Recently, crystallization and X-ray structure determination experiments of both the YadA and Hia C-terminal regions revealed a trimeric beta-barrel through which the coiled-coil linker region protrudes and could therefore confirm former biochemical structure predictions (26
). In the present study, we constructed hybrid proteins consisting of different Oca family TLDs (i.e., a linking region and the transmembrane beta-barrel region) and the YadA passenger domain to address two issues. First, we wanted to find proof for our assumption that the TLD is an autonomous functional translocator unit by exchanging the YadA TLD with related TLDs of Oca family members. Second, we wanted to reveal the contribution of the TLDs to serum resistance and virulence in the mouse.
All three constructed YadA-hybrid proteins were translocated across the OM and exposed their trimeric YadA passenger domain, as shown by detection with the YadA MAb 8D1. However, the YadA-UspA1 hybrid protein showed ca. 20% less MAb 8D1 reactivity, as demonstrated by ELISA and immunoblotting data, suggesting less surface exposure in comparison to wild-type YadA. In spite of this slight difference it can be concluded that the TLDs of YadA, EibA, Hia, and UspA1 are able to translocate the YadA passenger domain, which means that they can replace each other without a significant loss of autotransporter function. Upon comparing the amino acid sequence of the TLD regions of EibA, Hia, and UspA1 with that of YadA, we found homologies of only 44, 23, and 18%, respectively. In spite of this low degree of relatedness, YadA passenger domain translocation was efficient, indicating that the TLDs had similar structures. This finding is in accordance with crystal structure studies on the beta-barrels of monomeric autotransporters NalP and EspP, which showed almost superimposable structures by a homology of only 15% (5
). Since the two independently analyzed crystallographic structures of YadA and Hia membrane anchors also showed a high degree of similarity (26
), we conclude from our results that processes of membrane insertion and passenger domain translocation should be very similar and conserved in the Oca protein family.
Recently, the involvement of Omp85/YaeT in autotransporter assembly in the OM has been described, suggesting that C-terminal sequences of OM proteins might possess species-specific recognition sites for Omp85/YaeT, an effect that was observed when trying to express meningococcal porin PorA in E. coli
). Considering the involvement of Omp85/YaeT, our results with YadA-hybrid proteins are interesting, because we have not found strong evidence for species specificity in the function of trimeric autotransport. Possibly, this species specificity of Omp85/YaeT observed for porin proteins does not apply to Oca family proteins.
To study the promiscuous autotransporter capability of Oca family TLDs in more detail, we constructed two additional YadA-UspA1 hybrids, YadA-UspA1-3 and YadA-UspA1-2, by replacing the entire UspA1 linker region or only its proximal coiled-coil part by the homologous YadA region, respectively. In contrast to the functional chimeric YadA-UspA1 autotransporter, the YadA-UspA1-3 and YadA-UspA1-2 chimeras were not detectable on the bacterial surface, in the OM fraction, or in bacterial whole-cell lysates. Previous work has shown that a full-length YadA with specific deletions of either the proximal coiled-coil or distal hairpin-loop segment of the linker region is not functionally expressed and cannot be detected in the cytosol or the membrane fraction (42
). Furthermore, the crystallographic structure of the YadA C terminus displayed the trimeric alpha-helical coiled-coil traversing the beta-barrel pore, indicating a structural and functional unit of the TLD (56
). On the other hand, however, it could also be shown in previous work that the hairpin-loop region of the linker alone is sufficient to allow oligomerization and OM insertion of a FLAG-tagged truncated YadA membrane anchor (42
). The results of the three YadA-UspA1 hybrid proteins demonstrate that the UspA1 beta-barrel absolutely requires its cognate UspA1 linker region for translocation of the YadA passenger domain. This again indicates that the linker region and the four transmembrane beta-barrel strands form a coherent autotransporter module, i.e., a functional TLD, with translocation competence for foreign passenger proteins. This demonstrates also that the function of TLDs is sensitive to changes in amino acid sequence, as has also been shown by Grosskinsky et al.; in that study, the exchange of the highly conserved glycine residue G389 in the YadA TLD led to a severe impairment of YadA translocation (16
). Furthermore, Meng et al. could show that amino acid exchanges in the hairpin loop of the Hia TLD led to changes in structural stability (26
For Oca family members a temperature-sensitive oligomerization stability is known, which can be demonstrated by SDS-PAGE: UspA1 depolymerizes completely after the OM sample is boiled for 5 min (12
), whereas for Hia harsh formic acid pretreatment is required for disintegration of the trimers in SDS-PAGE (13
). The EibA oligomer has also been shown to remain completely stable after boiling (46
