Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway.
The Iga beta autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga beta of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga beta-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga beta-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga beta, whether or not they contain cysteine residues.
Jose
J
J
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Krämer
J
J
Klauser
T
T
Pohlner
J
J
Meyer
T F
TF
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins; D019704:Protein Disulfide-Isomerases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000235:genetics
D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D010088:Oxidoreductases; C077913:periplasmic protein disulfide oxidoreductase; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase; C057840:omptin outer membrane protease; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine