Infection of epithelial cells with Y. enterocolitica
triggers the expression of a high number of genes, including genes encoding proinflammatory molecules, such as IL-8 (4
). Inv was shown to be a major inductor of IL-8 expression and secretion. Plasmid-encoded factors, such as YopP, are able to counteract this response (4
). The induction of a proinflammatory response by Inv is known to be dependent on adhesion to β1 integrins. Taking into account that β1 integrins are also involved in adhesion via YadA, we hypothesized that YadA may also be involved in triggering a proinflammatory host response. Experiments in this study confirmed this hypothesis and demonstrated that YadA can mediate IL-8 secretion. Moreover, we showed that, like Inv, β1 integrins are involved in this process. Furthermore, the downstream signaling events triggered by YadA involve the same components as in Inv-mediated signaling, namely, Rho GTPases and the MAP kinases MEK1, JNK, and p38.
YadA was expressed either in E. coli strains or in plasmid-cured Yersinia strains. These strains do not express any of the virulence factors (e.g., YopP) that are known to counteract a proinflammatory response induced by Y. enterocolitica in vitro. In this setting, YadA and Inv are similarly effective in mediating adhesion to GD25-β1A fibroblasts and even more effective in mediating adhesion to HeLa cells, as proven with E. coli overexpressing YadA and Inv by the same vector system. The different efficacies of YadA and Inv in adhering to HeLa and GD25-β1A cells may be due to cell-type-specific differences.
By using the fibroblast cell line GD25 (18
), which lacks β1 integrin expression in comparison with the fibroblast cell line GD25-β1A overexpressing β1 integrins (57
), we demonstrated that β1 integrins are crucial for YadA-mediated adhesion of Yersinia
to cells. The data presented here are in line with previous reports (3
) that β1 integrins are required for YadA-mediated binding to cells. Eitel and Dersch (16
) showed that YadA of Y. pseudotuberculosis
can mediate highly efficient uptake into epithelial cells when overexpressed in E. coli
or when Y. pseudotuberculosis
is grown under specific growth conditions. This process can be blocked using neutralizing antibodies against fibronectin and β1 integrins, which led to the suggestion that internalization mediated by YadA occurs via extracellular-matrix-dependent bridging between YadA and the host cell β1 integrin receptors (16
In line with previous data (23
) and in contrast to YadA of Y. pseudotuberculosis
, even overexpression of Y. enterocolitica
YadA can only weakly mediate uptake of Y. enterocolitica
into epithelial cells. Thus, YadA of Y. enterocolitica
is much less efficient (18-fold) in mediating uptake than Inv of Y. enterocolitica
. The reason why YadA of Y. enterocolitica
may be less efficient in mediating uptake than Y. pseudotuberulosis
is unclear. Although the amino acid sequence of YadA is highly homologous between the two species, there are two additional amino acid sequences in the head domain of Y. pseudotuberculosis
and one additional amino acid sequence in the stalk domain of Y. enterocolitica
. One can speculate that these sequences could be responsible for the different ability to invade cells mediated by YadA. Further studies will be necessary to address this question.
The interaction between the Yersinia
Inv protein and the β1 integrin receptor permits bacterial attachment to the host cell and triggers internalization (27
). Previous studies uncovered a role for Inv-mediated adhesion in the production of IL-8 in epithelial cells infected with Y. enterocolitica
). In this study, we demonstrate that YadA expressed in E. coli
in the same vector system as Inv also induces IL-8 secretion. Since both YadA and Inv mediate adhesion but only Inv efficiently mediates uptake, these data support previous studies showing that adhesion, but not internalization, of Yersinia
is essential for triggering IL-8 secretion via β1 integrins (4
Tahir et al. (52
) reported that NSVAIG-S motifs in the N-terminal half of YadA are required for collagen binding. A recent study revealed that mutations of these putative collagen binding domains are located in the center of the YadA trimers and therefore cannot be directly involved in collagen binding. Mutation of these domains led to conformational changes of the head domain of YadA, which abolishes collagen binding. Interestingly, it was also shown that binding of Y. enterocolitica
to fibronectin and laminin is not affected by these mutations (52
). Using Y. enterocolitica
-expressing YadA mutants in which different NSVAIG-S motifs were mutated, we demonstrated that these mutations were sufficient to abolish YadA-mediated adhesion and subsequently IL-8 secretion. This is in keeping with the idea that collagen binding could be involved in adhesion and thus in subsequent IL-8 secretion.
Adhesion mediated by YadA and Inv is a prerequisite for IL-8 secretion. At least for HeLa cells, YadA mediates stronger adhesion than Inv. In contrast, the time delay of IL-8 secretion mediated by YadA compared to that mediated by Inv indicates that YadA is less effective in mediating IL-8 secretion than Inv. Moreover, YadA-mediated adhesion to and IL-8 secretion by HeLa cells can be blocked more efficiently by anti-β1 integrin antibodies than Inv-mediated adhesion and IL-8 secretion. Previously obtained results showed high-affinity binding of Inv to integrins (13
), which may explain why blocking of adhesion and IL-8 secretion with antibodies is not effective. In contrast, there is evidence that the binding of YadA to β1 integrins may be an indirect effect via bridging to extracellular matrix proteins, such as fibronectin (16
). Low-affinity binding to β1 integrins via extracellular matrix proteins may explain the more efficient adhesion and the delayed onset of IL-8 secretion, as well as the higher blocking capacities of anti-β1 integrin antibodies.
The involvement of β1 integrins in Inv- and YadA-triggered IL-8 secretion also indicated a common signaling pathway for YadA- and Inv-triggered proinflammatory responses. By cross-linking β1 integrins with antibodies, it was clearly shown that β1 integrins are directly involved in a signal cascade leading to IL-8 secretion via Rac1 and MAP kinase-p38 signaling pathways (32
). Similarly, for Inv-mediated IL-8 secretion, it was shown that Rac1 is required for activation of NF-κB (20
) and that Inv activates the MAP kinases p38, MEK1, and JNK (20
). The comparison of Inv- and YadA-mediated signaling presented here confirmed that the factors involved in Inv-mediated signaling leading to IL-8 secretion are also required for YadA-mediated signaling.
Taking these data together, we can conclude that YadA, in addition to a number of other biological properties, such as serum resistance (2
), arithrogenicity (22
), and epithelial-cell adhesion (23
), contributes to the proinflammatory response of the host to infection with Y. enterocolitica
. Our results and recent observations suggest that factors of Yersinia
which can induce a proinflammatory response involving NF-κB activation seem to be very redundant. Thus, Viboud et al. (55
) demonstrated for Y. pseudotuberculosis
that YopB can also induce IL-8 by activation of the small GTPase Ras and NF-κB. However, the actual contributions of these various factors, like Inv, YadA, and YopB, as well as lipopolysaccharide, to triggering a proinflammatory response in vivo in different cell types in concert with the mechanisms of Yersinia
which counteract the proinflammatory response in vivo are still unclear and will have to be addressed in future studies.