The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity.

Abstract

During infection of cultured epithelial cells, surface‐located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non‐polar yopB mutant strain displays a wild‐type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage‐like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia‐induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB‐dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell‐to‐cell transfer of Yop effector proteins.

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane

“…As expected from the overexpression of YopK, the intensity of the bacteria-associated staining was higher. These results are similar to what was previously found for HeLa cells infected with a multiple-yopmutant strain overexpressing YopB (Hå kansson et al, 1996b). No YopB could be detected in the cytosol, whereas the protein was readily detected in the vicinity of cell-associated bacteria.…”

“…3,C and F). This is similar to what has been shown for yopB-and yopDmutant strains, which are blocked for translocation (Rosqvist et al, 1994;Persson et al, 1995;Hå kansson et al, 1996b). Because the overexpression of YopK did not reduce YopE and YopH expression (Fig.…”

“…A multiple yop-mutant strain showed a YopB-dependent lytic activity, whereas this effect was strongly reduced for a strain expressing and translocating Yops. Taken together, these results argue for a model where translocation occurs through the YopB-induced pore (Hå kansson et al, 1996b).…”

“…At least 11 Yop proteins are secreted by the yscencoded type III secretion system of Yersinia. Of these, YopE, YopH, YopM, and YpkA are translocated into the cytosol of the eukaryotic cells (Rosqvist et al, 1994;Sory and Cornelis, 1994;Persson et al, 1995;Sory et al, 1995;Boland et al, 1996;Hå kansson et al, 1996a), and YopB and YopD are essential for the targeting of the Yop effectors into the cells (Rosqvist et al, 1991;Sory and Cornelis, 1994;Persson et al, 1995;Sory et al, 1995;Hå kansson et al, 1996b). YopK is expressed at a much lower level compared to most other Yops, with the exception of YpkA (Holmströ m et al, 1995).…”

“…Contact-haemolytic assays in the presence of carbohydrates were performed essentially as described by Hå kansson et al (1996b). The washed erythrocytes were resuspended in culture medium containing 60 mM of the respective carbohydrate to yield a final concentration of 30 mM in the assay-well and the PBS added after the second centrifugation contained 30 mM of the carbohydrate of interest.…”