INTRODUCTION

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Invasive pathogenic bacteria have in common the capacity to overcome the defense mechanisms of their animal host and to proliferate in its tissues. They each have their own life-style and target organs and cause a variety of symptoms and diseases, which suggested the existence of great diversity among the bacterial virulence strategies. However, recent data contradict this view and reveal the existence of major virulence mechanisms in various pathogenic bacteria. One is the release of A-B toxins as exemplified by Bordetella pertussis and Bacillus anthracis. Another was discovered more recently in a number of bacterial pathogens. By this mechanism, sometimes referred to as type III, extracellular bacteria that are in close contact with a eukaryotic cell deliver bacterial proteins into the cytosol of this cell. The Yop system of Yersinia spp., which we describe in this review, represents an archetype for this new mechanism. The other animal pathogens with related systems are Salmonella spp., Shigella spp., enteropathogenic Escherichia coli (EPEC), Pseudomonas aeruginosa, Chlamydia psittaci (165), and Bordetella spp. (383a). Related systems are also found in the plant pathogens that elicit the so-called hypersensitive response, such as Erwinia amylovora, Pseudomonas syringae, Xanthomonas campestris, and Ralstonia solanacearum (for reviews, see references 4 and 351). The literature on all the type III systems is now so abundant that an exhaustive description could no longer fit in one review. This review is thus specifically dedicated to the Yersinia type III system. However, homologs of the various Yersinia proteins in the other bacteria are mentioned and even described when appropriate. To integrate the Yop virulon in the general context of cross talk between bacterial pathogens and their host, the reader may refer to broader reviews (94, 95, 107). More information on Yersinia virulence in general is also available in recent reviews (51, 255). Less exhaustive reviews dealing with the type III system (201, 352) or, more specifically, the Yop virulon (75, 98, 335-337) are also available.

The genus Yersinia includes three species that are pathogenic for rodents and humans; Yersinia pestis causes plague, Yersinia pseudotuberculosis causes mesenteric adenitis and septicemia, and Yersinia enterocolitica, the most prevalent in humans, causes gastrointestinal syndromes ranging from an acute enteritis to mesenteric lymphadenitis (76). Y. pestis is generally inoculated by a flea bite, while the two others are food-borne pathogens. In spite of these differences in the infection routes, all three have a common tropism for lymphoid tissues and a common capacity to resist the nonspecific immune response, in particular phagocytosis and killing by macrophages and polymorphonuclear leukocytes (PMNs). Y. pestis and Y. pseudotuberculosis are natural rodent pathogens. Although this does not seem to be the case for Y. enterocolitica, experimental infection of mice reproduces some of the symptoms observed in humans, in particular those related to invasion of the lymphoid tissues. After orogastric inoculation of mice, Y. enterocolitica selectively invades the Peyer's patches via M cells (15, 131, 140). This invasion leads to an enormous recruitment of PMNs, formation of microabscesses comprising extracellular Yersinia, and, finally, complete destruction of the cytoarchitecture of the Peyer's patches. Later, abscesses appear in mesenteric lymph nodes, suggesting that Y. enterocolitica disseminates via the lymphatic vessels (15). Anatomopathological examination of mice experimentally infected with Y. pseudotuberculosis also concluded that these bacteria are largely extracellular (309). In accordance with these in vivo observations, Yersinia manifests some resistance to phagocytosis in vitro, both by macrophages (87, 281) and by PMNs (53, 65, 291, 361). Once they are phagocytosed, Y. pseudotuberculosis and Y. enterocolitica generally do not survive. These observations led to the concept that Y. pseudotuberculosis and Y. enterocolitica are extracellular pathogens and that their survival strategy basically consists in avoiding the nonspecific immune response. Y. pestis has the same capacity as the other Yersinia spp. to resist phagocytosis. However, if it has been phagocytosed, it probably has a better capacity to resist killing. Early work by Straley (333, 334) showed that indeed Y. pestis can grow in the phagolysosome of cultured murine resident peritoneal macrophages. The reason for this capacity is not clearly established, but it does not depend on the type III system.

