New insights into the periplasmic redox pathways of Escherichia coli

(eng) The formation of a disulfide bond results from the oxidation of two cysteine thiol groups, with the concomitant release of two electrons. Disulfide bonds are important for the correct folding of many secreted proteins. In these proteins, disulfide bonds stabilize protein structure and protect the thiol group from over-oxidation. The formation of a disulfide bond is a rate-limiting step of the folding process of a protein. Therefore, although disulfides can form spontaneously in vitro, disulfide bond formation is a catalyzed process in vivo in order to allow the rapid folding of nascent proteins. During my PhD thesis, I studied the proteins that catalyze the formation of disulfide bonds in the Escherichia coli periplasm. These proteins belong to the “Dsb” family (Dsb stands for disulfide bond). The Dsb proteins are involved in two major pathways: an oxidation pathway (DsbA and DsbB) and an isomerization/reduction pathway (DsbC, DsbG, DsbD). First, I contributed to a phenotypic and proteomic analysis of the dsbAdsbC double mutant. Our results led us to revise the general view of the disulfide bond formation pathway. In particular, we showed that the oxidizing and reducing pathways are more intertwined than previously thought. Moreover, using two-dimensional liquid chromatographic mass-spec/mass spec analysis, we identified several DsbA and DsbC substrates. Second, I set out to discover the function of DsbG. When I started my work, DsbG was generally considered as a putative protein disulfide isomerase. Using a catalytic mutant of DsbG, I identified three substrates of this protein. Unexpectedly, the three DsbG substrates contained only a single cysteine residue. I showed that this residue was sensitive to reactive oxygen species and could be oxidized to a sulfenic acid (-SOH). Sulfenic acids are highly unstable groups that can be irreversibly modified to sulfinic and sulfonic acids. I found that the DsbG is a key player in a new periplasmic reducing pathway that regulates sulfenic acid formation on proteins containing single cysteine residues.