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Fig. 1. The Yop virulon. When Yersinia are placed at 37°C in a rich environment, the Ysc secretion channel is installed. Proteins YscD, -R, -S, -T, -U, and -V are localized in the inner membrane (IM), whereas YscC and YscP are exposed at the bacterial surface. Lipoprotein YscW stabilizes YscC. YscN belongs to the family of ATPases. A stock of Yop proteins is synthesized, and some of them are capped with their specific Syc chaperone. As long as there is no contact with a eukaryotic cell, a stop-valve, possibly made of YopN, TyeA, and LcrG, blocks the Ysc secretion channel. On contact with a eukaryotic target cell, the bacterium attaches tightly by interaction between its YadA and Inv adhesins and β-integrins, and the secretion channel opens. The Yops are then transported through the Ysc channel, and the Yop effectors are translocated across the plasma membrane, guided by the translocators YopB, YopD, and LcrV.

good agreement with the localization proposed for the homologous Ysc proteins. Thus, the similarity between the Ysc apparatus and the flagellum export apparatus resides in their most inner part. While the Salmonella and Shigella “injectisomes” can be visualized by electron microscopy (13, 14), such visualization is not yet the case for the Yersinia Ysc apparatus. Little is known about the actual mechanism of export, but it is generally assumed that the Ysc apparatus serves as a hollow conduit through which the exported proteins travel to cross the two membranes and the peptidoglycan barrier, in one step. Whether proteins travel folded or unfolded has not yet been demonstrated but, given the size of channel, it is likely that they travel at least partially unfolded.

Translocation of Effectors Across Animal Cell Membranes. Purified secreted Yops have no cytotoxic effect on cultured cells, although live extracellular Yersinia have such an activity. Cytotoxicity nevertheless depends on the capacity of the bacterium to secrete YopE and YopD, and YopE alone is cytotoxic when microinjected into the cells (15). This observation led to the hypothesis that YopE is a cytotoxin that needs to be injected into the eukaryotic cell's cytosol by a mechanism involving YopD to exert its effect (15). This hypothesis was demonstrated by confocal laser scanning microscopy (16) and by the adenylate cyclase reporter enzyme strategy, an approach that is now widely used in “type III secretion” (17): infection of eukaryotic cells with a recombinant Y. enterocolitica producing hybrid proteins consisting of the N terminus of various Yops (other than YopB and YopD) fused to the catalytic domain of a calmodulin-dependant adenylate cyclase (Yop-Cya proteins) leads to an accumulation of cyclic AMP (cAMP) in the cells. Since there is no calmodulin in the bacterial cell and culture medium, this accumulation of cAMP signifies the internalization of Yop-Cya into the cytosol of eukaryotic cells (17). The phenomenon is strictly dependant on the presence of YopD and YopB. Thus, extracellular Yersinia inject Yops into the cytosol of eukaryotic cells by a mechanism that involves at least YopD and YopB (18, 19). Yops are thus a collection of intracellular “effectors” (YopE, YopH, YopM, YpkA/YopO, YopP/YopJ, and YopT) and “translocators” (including YopB and YopD) which are required for the translocation of the effectors across the plasma membrane of eukaryotic cells ( 20).

This model of intracellular delivery of Yop effectors by extracellular adhering bacteria is now largely supported by a number of other results, including immunological observations. During a mouse infection by wild-type Y. enterocolitica, the epitope formed by amino acid residues 249–257 of the YopH effector protein is presented by MHC class I molecules, as cytosolic proteins are, and not by MHC class II molecules, as antigens are that are processed in phagocytic vacuoles (21).

A Pore Formed by Translocators. The translocators YopB and YopD have hydrophobic domains, suggesting that they could act as transmembrane proteins (16, 17 and 18, 22). In agreement with this possibility, Yersinia has a contact-dependent lytic activity on sheep erythrocytes, depending on YopB and YopD (19, 23), which suggests that the translocation apparatus involves some kind of a pore in the target cell membrane by which the Yop effectors pass through to reach the cytosol. This YopB- and YopD-dependent lytic activity is higher when the effector yop genes are deleted, suggesting that the pore is normally filled with effectors (19, 23). The idea of a translocation pore is further supported by the observation that the membrane of macrophage-like cells infected with an effector polymutant Y. enterocolitica becomes permeable to small dyes (23). If the macrophages are preloaded before the infection with a low-molecular weight fluorescent marker, they release the fluorescent marker but not cytosolic proteins, indicating that there is no membrane lysis but rather insertion of a small pore (diameter 16–23 Å) into the macrophage plasma membrane (23). The hypothesis of a channel is reinforced by the observation that artificial liposomes that have been incubated with Yersinia contain channels detectable by electrophysiology (24). All these events are dependent on the presence of the translocators YopB and YopD. These two hydrophobic Yops seem thus to be central for the translocation of the effectors and for the formation of a channel in lipid membranes. They presumably play different roles in pore formation. Indeed, YopB alone can disturb artificial membranes, whereas YopD cannot. Moreover, YopD has been shown to end up in the cytosol of eukaryotic cells (25).

YopB and YopD are encoded by a large operon that also encodes LcrV, LcrG, and the chaperone SycD. LcrV is a secreted Yop that has a different name for historical reasons. The fact that LcrG and LcrV are encoded together with translocators suggests that they could also be involved in translocation. Not surprisingly, LcrV interacts with YopB and YopD (26), is surfaceexposed before target cell contact (27), and is also required for translocation (26). In contrast with YopB, YopD, and LcrV, LcrG is not a released protein, but its exact localization in the bacterium remains elusive. It is required for efficient translocation of Yersinia Yop effector proteins into the eukaryotic cells but it is not required for pore formation. It binds to heparan sulfate proteoglycans (28), but the significance of this binding is not clear yet.

The Cytosolic Chaperones. Type III secretion often involves a new type of small cytosolic chaperone (29, 30 and 31) (Fig. 1). In Yersinia, these chaperones are called “Syc” (for specific Yop chaperone) (31). Generally, they are encoded by a gene that is located close to the gene encoding the Yop protein they serve, and this is a useful indication to recognize them. These chaperones may not form a single homogeneous group but rather could belong to two



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