The disulphide bond (Dsb) forming machinery and more specifically the DsbA disulphide bond catalysts, is a central mediator of bacterial pathogenesis as it promotes the assembly of multiple virulence factors required at different stages of the infection process. Extensive research in this area has focused in deciphering Dsb systems in pathogens, however to date little is known about how Dsb proteins interact with and fold virulence substrates. Interestingly, the pathogen Uropathogenic E. coli (UPEC), the main causative agent of urinary tract infections, possesses two DsbA enzymes, DsbA and DsbL, for virulence factor folding. Using a combination of molecular biology, structural biology and biophysics, this study focusses on dissecting the structural and molecular determinants directing substrate specificity of these two distinct DsbA homologues. Outcomes of this research will establish how bacterial pathogens use the Dsb system to generate virulence proteins, which is essential for understanding how bacteria cause disease, thus providing important information to current campaigns that aim at developing inhibitors of DsbA proteins as potential antivirulence agents.