It has been known since the mid-1950s that Y. pestis is unable to grow at 37°C in Ca²⁺-deprived media (157). It has also been known for decades that this unusual property can be lost and that its loss correlates with a loss of virulence. This Ca²⁺ dependency phenotype offered an extraordinary clue to the pathogenicity arsenal because nonvirulent mutants could be easily detected and even selected for. It appeared that virulence and Ca²⁺ dependency are encoded by a 70-kb plasmid (112, 390), sometimes called pYV (200). Under conditions of growth restriction, this plasmid governs the synthesis of a set of about 12 proteins called Yops (for "Yersinia outer membrane proteins"), which were originally designated by a letter, a number, or their molecular weight, according to the authors (42, 44, 73, 74, 97, 100, 110, 220, 256, 267, 330, 331, 377). The LcrV protein, an antigen of Y. pestis that had already been discovered in the mid-1950s (53), turned out to be one of these Yops (97, 239, 331). Most of the yop genes have been identified and sequenced, and they appeared to be almost identical in the three species. A uniform nomenclature has been introduced for YopB, YopD, YopE, YopH, YopM, and LcrV. YopN is sometimes still called LcrE (360). A few other Yops do not benefit from a common nomenclature because they were discovered or characterized more recently: YopO, YopP, and YopQ in Y. enterocolitica (74, 229) are called YpkA, YopJ, and YopK, respectively, in Y. pseudotuberculosis (111). The YopJ nomenclature is also used in Y. pestis (330). YopR (8) turned out to be the product of yscH. A Y. pestis YopL has been mentioned (329, 332), but its gene has not yet been identified and sequenced and it is not known whether it corresponds to a Yop described in Y. enterocolitica or Y. pseudotuberculosis. Finally, YopT was described only very recently (170). The "S" has been skipped to avoid confusion with Yop in the plural.

Although initially described as outer membrane proteins, the Yops could also be recovered from the culture supernatant (149, 151), and it was later found that they were actually secreted proteins (229). Their secretion occurs by a new pathway (now called type III) and requires a specific apparatus (called Ysc for "Yop secretion"), which is also encoded by the pYV plasmid (228, 229).

To trigger Yop secretion in vitro, Yersinia is generally grown at 28°C in a medium depleted of Ca²⁺ and then transferred to 37°C. Ca²⁺ depletion (or contact with a eukaryotic cell [see below]) and temperature both control transcription of the yop genes. The best-characterized regulator is VirF (LcrF in Y. pestis and Y. pseudotuberculosis), a transcriptional activator of the AraC family (72). It controls transcription of most of the genes involved in Yop synthesis and secretion (199).

Genetic analysis indicated that most of the Yop proteins are essential for virulence. In particular, YopE turned out to be responsible for a cytotoxic activity (282) that had been described earlier (119, 266). YopH was found to inhibit the phagocytosis of bacteria by macrophages (281) and later was shown to be a protein tyrosine phosphatase (PTPase) related to eukaryotic counterparts (132). However, three observations were enigmatic: (i) Yops form large and insoluble aggregates in the culture medium, which is unusual for virulence effectors; (ii) YopE has no toxic activity on its own (119, 287); and (iii) what would be the role of an extracellular PTPase?

A major advance occurred when Rosqvist et al. (283) showed that Yop preparations elicit a cytotoxic response when microinjected into HeLa cells, indicating that the target of the YopE protein was intracellular. A yopD mutant was unable to affect HeLa cells, while a preparation of Yops secreted by the very same mutant was cytotoxic when microinjected into the cytosol of HeLa cells. Rosqvist et al. logically concluded from this that the YopD protein should play a role in translocating the YopE protein across the plasma membrane of the eukaryotic target cell to reach the cytosolic compartment (283).

The evidence for YopD-mediated translocation of the YopE protein was essentially genetic. In 1994, this elegant hypothesis was confirmed by two different approaches. The first was based on immunofluorescence and confocal laser-scanning microscopy examinations. Rosqvist et al. (285) showed that the YopE protein appeared in the cytosol of HeLa cells infected with wild-type Y. pseudotuberculosis. In contrast, when cells were infected with a mutant strain of Y. pseudotuberculosis unable to produce YopD, YopE was no longer internalized, showing that the YopD protein was essential for the translocation of YopE across the target cell membrane (285). The second approach was based on a reporter enzyme strategy introduced by Sory and Cornelis (321) (Fig. 1). The reporter system consisted of the calmodulin-activated adenylate cyclase domain (called Cya) of the Bordetella pertussis cyclolysin (118). The rationale was as follows: the Yop-Cya hybrid enzyme introduced into the cytosol of eukaryotic cells would produce cyclic AMP (cAMP) while the intrabacterial Yop-Cya hybrid would not, because of the absence of calmodulin in the bacterial cytoplasm. Since the catalytic domain of cyclolysin is unable to enter eukaryotic cells by itself, accumulation of cAMP would essentially reflect Yop internalization. Infection of HeLa cells with recombinant Y. enterocolitica producing a hybrid YopE-Cya protein resulted in a marked increase in the level of cAMP even when internalization of the bacteria themselves was prevented by cytochalasin D. Infection with a Y. enterocolitica mutant unable to produce both the YopD and YopB proteins did not lead to cAMP accumulation, confirming the involvement of YopD and/or YopB in translocation of the YopE protein across eukaryotic membranes (321).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   The Yop-Cya reporter strategy used to study translocation of Yop proteins into the cytosol of eukaryotic cells. Reprinted from reference 321 with permission of the publisher.

