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 <i>Escherichia coli dsb</i> mutants. Our analysis revealed a VKOR homologue, a VKOR-DsbA fusion and three DsbA homologues capable of complementing <i>E. coli dsb</i> mutants. Notably, <i>Clostridium botulinum</i> DsbA functioned independently of a regenerating partner, with its activity recycled by glutathione disulphide or ergothioneine. In contrast, <i>Clostridium tetani</i> and <i>Clostridioides difficile</i> DsbA proteins required <i>E. coli</i> DsbB for regeneration, suggesting reliance on distinct thiol or enzyme partners. Understanding oxidative protein folding in Clostridia could reveal new targets for antibacterial intervention.