent. Functional assays revealed that DdD is not necessary for EntDD14 synthesis and export but is essential for self-immunity. The ΔddD mutants exhibited reduced resistance to EntDD14, when it was provided extracellularly, and impaired growth when it was produced intracellularly. Expression of ddD in a multicopy plasmid restored full resistance, confirming its role in self-protection. Thus, DdD emerges as a novel immunity determinant of the EntDD14 system, expanding the repertoire of accessory proteins required for LBB self-resistance.