In light of these results, a coherent model could be established. According to this model (Fig. 2), the Yops form two distinct groups of proteins. Some Yops are intracellular effectors delivered inside eukaryotic cells by extracellular Yersinia organisms adhering at the cell surface, while other Yops (translocator Yops) form a delivery apparatus. This model is now largely supported by a number of other results that will be presented in this review. Among others, it is supported by immunological observations. While antigens processed in phagocytic vacuoles of phagocytes are cleaved and presented by major histocompatibility complex class II molecules, epitope 249-257 of YopH produced by Y. enterocolitica during a mouse infection is presented by major histocompatibility complex class I molecules, like cytosolic proteins (328).

View larger version (55K): [in this window] [in a new window]   FIG. 2.   A tentative model for the interaction between Yersinia and a macrophage. When Yersinia is placed at 37°C in a rich environment, the Ysc secretion apparatus is installed and a stock of Yop proteins is synthesized. Some of these proteins are capped with their specific Syc chaperones, which presumably prevent premature associations. As long as there is no contact with a eukaryotic cell, the YopN-TyeA-LcrG plug blocks the Ysc secretion channel. Upon Ca2+ depletion or contact with the eukaryotic target cell, the secretion channel opens and the YopB translocator inserts in the eukaryotic cell with the help of YopD and LcrV. The Yop effectors (YopE, YopH, YopM, YopO/YpkA, YopP/YopJ, and YopT) are then transported through the secretion channel and translocated across the plasma membrane, guided by the translocators. YopE and YopT act on the cytoskeleton, while YopP/YopJ induces apoptosis and inhibits the release of TNF-.

The virulence plasmid thus encodes an integrated antihost system allowing the delivery of a set of effector Yops into the cytosol of eukaryotic cells by a delivery apparatus and a specialized secretion system. The virulence plasmid has now been completely sequenced in Y. enterocolitica W22703 (pYV227) (171) and in Y. pestis KIM (pCD1) (165a, 257a). Most of the sequence of plasmid piB1 from Y. pseudotuberculosis YPIII is also available. The genetic maps are given in Fig. 3. About 50 genes are involved in virulence, and they occupy three-quarters of the plasmid. A total of 35 genes encoding the secretion and translocation machineries form a continuous block flanked on both sides by more dispersed genes encoding effectors and their chaperones.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   The genetic maps of pYV227 from Y. enterocolitica W227 (serotype O:9) (redrawn from reference 170), pIB1 from Y. pseudotuberculosis YPIII (redrawn from reference 259), and pCD1 from Y. pestis KIM (redrawn from references 165a and 257a). Note that none of these maps is complete. For pCD1, the plasmid has been sequenced twice (165a, 257a) and only the genes that are identified in the two sequences or in one sequence and in Y. enterocolitica are shown. Plasmid pYV227 has also been completely sequenced (171), but only the genes described in this review are included here. Shading of the genes has been done on the basis of the data presented in this review.

We first review the effects of this virulence apparatus on eukaryotic cells and then analyze in detail the fate of the Yops, from secretion to delivery and action in eukaryotic cells. We then describe the adhesin YadA and, finally, review the genetic aspects, regulation of gene expression, and plasmid organization.

EFFECTS ON HOST CELLS

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Macrophages

Inhibition of phagocytosis. One of the simplest ways to resist killing by macrophages is to avoid being ingested. It has been known for a long time that Yersinia spp. are endowed with the capacity to resist phagocytosis by macrophages and that this property depends on the presence of the pYV plasmid (58, 59).

Inhibition of the respiratory burst. It was suspected for a long time that Yersinia interferes with the normal respiratory burst of macrophages, since the oxidative burst occurring after interaction with Y. pestis is much lower than that seen after phagocytosis of E. coli (59). More recently, Hartland et al. (147) infected bone marrow-derived macrophages with various Y. enterocolitica mutant strains before stimulation of the respiratory burst by the addition of zymosan, which triggers the CR3 receptor. They measured the intensity of the respiratory burst by assaying the amount of reduced cytochrome c produced during the generation of O₂ (127). This showed that Y. enterocolitica also has the capacity to inhibit the respiratory burst and that this capacity depends on the pYV plasmid. Loss of the effectors YopE, YopH, and YopO/YpkA did not affect this capacity, but loss of the translocator YopD did. This property thus probably depends on an effector different from YopE, YopH, and YopO.

