Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain

The mechanism of protein secretion mediated by the beta-domain of the Neiss eria gonorrhoeae IgA protease, a paradigm of a family of secreted polypepti des of Gram-negative bacteria called autotransporters, has been examined us ing a single-chain antibody (scFv) as a reporter passenger domain to monito r the translocation process. Fusion of a scFv to the beta-module of the IgA protease allowed us to investigate the passage of the chimeric protein thr ough the periplasm, its insertion into the outer membrane and the movement of the N-terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formatio n of S-S bridges could be analysed in detail. In contrast to the current no tion that only an unfolded N-passenger domain can be translocated through t he beta-domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, In an active conformat ion with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re-evaluation of the prevailing model for secretion of the N-domain of autotransporters.