Abstract
BrkA is an autotransporter native to Bordetella pertussis that can be utilized to display proteins of interest in its passenger domain on the surface of Escherichia coli. However, cysteine-rich recombinant passengers such as EC10 may introduce disulfide bonding, which may obstruct translocation through the β-barrel and reduce surface expression. Prior research on autotransporter secretion implicated presence of periplasmic DsbA chaperone and its role in introducing disulfide bonds as a possible explanation for reduced secretion efficiency of the passenger. Though the periplasmic transit process remains a contested area, this study aims to elucidate the role of DsbA, a protein native to E. coli, in the inhibition of BrkA-EC10 translocation in order to optimize the BrkA autodisplay system for cysteine-rich proteins. To compare and evaluate expression of BrkA and BrkA-EC10 in wild-type BW25113 K-12 E. coli (WT) and JW3832 K-12 E. coli (ΔdsbA), as well as any possible pleiotropic effects, we employed a growth curve assay, a trypsin accessibility assay, and a Western blot. Ultimately, our results indicated that ΔdsbA did not impair or enhance the translocation of the BrkA-EC10, failing to restore expression to levels observed for BrkA in our WT strain. Additionally, we found BrkA-EC10 expressed in the ΔdsbA strain exhibited reduced cell viability. These findings suggest there may be more factors at play for the translocation of BrkA-EC10 beyond DsbA and disulfide bonding.