Induction of apoptosis. In 1986, Goguen et al. (119) reported that Y. pestis and Y. pseudotuberculosis have a cytotoxic effect on the mouse macrophage cell lines IC21 and P388D1 as well as on mouse resident peritoneal macrophages. They observed that cells infected with a wild-type strain change shape, acquire a granular aspect, and detach easily from the culture dish. This effect, which was dependent on the presence of the pYV plasmid, evokes apoptosis, although it was not described as such at that time. Recently, three groups, two working with Y. enterocolitica (232, 290) and one working with Y. pseudotuberculosis (237), showed that Yersinia triggers apoptosis of cultured macrophages. Infected macrophages displayed general features of apoptosis, such as membrane blebbing (apoptotic body formation), cellular shrinkage (232, 290), and DNA fragmentation (Fig. 4). Infection of macrophages with secretion and translocation mutants of Y. enterocolitica did not lead to apoptosis, showing that a translocated Yop effector is involved. Screening of a library of yop mutants showed that the YopE cytotoxin is not involved and identified YopP as the effector responsible for apoptosis (232) (Fig. 4). In an independent study, Monack et al. (237) came to the conclusion that YopJ, the Y. pseudotuberculosis homolog of YopP, is required for the induction of the cell death process. The phenomenon displays some cell specificity, since epithelial cells (232, 237, 290) and fibroblasts (237) do not undergo apoptosis upon infection with Yersinia. The mechanism by which Yersinia induces macrophage apoptosis remains to be elucidated, but it parallels that used by cytotoxic T lymphocytes to kill their target cells; cytotoxic T cells inject granzyme B into the cytosol of their target cells, thereby inducing apoptosis (308). One of the virulence functions of Yersinia organisms thus appears to mimic a physiological process of their host.

View larger version (154K): [in this window] [in a new window]   FIG. 4.   YopP-induced apoptosis. Semithin sections were stained with toluidine blue and examined by light microscopy (a to d). (a) Wild-type Y. enterocolitica E40. Apoptotic nuclei (arrows) and cell surface-associated bacteria (brown particles, arrowhead) are visible. (b) yscN secretion mutant. No apoptotic cells are detected. Internalized bacteria either in tight (single arrowhead) or spacious (double arrowhead) phagosomes are abundant. (c) yopP effector mutant. No apoptotic cells are detected. Bacteria are seen at the cell surface (arrowhead). (d) yopP+++ (yopP cloned in a multicopy vector). Apoptotic cells are visible (arrows). (e and f) Ultrastructural analysis of cells infected with yopP+++ from panel d is shown. Typical features of apoptosis include (i) peripheral chromatin condensation in crescents, except in the vicinity of nuclear pores (large arrows); (ii) bulging of nuclear crescents into the cytoplasm (best seen in panel e); and (iii) appearance of central clusters of small particles of unknown nature, typical of apoptosis (small arrow in panel f). Nuclear and plasma membrane alterations contrast with a good ultrastructural preservation of cytoplasm, particularly of endoplasmic reticulum and mitochondria. Bars, 10 µm (a to d) and 2 µm (e and f). Reprinted from reference 232 with permission of the publisher.

Inhibition of TNF- and IFN- release. TNF- is a proinflammatory cytokine that plays a central role in the development of the immune and inflammatory responses to infection. Secreted mainly by macrophages, TNF- acts on various cell types involved in the host defense mechanisms. It stimulates both macrophage and PMN microbicidal activity and acts on natural killer cells together with IL-12 to provoke the release of gamma interferon (IFN-), which further increases the microbicidal activity of macrophages. Moreover, TNF- induces expression of adhesion molecules on endothelial cells and is chemotactic for monocytes, thus contributing to the amplification of the inflammatory response (for a review, see reference 356). The importance of the cytokines TNF- and IFN- in the host immune response against a Yersinia infection was first illustrated by the fact that treatment of mice with antibodies directed against TNF- or IFN- exacerbates infection by Y. enterocolitica (16). Moreover, an immunohistological study showed that administration of anti-TNF- antibodies to mice before and after orogastric infection with Y. enterocolitica leads to complete destruction of Peyer's patches and to a dramatic increase of bacterial counts in Peyer's patches, mesenteric lymph nodes, and spleen, even though phagocytes were normally recruited in Peyer's patches and mesenteric lymph nodes (17). This suggests that TNF- plays an essential role in the local host defense mechanism in the intestinal tissues, possibly by activating phagocytes (17).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Model showing the effects of Yersinia spp. on the macrophage intracellular cascades. Lipopolysaccharide (LPS) activates the ERK1/2, JNK, and p38 MAPK pathways, leading to increased TNF- production. Activated MAPKs can lead to NF-B activation; activated NF-B can, in turn, enhance TNF- transcription. Translocated YopP/YopJ induces macrophage apoptosis by a mechanism involving caspase activation. It also downregulates MAPKs and impairs NF-B activation, two effects that could explain the YopP/YopJ-induced reduction of TNF- production. See the text for details and references.

