Arrangement of the translocator of the autotransporter adhesin involved in diffuse adherence on the bacterial surface

Abstract

Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal alpha-domain ("passenger") fused to a C-terminal beta-domain ("translocator"). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted "hairpin model" the passenger proteins are translocated through a channel formed by the beta-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six beta-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

FIG. 1.

Schematic representation of the modular organization of the AIDA autotransporter. The domains incorporated into fusion proteins CB3, DT1, and DT2 are indicated. In MK25 the HA epitope (YPYDVPDYA) was inserted at position 859 (22, 24).

FIG. 2.

Purification of AIDA translocator fusion proteins DT1 and DT2. The proteins were purified by IMAC as described in Materials and Methods. The final elution was performed with 250 mM imidazole in TNO-0.5. (A) The fractions were treated with sample buffer, analyzed by gradient SDS-PAGE (5 to 11% polyacrylamide), and stained with Coomassie brilliant blue or, after Western blotting, detected with antibodies directed against the AIDA translocator (anti-fp12). (B) Treatment with trypsin of the refolded DT1 and DT2 proteins generates the protease-resistant core region of the AIDA translocator (22, 24).

FIG. 3.

Elution profile of the AIDA translocator fusion proteins. Purified AIDA translocator fusion proteins DT1 (57 kDa) and DT2 (49 kDa) were loaded onto a 180-ml Sephacryl S-300 high-resolution gel filtration column. The fractions were analyzed by Western blotting and were quantified by measuring the band intensities using the LUMI-Analyst software. The positions of standard proteins are indicated at the top. The elution of proteins was monitored by A₂₈₀ (OD 280 nm), which is shown for the DT2 protein.

FIG. 4.

Use of electron microscopy to visualize putative oligomeric structures. (A) Isolated DT2 protein on mica plates was coal-platinum coated and evaluated on copper grids (magnification, ×38,000). (B and C) Immunogold electron microscopy (12-nm gold particles) was used to visualize the distribution of the authentic AIDA-I adhesin (B) and of the HA-tagged AIDA translocator (MK25 fusion protein) (C) on the bacterial surface as described in the text. Bars = 300 nm. No ordered structures are discernible.

FIG. 5.

Coexpression of AIDA-I and OspG on the bacterial surface. pIB264 encoding the authentic AIDA-I passenger (∼100 kDa) and pCB3 encoding the OspG-AIDA translocator fusion protein harboring the 22-kDa OspG passenger protein were transformed into E. coli UT5600. (A) Surface expression of AIDA was detected by using anti-AIDA antiserum and a goat anti-rabbit secondary antibody labeled with Cy2 (green). (B) OspG was visualized with a specific anti-OspG antiserum raised in mice and was detected with a secondary goat anti-mouse antibody labeled with Cy3 (red). (C and D) Merged immunofluorescence (C) and phase-contrast (D) micrographs. All bacteria expressing OspG also express AIDA-I. Bars = 10 μm.

FIG. 6.

Cross-linking experiments for detection of dimerization of N-terminal AIDA fusion proteins. E. coli UT5600 cells carrying pDM1, coding for unprocessed autotransporter adhesin heptosyltransferase-modified whole AIDA (α-domain and translocator), pIB264, coding for processed and modified whole AIDA, and pDT1, coding for the AIDA translocator fusion protein DT1, were incubated with the cross-linker DSP. To release AIDA-I (α-domain) the bacteria were incubated at 60°C for 20 min and washed thoroughly prior to addition of the cross-linker. 2-ME, 2-mercaptoethanol.