). Interestingly, this oligomer stability could also be observed for the corresponding YadA chimeras. Although, the YadA oligomer could be separated into trimer and monomer bands after boiling and SDS-PAGE, the YadA-UspA1 hybrid disintegrated completely into its monomeric form. Strikingly, YadA-EibA and YadA-Hia hybrids completely remained in their oligomeric form after boiling. Treatment of the samples with 8 M urea disintegrated YadA and YadA-UspA1 completely but not the YadA-EibA and YadA-Hia hybrids (results not shown). From this we conclude that the TLDs of these trimeric autotransporters determine the heat stability of the oligomeric form. The oligomers also remained stable after a mild tryptic digestion, indicating that oligomerization is controlled by the TLDs and not by the passenger domain. Recently, it was demonstrated for Oca family member Hia that a stably oligomerized passenger domain is required for cell adhesion (13
). Therefore, yersiniae producing YadA chimeras were tested for adherence capability to collagen and HEp-2 cells. Interestingly, the YadA hybrids showed no significant differences in the two adherence tests. These results do not exclude that the YadA hybrid proteins might differ in their passenger domain structure (e.g., in their packing density). Meng et al. could show that amino acid exchanges in the hairpin loop of the Hia linker region lead to changes in structural stability of Hia but not in adhesion ability for cells or ECM (26
). Furthermore, bacterial AA, a YadA-dependent phenotype, could not be observed for the YadA-UspA1 hybrid producing yersiniae. The ELISA data and the immunoblot analysis indicated slightly reduced surface concentrations of YadA-UspA1 hybrid protein in the OM. This might suggest an impaired formation or reduced stability of the trimeric YadA head structure in YadA-UspA1, which could lead to the loss of AA.
Another feature of the YadA molecule and several other Oca family members (e.g., UspA1, UspA2, EibA, and DsrA) is their ability to confer resistance to serum-mediated killing (38
). However, the exact mechanism of serum resistance mediated by Oca family members is still unclear. For example, it was possible to demonstrate that the passenger domains of UspA1 and UspA2 are involved in the binding of complement inhibitor factors C4BP, C3, and vitronectin, but the physiological relevance of these binding activities still needs to be fully elucidated (3
). Also, the binding of the complement inhibitor factor H through YadA has been under debate, but until now these findings could not be corroborated (7
). In contrast to EibA, the Hia protein does not confer serum resistance. However, a detailed analysis of EibA-mediated serum resistance is lacking. Previously, we could demonstrate that the YadA head-neck region is not necessary for conferring serum resistance, but we were not able to exactly localize it to the stalk or TLD of YadA (42
). Therefore, we presumed that the YadA TLD could be important for this process, since beta-barrel pores from other OM proteins have already been shown to be involved in serum resistance, such as, for example, Ail, another Yersinia
OM protein, where probably complement inhibiting factors such as factor H or C4b-binding protein bind to these loops (27
). Interestingly, all strains expressing YadA hybrid genes showed reduced resistance to 50% normal human serum. This supports our previous conclusion that the TLD of YadA could contribute to serum resistance. Possibly, the linker region of YadA or the surface-exposed loops linking the transmembrane beta-strands are involved in this phenomenon by binding a complement inhibitor factor, such as, for example, factor H. Although the surface- exposed loops of the TLD of the trimeric autotransporters are shorter than those of Ail or OmpX, the contribution of TLD to serum resistance cannot be excluded. The YadA stalk region could also be involved in the binding of factor H or C4BP. Thus, distortion of the coiled-coil structure of the stalk of YadA due to the fused non-YadA TLD may affect the binding of complement inhibitors and favor complement activation.
Finally, we were interested to find out whether the degree of YadA hybrid protein-mediated serum resistance correlated with the degree of mouse virulence. A comparison of bacterial loads in the Peyer's patches, spleen, and liver after peroral challenge and in the spleen and liver after intravenous or intraperitoneal challenge of BALB/c mice clearly showed a highly significant attenuation of all YadA-hybrid producing strains compared to the wild-type YadA producing WA(pYVO8-A1). Interestingly, the chimeric YadA-UspA1 producing strain seemed to be even more attenuated than the YadA mutant strain. Although we have no convincing explanation for this yet, it is conceivable that chimeric YadA-UspA1 transport and insertion into the OM could cause more envelope stress than the other YadA chimeras, which might affect the type 3 protein secretion apparatus in contrast to the YadA-negative mutant.
In summary, nonautologous TLDs fused to the N-terminal YadA might lead to structural changes or distortions of the YadA stalk and head region with concomitant attenuation of virulence function. Moreover, serum resistance seems to contribute essentially to the virulence function of YadA in the mouse model.