Resistance to phagocytosis and killing. The interaction between Y. enterocolitica and PMNs was first studied by monitoring the luminol-enhanced chemiluminescence (CL) response (211), which is a measure of the intensity of the oxidative burst (82). A pYV⁺ Y. enterocolitica strain grown at 37°C (Yop-inducing conditions) induced four- to sixfold less CL than did the same strain grown at 25°C or a plasmidless, isogenic strain grown at either temperature. This demonstrated for the first time the involvement of pYV-encoded proteins in the inhibition of the PMN oxidative burst (211). Since the CL response is a sensitive, indirect measure of the degree of phagocytosis in human neutrophils (126), this also suggested that Y. enterocolitica may resist phagocytosis by PMNs. Indeed, Lian et al. (210) showed that wild-type pYV⁺ bacteria are resistant to phagocytosis by PMNs while pYV bacteria are not. This effect was seen not only in vitro but also in vivo; after intradermal inoculation into rabbits, histological examination of the inflammatory lesions by light or electron microscopy revealed that numerous bacteria of the pYV strain were located intracellularly in vacuoles of PMNs and mononuclear cells while pYV⁺ bacteria were extracellular and surrounded by inflammatory cells without being phagocytosed (209).

Resistance to antimicrobial peptides. As described above, pYV⁺ Y. enterocolitica strains impede to some extent their phagocytosis by PMNs. However, when ingested, most of the pYV⁺ bacteria are not killed whereas pYV bacteria are killed almost instantly (86, 362), implying that plasmid-encoded factors can interfere with the killing mechanisms. These involve oxygen-dependent mechanisms (oxidative burst) and oxygen-independent mechanisms, which include acidification of the phagosome and attack by antimicrobial polypeptides. Antimicrobial polypeptides present in azurophilic granules of human granulocytes include bactericidal permeability-increasing protein, cathepsin G, elastase, proteinase 3, azurocidin, lysozyme, and defensins. These antimicrobial polypeptides are released into the phagolysosome through fusion of cytoplasmic granules with the phagosomes. Using a gel overlay assay (205), Visser et al. (362) showed that pYV Y. enterocolitica strains are more susceptible to these granule-antimicrobial polypeptides than are wild-type Yersinia strains. Similarly, a yadA mutant was also more sensitive than wild-type bacteria, and introduction of a plasmid encoding only YadA in a pYV strain restored, at least partially, the bacterial protection against the microbicidal activity of the granule extracts. YadA is thus involved in the resistance of Y. enterocolitica to the antimicrobial activity of polypeptides from human granulocytes, although the involvement of other plasmid-encoded factors could not be completely ruled out (362).

Cytotoxicity. HeLa cells have been very important in the discovery of injection of Yop effectors inside eukaryotic cells by extracellular adhering bacteria (137, 259, 285, 321). HEp-2 cells (267, 358, 359) and HeLa cells (287) are very sensitive to the cytotoxic effect of YopE. This cytotoxic effect consists in rounding up of the cells and detachment from the extracellular matrix (119, 282). Rosqvist et al. (283) showed that the YopE-induced cytotoxicity is due to disruption of the actin microfilament structures of the target cell and that this effect is mediated by intracellularly located YopE. In addition to YopE, three other Yops, namely YopH, YopO, and YopT, have a cytotoxic effect on cultured epithelial cells (see "Yop effectors and their targets" for details).

