Mechanistic insights into enterocin C targeting the undecaprenyl phosphate recycling protein BacA

Enterococcus faecalis is an important opportunistic pathogen responsible for healthcare-associated infections. It is intrinsically resistant to various antibiotics, particularly to cephalosporins and vancomycin, creating an urgent need for alternative therapeutics. In this context, bacteriocins warrant investigations as a potential source of medically useful antibiotics. Herein, we demonstrate that Enterocin C, a class IIb two-peptide bacteriocin, specifically targets the membrane-embedded undecaprenyl phosphate recycling protein BacA from enterococci as a cell surface receptor. Using biochemical and biophysical methods, supported by computer modeling and mutagenesis, we deciphered the EntC's molecular interaction pattern with its target, marking the first mechanistic insight of a two-peptide bacteriocin. The two peptides act cooperatively at nanomolar concentrations to interact with the outward-open catalytic pocket of BacA: the peptide EntC1 docks deeply into the catalytic site, inhibits BacA's enzymatic activity, and enables the binding of peptide EntC2, eliciting membrane permeabilization, eventually leading to cell death. This work paves the way for the bioengineering of BacA-targeting bacteriocins to develop tailored antimicrobial strategies.