The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1.

Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity. Mutational effects were assessed by studying EntK1 sensitivity and binding with strains of naturally EntK1-insensitive Lactiplantibacillus plantarum-expressing various RseP variants. We determined that site-directed mutations in conserved sequence motifs related to catalysis and substrate binding, and even deletion of two such motifs known to be involved in substrate binding, did not abolish bacteriocin sensitivity, with one exception. A mutation of a highly conserved asparagine, Asn359, in the extended so-called LDG motif abolished both binding of and killing by EntK1. By constructing various hybrids of the RseP proteins from sensitive E. faecium and insensitive L. plantarum, we showed that the extracellular PDZ domain is the key determinant of EntK1 sensitivity. Taken together, these data may provide valuable insight for guided construction of novel bacteriocins and may contribute to establishing RseP as an antibacterial target.