Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway.

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.