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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Gram-negative enteropathogen Yersinia pseudotuberculosis causes infections in human and animals. Infections are characterized by gastrointestinal symptoms, such as diarrhoea, abdominal pain and fever (Cover and Aber, 1989). Infections in humans are usually self-limiting, but Y. pseudotuberculosis can cause lethal infections in rodents. The pathological changes caused by Y. pseudotuberculosis infections in mice resemble those caused by the closely related species Y. pestis in humans. Yersinia proliferates in lymphoid tissues and is eventually disseminated via the lymphatic system throughout the body. Dissemination in the bloodstream cause acute bacteriaemia and septicaemia. As a result of widespread infection to many organs, necrotic and haemorrhagic lesions lead to the death of the human or animal host.

Yersinia species have the ability to resist host defence mechanisms by inhibition of phagocytosis, cytokine release and oxidative burst responses (Rosqvist et al., 1988; 1990; Bliska and Black, 1995; Fällman et al., 1995; Schesser et al., 1998). The property of evading the host defence is based on Yersinia outer proteins (Yops), which are encoded on an ≈ 70 kb virulence plasmid common to all virulent Yersinia strains (Portnoy et al., 1983; Heeseman et al., 1984). Upon bacterial contact with the target cell, Yop effector proteins are translocated into the cytosol of the eukaryotic host cell, while the bacteria remain extracellular (Rosqvist et al., 1994; Persson et al., 1995; Boland et al., 1996; Håkansson et al., 1996). The Yop effectors responsible for inhibition of phagocytic processes are YopH, a protein tyrosine phosphatase, and YopE, characterized as a contact-dependent cytotoxin (Rosqvist et al., 1988; 1990; 1994; Fällman et al., 1995; Persson et al., 1997). YopH has been shown to dephosphorylate focal adhesion kinase (FAK) and p130CAS and was suggested to act through a specific disruption of b1 integrin containing focal adhesion complexes (Black and Bliska, 1997; Persson et al., 1997). The specific localization of YopH to focal adhesions has been demonstrated to be essential for antiphagocytosis and for virulence during Yersinia infection (Persson et al., 1999)

Experiments using HeLa cells have demonstrated that YopE causes the disruption of actin microfilament stress fibres (Rosqvist et al., 1991). YopE did not have any effect on DNA or protein synthesis. Levels of Ca2+ or cyclic AMP remained unchanged in the eukaryotic cell, and no effect on the ADP ribosylating capacity of the cells could be observed. As in vitro-polymerized actin filaments were unaffected by YopE, it was suggested that YopE does not affect F-actin itself, but rather affects mechanisms involved in the regulation of the actin cytoskeleton (Rosqvist et al., 1991).

In mammalian cells, the Rho subfamily of small GTPases is involved in the rearrangement of the actin cytoskeleton. Microinjection of activated Cdc42, Rac or Rho proteins induces the polymerization of actin into particular structures. Activated Cdc42 leads to the formation of filopodia and microspikes (Kozma et al., 1995), while microinjection of Rac generates lamellipodia and membrane ruffles (Ridley et al., 1992).

Rho has been demonstrated to be directly involved in actin stress fibre formation. In fibroblasts, the dominant activated mutant protein RhoV14 induces stress fibres upon microinjection, and lysophosphatidic acid (LPA)-induced stress fibre formation is mediated by Rho (Ridley and Hall, 1992).

Furthermore, Cdc42 can induce lamellipodia and membrane ruffles through the activation of Rac (Kozma et al., 1995; Nobes and Hall, 1995) and, likewise, activated Rac can induce stress fibres through the stimulation of Rho activity (Ridley et al., 1992; Nobes and Hall, 1995). YopE has been shown to cause disruption of stress fibres (Rosqvist et al., 1991) and also to inhibit the Rho-dependent invasion of Shigella (Mecsas et al., 1998); therefore, we wanted to investigate the Rho pathway as a putative target for YopE in order to understand its mode of action. As a model system, we have chosen to use yeast, as we found earlier that, when YopE is expressed in Saccharomyces cerevisiae, growth is inhibited.

