Disulphide bond-forming enzymes in clostridial species.

Disulphide bond formation is critical for the folding and stability of proteins involved in bacterial cell envelope processes yet remains understudied in clostridial pathogens. While a few Clostridia-derived toxins and virulence factors are known to depend on disulphide bonds, the enzymes catalysing their formation are poorly characterized. Here, we performed a bioinformatic search to identify ten putative disulphide bond-forming enzymes in Clostridia. We cloned and codon-optimized these genes, testing their ability to complement Escherichia coli dsb mutants. Our analysis revealed a VKOR homologue, a VKOR-DsbA fusion and three DsbA homologues capable of complementing E. coli dsb mutants. Notably, Clostridium botulinum DsbA functioned independently of a regenerating partner, with its activity recycled by glutathione disulphide or ergothioneine. In contrast, Clostridium tetani and Clostridioides difficile DsbA proteins required E. coli DsbB for regeneration, suggesting reliance on distinct thiol or enzyme partners. Understanding oxidative protein folding in Clostridia could reveal new targets for antibacterial intervention.