Cytokine response. The cytokine response of epithelial cells to Yersinia infection has been investigated by using the HEp-2 human laryngeal epithelium cell line (13) and various human colon epithelial cell lines (181, 303). The capacity of HEp-2 cells to release cytokines is modified upon Yersinia infection, and although these cells do not originate from the gastrointestinal tract, this observation suggests that epithelial cells may participate in the modulation of the immune response against infection by Yersinia via the release of cytokines (13). In agreement with this idea, infection of monolayers of human colon epithelial cells (T84, HT29, and Caco-2) with invasive bacteria, including Y. enterocolitica, results in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, namely, IL-8, monocyte chemotactic protein-1, granulocyte-macrophage colony-stimulating factor, and TNF-, as assessed by mRNA levels and cytokine secretion (181). The same cytokines, as well as IL-6, are also expressed by freshly isolated human colon epithelial cells and upregulated upon infection with invasive bacteria including Y. enterocolitica (181). These cytokines play a role in the initiation or amplification of the inflammatory response; IL-8 and monocyte chemotactic protein-1 act as potent chemoattractants and activators of neutrophils and monocytes, respectively; TNF- activates neutrophils and mononuclear phagocytes, while granulocyte-macrophage colony-stimulating factor prolongs the survival of neutrophils and monocytes and increases the response of those cells to other proinflammatory stimuli, which can further amplify the inflammatory response. Colon epithelial cells thus appear to be programmed to provide a set of chemotactic and activating signals to adjacent and underlying immune and inflammatory cells in the earliest phases after microbial infection (181). Interestingly, virulent Y. enterocolitica strains induce a significantly lower level of IL-8 secretion by T84 cells than do nonvirulent Y. enterocolitica strains and the YopB and YopD proteins are required for this suppressive effect (303). It is easily conceivable that this effect favors Yersinia, especially during the early phase of infection, by delaying a massive influx of PMNs into the site of infection.

YOP SECRETION

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Yop Secretion Pathway

Discovery of Yop secretion. The Yops were initially described as outer membrane proteins (44, 267, 331). Later, Heesemann et al. (149, 151) showed that Yops could also be recovered from the culture supernatant. Some of the Yops (LcrV, YopM, YopQ/YopK, and YopR) are soluble in the culture supernatant, but others (YopH, YopE, YopO/YpkA, YopB, YopD, YopP/YopJ, and YopN/LcrE) have a propensity to aggregate as visible filaments (229) (Fig. 6). This led Michiels et al. (229) to question the outer membrane localization of the Yops. These authors studied the kinetics of transcription and appearance of the Yops in the different compartments and observed the following. (i) Yops are detected first in the supernatant and later in the membrane fraction. (ii) The appearance of Yops in the membrane fraction is concomitant with the decrease of the corresponding protein in the supernatant. (iii) Disappearance of the less soluble Yops from the supernatant is not a consequence of degradation. (iv) There is a correlation between the propensity of a given Yop to aggregate in the supernatant and the presence of that Yop in the membrane fraction. (v) Yops still accumulate in the membrane fraction after 3 h of induction, whereas transcription of the yop genes at that time is dramatically reduced. (vi) Yops are separated from the cell fraction upon treatment with hydrophobic agents such as xylene or hexadecane, whereas chromosome-encoded integral membrane proteins and YadA are not. On the basis of these observations, Michiels et al. (229) concluded that Yops are not membrane-anchored proteins but true secreted proteins that copurify with membranes when they are prepared as centrifugation pellets. The name YOP, introduced by the group of Wolf-Watz (44) for yersinia outer membrane protein, could thus be questioned, but it is so popular that it was decided, during the Keystone 1990 meeting on Yersinia, to keep it but to write it Yop(s) rather than YOP(s) to indicate that it is not a set of initials. The name "Yersinia outer proteins" fits with the acronym but is not particularly elegant.

View larger version (67K): [in this window] [in a new window]   FIG. 6.   Yops secreted by Y. enterocolitica W22703. Bacteria were grown at 28°C in a conical flask (seen from the top) containing oxalated brain heart infusion and then transferred to 37°C. The photograph showing the Yop filaments was taken 4 h after the temperature shift. SDS-PAGE of the filaments (right lane) and of Yops precipitated from the supernatant by ammonium sulfate (left lane) is shown on the right. Adapted from reference 229.

No classical signal sequence is cleaved off. When Bölin and Wolf-Watz (46) and Michiels and Cornelis (226) sequenced the yopH gene (then known as yop2b and yop51), they noticed that the N-terminal end of the predicted YopH protein does not resemble a typical signal sequence. In 1990, Michiels et al. (229) determined the sequence of the N terminus of the secreted YopH and found the same sequence as that deduced from translation of the 5' end of the gene, including the terminal methionine. Reisner and Straley (274) showed that the 13 N-terminal residues of YopM are also identical to those deduced from the nucleic sequence. The same observation was made later for YopN by Forsberg et al. (99): residues 2 to 9 obtained by the Edman degradation procedure were those encoded by codons 2 to 9. Håkansson et al. (136) reported the same for YopD. Finally, the 7 N-terminal residues of YpkA/YopO and the 11 N-terminal residues of YopJ/YopP have also been found to match the translated nucleic sequence (111). Hence, secretion of YopH, YopN, YopP/YopJ, YpkA/YopO and YopD occurs without removal of an N-terminal signal sequence. This presumably also applies to the other Yops. Indeed, no typical signal was found in the sequence of YopE (100, 229), YopQ (229), YopM (208), LcrV (25), YopB (136), YopR (8), or YopT (170).

