The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation

Abstract

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.

Fig 1

Models of passenger domain translocation across the OM. (A) Protein with folded β-barrel and inserted into the OM, before translocation and folding of the passenger domain. Proposed models for passenger translocation are also shown: threading model (a submodel of the single-chain model), in which the N terminus is translocated first and then the rest of the protein is translocated with an N-to-C polarity (B); hairpin model (second submodel of the single-chain models), where the C terminus of the passenger domain is first inserted into the pore of the β-barrel so that a hairpin structure is created and the rest of the protein is translocated C to N (C); and a Bam complex-assisted model in which the Bam complex is involved not only in β-barrel insertion into the OM but also in passenger domain translocation (D). Panel E presents the folded structure of EibD, with protein domains marked on the right-hand side (24).

Fig 2

Eib proteins form heterotrimers. Expression and outer membrane purification show formation of heterotrimers between EibA and EibD and between EibC and EibD. As a negative control, OM proteins purified from cultures transformed with empty pETDuet-S vector were used (marked Φ), and, as positive controls, strains expressing only EibA, EibC, or EibD were used. M, molecular mass marker (kDa). (A) Coomassie-stained SDS-PAGE. (B) Western blots with StrepTactin that binds EibA and EibC.(C) Ni-NTA conjugates that bind EibD. For better comparison of gel and blot results, two figure sets were composed for heterotrimers EibAD (D) and heterotrimers EibCD (E). In panels D and E, the letters above the lanes (a to i) show which lanes from the original gel and blots shown in panels A to C were used; in panel D numbers I to IV mark heterotrimers, as follows: IV, EibA₃; III, EibA₂/EibD; II, EibA/EibD₂; I, EibD₃. Surface expression of heterotrimers was confirmed by proteinase K assay (F). Arrows mark bands corresponding to full-length proteins, and arrowheads mark bands corresponding to translocation domains of the proteins. −, untreated samples; +,samples treated with proteinase K. (G) A functional assay with Fc IgG binding was also made. For further confirmation of our interpretation, bands marked with numbers 1 to 7 in panel A were cut from the gel and sent for mass spectrometry measurements (Table 2).

Fig 3

YadA and EibA do not heterotrimerize. Based on coexpression in pETDuet-S vector of distinct TAAs, EibA and YadA showed no heterotrimer formation. The figure shows a Coomassie-stained SDS-PAGE gel after OM protein purification from three cultures with different protein expression levels (EibA/YadA lanes 1, 2, and 3). As a negative control OM proteins purified from cultures transformed with empty vector pETDuet-S (marked Φ) were used, and, as a positive control, homotrimers of EibA and YadA were used. M, molecular mass marker (kDa). Protein expression was done at 37°C, where YadA expression is higher than expression of EibA.

Fig 4

The translocation domain is responsible for trimerization and translocation. Coexpression and OM purification of chimeric proteins with EibA or YadA prove sufficiency of β-barrel for trimerization and translocation of passenger domain. For all panels, lanes are as follows: lane Φ, control sample OM proteins from expression of empty vector pETDuet-S; lane 1, EibA; lane 2, YadA; lane 3, EibA-YadA; lane 4, YadA-EibA; lanes 5 and 6, investigated samples EibA/YadA-EibA and YadA/EibA-YadA, respectively, that form heterotrimers; lane M molecular mass marker (kDa). (A) Coomassie-stained SDS-PAGE gel. Western blotting with StrepTactin, which binds EibA (B), and with Ni-NTA conjugate, which binds YadA (C) showed heterotrimer formation. (D) Surface expression of heterotrimers was confirmed with proteinase K assay. Arrows mark bands corresponding to full-length proteins, and arrowheads mark bands corresponding to translocation domains of the proteins.−, untreated samples; +, samples treated with proteinase K. Functional assays with Fc IgG binding (E) and with collagen (F) were performed showing that heterotrimers still posses the activity of comprised proteins. Finally, for further confirmation of our interpretation, bands marked with numbers 1 to 13 in panel A were cut from the gel and sent for mass spectrometry measurements (Table 3).

Fig 5

The comparison of precursor MS spectra: baseline creation. Spectra of two sequences, TTLETAEEHANSVAR (A) and SSSVLGIANNYTDSK (B), the only unique sequences found for the YadA passenger domain, are shown. Spectra for samples containing full-length YadA protein (bold dashed line, sample 9; bold solid line, sample 10) are compared with samples that should not contain a YadA passenger domain unless contaminated (unbolded line, samples 11, 12, and 13).

Fig 6

The outer membrane is intact. Expression of chimeric proteins with EibA or YadA in minimal medium supplemented with maltose followed by proteinase K treatment and MBP detection proves integrity of OM. MBP levels are shown for the empty pETDuet-S vector (Φ; positive control for MBP level),wild-type proteins EibA and YadA, chimeric EibA-YadA and YadA-EibA, and coexpressed EibA/YadA-EibA (E/Y-E) and YadA/EibA-YadA (Y/E-Y). Lane M, molecular mass marker (kDa); −, untreated sample; + sample treated with proteinase K. The MBP band is boxed. Results were similar for the EibA, -C, and -D heterotrimers (data not shown).