Production of the Bacteriocin Antibiotic Thuricin CD

Abstract

Bacteriocins are ribosomally produced peptide antibiotics produced by bacteria as highly effective antimicrobial natural products. Many bacteriocins are thought to function by selective insertion of multimers into the cell membrane, generating unregulated ion pores that ultimately trigger cell lysis. Bacteriocins often contain stabilizing post-translational modifications including N- and C-terminal additions, epimerized D-amino acids, chemically modified amino acids, and disulfide and thioether crosslinks. Thuricin CD is a two-component antimicrobial consisting of peptides, Trnα and Trnβ, produced by Bacillus thuringiensis, that specifically targets the bacterium Clostridium difficile. Each peptide contains three thioether crosslinks in which cysteine residues are chemically bonded to the α-carbon of distal amino acid residues; the resulting compact structure is highly resistant to proteolysis. The thioether crosslinks are proposed to be introduced by the radical SAM enzymes TrnC and/or TrnD. In this work, we develop a fusion protein strategy for producing the propeptides TrnA and TrnB and further demonstrate that TrnC alone is capable of introducing two of the thioether crosslinks. The successful in vitro production of thuricin CD could provide a potential lead for novel antimicrobials targeting C. difficile infections.