The N-terminal (or 5' mRNA) secretion signal. Analysis of the secretion of hybrid proteins composed of the N terminus of YopH or YopE and various prokaryotic or even eukaryotic proteins indicated that the information necessary for Yop secretion is nevertheless contained in the N terminus (227, 321-323). The minimal region shown to be sufficient for secretion of YopH was gradually reduced from 48 residues in a YopH-PhoA hybrid (227) to 17 residues in a YopH-Cya hybrid (320). Similarly, the minimal sequence required for secretion of YopE was reduced to 15 residues by gradual deletions of YopE-Cya hybrids (320) and later even to 11 residues, still by the same approach (300). By analysis of translational fusions to neomycin phosphotransferase (Npt), Anderson and Schneewind (10) localized the YopN secretion signal in the first 15 codons of the gene. The minimal domain of YopM sufficient for secretion of YopM-Cya was found to be shorter than 40 residues (41). For YopO/YpkA and YopP/YopJ, it is shorter than 77 and 43 residues, respectively (324).

A second secretion signal? The experiments of Sory et al. (320) demonstrated that the first 15 codons of YopE contain a signal that is sufficient to promote secretion in rich culture medium. They did not show that this N-terminal signal is absolutely required for YopE secretion. To address this question, Cheng et al. (63) deleted codons 2 to 15 and monitored secretion of the hybrid YopE-Npt. They observed that 10% of the hybrid proteins deprived of the N-terminal secretion signal were still secreted in M9 medium supplemented with 1% Casamino Acids. They inferred that there is a second secretion signal and showed that this second, weaker secretion signal corresponds to the SycE-binding site (see below). Not surprisingly, it is functional only in the presence of the SycE chaperone (63), rejuvenating the pilot hypothesis of Wattiau and Cornelis (366) for SycE. As discussed below, this second signal, binding the chaperone, is required for translocation of YopE into eukaryotic cells (204a, 320).

Conclusion. There are two different signals driving the export of YopE by the type III secretion apparatus. The first is the structure of the 5' mRNA, and the second, built into the protein, uses the chaperone as a pilot. The same could apply to the effectors YopH and YopT. Some other effector Yops do not seem to have a chaperone, in which case they would be recognized only by their N- or 5'-terminal signal. Finally, it must be stressed that we know less about secretion of the translocators. No signal sequence is removed from YopB, YopD, and LcrV, but their secretion signal has not yet been identified. Some observations tend to suggest that secretion of YopB and YopD could proceed by a mechanism slightly different from that used by the effector Yops. First, LcrV appears to be necessary for secretion of YopB and YopD (294). Second, mutations in some genes such as virG (7), yscF (8), or yscM/lcrQ (275, 327) lead to phenotypes in which YopB, YopD, and LcrV are secreted differently from the other Yops.

In 1991, Michiels et al. (228) established that, like the Yops, the Yop secretion apparatus is encoded by the pYV plasmid itself and in particular by genes that they called ysc (for "Yop secretion"). Some of these genes had previously been considered regulatory genes; this misinterpretation can be explained by the fact that there is a strong regulatory feedback that blocks Yop synthesis as soon as secretion is compromised (see "Regulation of transcription of the virulon genes," below).

The ysc genes are contained in four contiguous loci that were initially called virA, virB, virG, and virC (for "virulence") in Y. enterocolitica (73) (Fig. 3). lcrD (for "low calcium response"), initially described in Y. pestis (263), turned out later to be one of these secretion genes (265), and it will probably be called yscV in the future. In total, 28 genes have been identified within these loci. Knockout mutants have been constructed for most but not all of them. The information available on these genes and their products is detailed in the next paragraphs and summarized in Table 1. For the sake of clarity, the four loci are treated separately.

