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 p athway, which does not have a periplasmic intermediate of the secreted prot ein, 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 dis ulphide bond and protein-folding reporter, as it contains two disulphide br idges that are required for its correct folding (i.e. to preserve its antig en-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular mediu m with the disulphide bonds correctly formed, Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmi c thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation, Howev er, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a spec ific inhibitory effect on the Hly-dependent secretion of disulphide-contain ing proteins. These data suggest that premature cytoplasmic oxidation of th e substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of di sulphide-containing proteins, but also that disulphide bonds are specifical ly formed during the passage of the polypeptide through the export conduit.