Identified from the pathogen Bacillus cereus SJ1, the two-component lantibiotic bicereucin is featured by the presence of a series of nonproteogenic amino acids and exhibits potent synergistic activity against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, as well as hemolytic activity against mammalian cells. In this study, we performed site-directed mutagenesis on the nonproteogenic amino acids as well as truncation of dehydrobutyrine-rich N-terminal residues and evaluated the effects on both biological activities. We identified that D-Ala21 and D-Ala26 of Bsjα and D-Ala23 and D-Ala28 of Bsjβ play an essential role in the antimicrobial activity, while the N-termini of both peptides are important for both activities. We also determined that the integrity of both subunits is essential for hemolytic activity. Finally, we obtained two variants BsjαtS17A+Bsjβ and BsjαS30A+BsjβT19A, which retained the antimicrobial activity and exhibited greatly decreased hemolytic toxicity. Overall, our results provide a comprehensive understanding of the structure-activity relationships of bicereucin and insights into the mechanism of action thereof, facilitating the further exploration of the molecular basis of the binding receptor of bicereucin and genome mining of potential novel two-component lantibiotics.
Keywords: RiPPs; antimicrobial activity; d-amino acid; hemolysis; lantibiotics; structure−activity relationship.