YscC secretin and other products of the virC operon. The virC locus of Y. enterocolitica consists of a large operon, yscABCDEFGHIJKLM, encoding 13 proteins (228, 327). Parts of the virC locus have also been analyzed in Y. pestis (134) and Y. pseudotuberculosis (275), where the counterparts of yscH, yscI, yscJ, yscK, yscL, and yscM have been initially called lcrP, lcrO, lcrKa, lcrKb, lcrKc, and lcrQ, respectively. Apart from yscM1, which is called lcrQ in Y. pseudotuberculosis (275), the ysc nomenclature has now been adopted in the three species. Nonpolar mutations in yscC, yscD, yscE, yscF, yscG, yscI, yscJ, yscK, and yscL completely abolish Yop secretion (8, 265). In contrast, nonpolar yscA, yscH, and yscM mutants are not impaired in Yop secretion (8, 296).

VirG/YscW lipoprotein. virG is a small, monocistronic gene situated immediately upstream from the virC operon and downstream from the regulatory gene virF (7) (Fig. 3). It encodes a polypeptide of 131 amino acids with a predicted molecular mass of 14.7 kDa and a calculated isoelectric point of 11.1. The signal sequence of VirG ends with Leu-Xaa-Gly-Cys, a motif characteristic of the processing site of lipoproteins. While attempting to show that VirG is a lipoprotein that can be labelled by [³H]palmitate, Allaoui et al. (7) encountered the difficulty that gram-negative bacteria produce several lipoproteins in the range of 10 to 30 kDa. To circumvent this, they labeled three strains containing different virG-phoA gene fusions and detected the larger VirG-PhoA hybrid proteins among the proteins labelled with [³H]palmitate. VirG is thus a small lipoprotein. Allaoui et al. (7) constructed a nonpolar virG mutant and observed that secretion of some Yops, in particular YopB, YopD and LcrV, was severely impaired. The function of VirG became more clear when the YscC secretin was characterized by Koster et al. (194). It appeared that VirG is required for proper insertion of YscC in the outer membrane, but more work is needed for an understanding of its exact function (194). The correlation between the role of VirG in the installation of the secretin and the requirement of VirG for secretion of YopB, YopD, and LcrV suggests that these Yops could be the most bulky ones to be transported through the YscC channel. Since lipoprotein VirG belongs to the Ysc secretion apparatus, we suggest that it be renamed YscW.

Products of the virB operon. The virB operon consists of eight genes, yscN to yscU (24, 93). Among the proteins encoded by these genes, YscN, YscR, and YscU are the best characterized so far. YscN is a 47.8-kDa protein with ATP-binding motifs (Walker boxes A and B) resembling the  catalytic subunit of F₀F₁ proton translocase and related ATPases. A pYV derivative encoding an YscN protein deprived of Walker box A is impaired in Yop secretion, indicating that YscN is a necessary component of the secretion machinery and possibly acts as an energizer (375). It has not been shown that YscN is an ATPase, but this was shown for InvC, the YscN homolog in S. typhimurium (83). It is thus reasonable to assume that YscN acts as an ATPase. YscR is a 24.4-kDa inner membrane protein with four transmembrane regions and a large central hydrophilic domain, as suggested by the analysis of yscR-phoA translational gene fusions (93). The 40.3-kDa YscU is a second inner membrane protein with four transmembrane segments anchoring a large cytoplasmic C-terminal domain (9). Not surprisingly, mutations in yscR and yscU abolish Yop secretion (9, 93). Interestingly, the products of yscO (251) and yscP (252a, 326a) are secreted by the Ysc apparatus under low-Ca²⁺ conditions. YscO (251) is required for secretion of all the Yops, while YscP (252a) is required for normal secretion of some Yops. Less is known about YscQ (93) and YscS (261), but they have been shown to be required for Yop secretion. Finally, the importance of YscT has not been determined.

virA locus: yopN, tyeA, sycN, yscXY, lcrD/yscV, and lcrR. The virA locus encodes first YopN, TyeA, and SycN, which are described later in this review. The next two genes, yscX and yscY, encode small proteins that are required for Yop secretion (170a). The next gene, lcrD/yscV, encodes a 77-kDa inner membrane protein that is required for Yop secretion (263, 264). LcrD/YscV is the archetype of a family of proteins encountered in every known type III system (Table 2). The predicted overall secondary structure of these proteins is quite well conserved and consists of a hydrophobic N terminus with six to eight potential transmembrane domains and a hydrophilic C terminus protruding into the cytoplasm. All the members of this family can be aligned over the entire length of the amino acid sequence, with the highest degree of homology occurring in the N terminus (108, 263, 264). At least some of the members have interchangeable functions. For instance, MxiA from Shigella is able to complement the eukaryotic cell entry defect of a Salmonella invA mutant, but LcrD/YscV from Yersinia canno