Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions

Abstract

The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region. The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted beta-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.

FIG. 1

(A) Scheme of the composition of the various CTB–AIDA-I fusion proteins. The orientation and location of primers are indicated, as are the sites used for cloning and amino acid positions according to the native AIDA-I sequence. Total lengths of the various fusion proteins and the respective plasmids are shown. The arrangement of β-sheets in the proposed β-barrel and their numbering are given. LR, linking region; SP, signal peptide. (B) Topological model of the organization of the AIDA-I TU and the CTB fusion proteins. β-Barrel sequences (in italics) and linker sequences are shown relative to the lipid/LPS bilayer of the outer membrane (depicted by the horizontal lines). The fusion points of CTB (and any cloning-derived residues) in the respective CTB–AIDA-I fusion proteins are indicated by connecting lines. Arrows represent the predicted amphipathic β-sheets and their assumed directions relative to the (horizontal) plane of the outer membrane.

FIG. 2

Expression and trypsin sensitivity of CTB–AIDA-I fusion proteins. (A) Equal amounts of whole-cell protein from E. coli JK321 harboring the indicated plasmids were separated by SDS-PAGE and Western blotted with anti-CTB serum AK55. Specific CTB-derived bands are indicated with arrowheads, and the migration of molecular size markers is indicated in kilodaltons. (B) Cells of E. coli JK321 were subjected to trypsin treatment, and subsequently treated as described for panel A. (C) E. coli UT5600 harboring pJM1141 (or pJM7 as a control) was treated as described for panels A and B. Panels A to C employed equivalent cell numbers.

FIG. 3

Heat modification of AIDA-I. An outer membrane preparation of E. coli UT5600(pJM1013) was prepared by using the Sarkosyl method as described in Materials and Methods. Aliquots were incubated for 10 min at the indicated temperature. (A and C) Western blots probed with anti-OmpA serum AK57 (A) or anti-CTB-serum AK55 (C). (B) SDS-PAGE gel stained with Coomassie brilliant blue R250. Arrowheads pointing to the right indicate heat-modified (h) forms of the respective proteins, while arrowheads pointing to the left indicate not-heat-modified (nh) forms of the proteins.

FIG. 4

Heat modification of CTB–AIDA-I fusion proteins. Cell envelopes from recombinant E. coli UT5600 were prepared. After incubation at the indicated temperatures, aliquots corresponding to 3.0 ml of culture with an OD₅₇₈ of 1.0 for E. coli UT5600 containing either plasmid pJM1141, pJM918, or pJM920, 3.5 ml for pJM967, 0.7 ml for pJM942 and pJM964, and, finally, 0.2 ml for pJM7 were Western blotted with anti-CTB serum AK55.

FIG. 5

Schematic model and features of CTB–AIDA-I fusion proteins. The assumed positions, export, and folding behavior of the β-barrel of the various CTB–AIDA-I fusion proteins are visualized. OM, outer membrane; PP, periplasm.