In this study, we show that the YopE cytotoxin targets the Rho pathway. We show that YopE is a GTPase-activating protein (GAP) of small RhoGTPases in vitro and that the GAP activity is essential for the cytotoxic effect mediated by YopE.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

YopE expression blocks cell growth of S. cerevisiae

We investigated the effect of heterologous expression of the Y. pseudotuberculosis YopE protein in S. cerevisiae. A 0.9 kb BamHI–HindIII fragment of plasmid pTB1 (Rosqvist et al., 1990), carrying a promoterless yopE gene was cloned under the control of the methionine-inducible MET3 promoter. The MET3 promoter is only expressed when cells are grown in media lacking methionine, while expression is repressed by the presence of methionine. The MET3–yopE construct was introduced into plasmid pRS415 (Christianson et al., 1992) and transformed into yeast strain FY24. Controls were plasmids pRS415M without insert and pRS415M-yopE191. Plasmid pRS415M-yopE191 contains a yopE mutant gene truncated for the 28 most C-terminal amino acids, and this construct is not able to mediate a cytotoxic effect on HeLa cells (data not shown). Transformants were restreaked onto SC plates lacking either leucine (to select for the plasmid) or leucine and methionine, and incubated at 30°C. As expected, we found that all strains were growing on SC-leu plates. In contrast, only strains expressing wild-type yopE were unable to form colonies upon induction of the MET3 promoter on plates lacking methionine (Fig. 1). As the levels of expression from plasmids pRS415M-yopE and pRS415-yopE191 were similar, as determined by Western blotting (data not shown), these results show that expression of wild-type YopE is inhibitory for yeast cell growth. Furthermore, expression of the protein tyrosine phosphatase YopH, or expression of YopD, which is essential for the translocation of Yop effector proteins, did not affect the growth of S. cerevisiae strains (data not shown). Thus, the YopE protein of Y. pseudotuberculosis mediates a specific cytostatic effect on S. cerevisiae cells.

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Figure 1. YopE expression blocks cell growth of S. cerevisiae. The Y. pseudotuberculosis YopE protein was cloned under the control of the methionine-inducible MET3 promoter and transformed into yeast strain FY24 (MATα ura3-52 trp1Δ63 leu2Δ1). Controls are plasmids pRS415M without insert and pRS415M-yopE191. Cells expressing wild-type yopE were unable to form colonies upon induction of the MET3 promoter on plates lacking methionine.

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Mutants defective in any of the components of the Rho1 Pkc1 pathway undergo cell lysis at higher temperatures, a phenotype that can often be rescued on osmotically stabilized media. If the molecular target of YopE were part of the Rho1 Pkc1 signalling pathway and YopE interfered with the function of that component, one might expect that the inhibitory effect of YopE could be suppressed on osmotically stabilized media. Strain FY24 (pRS415MyopE) was streaked onto SC-leu-met plates supplemented with 1 M sorbitol, 100 mM CaCl2, 100 mM MgCl2 or 500 mM NaCl. However, osmotically stabilized media did not allow YopE-expressing strains to grow on plates lacking methionine (Fig. 3, see pRS414 control). Thus, osmotic stabilizing agents cannot suppress the cytostatic effect of YopE expression.

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Figure 3. Effect of different proteins on the survival of S. cerevisiae cells expressing YopE. All strains are FY24 (MATα ura3 52 trp1Δ63 leu2Δ1) carrying either pRS415MYopE or pRG416MYopE and the indicated plasmids.

C. Growth on SC plates lacking leucine and uracil (left) is compared with growth on SC plates lacking methionine, cysteine, leucine and uracil (right). All plates are supplemented with 0.8 M sorbitol as an osmotic stabilizer. For plasmid designations, see Table 1.

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Components of the Rho1 pathway suppress the cytostatic effect of YopE

To investigate whether co-expression of components of the Rho1 pathway (Fig. 2) could titrate the cytostatic effect of YopE in yeast, we transformed strain FY24 carrying either pRS415MyopE or pRG416MyopE with low-copy-number plasmids containing the Rho1-GEF ROM2, RHO1, PKC1, BCK1 (a MEKK kinase homologue, activated by Pkc1p) or ACT1, encoding actin in yeast. In addition, we tested the PI-3 kinase homologue TOR2, and SAC7 and BEM2, two Rho1-GTP activating proteins (GAPs), on high-copy-number plasmids, as well as PKC1 overexpressed from the strong inducible GAL1 promoter.

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Figure 2. Schematic representation of the RHO1-PKC pathway in S. cerevisiae. MAPK kinase cascades are shown in boxes. Relevant interacting protein components are shown in black. Only proteins of importance for the present study are shown, and the overview is not intended to be comprehensive.

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As the Rho1 pathway also regulates the cell wall integrity of yeast cells (see above), transformants were streaked onto plates supplemented with sorbitol to uncouple the cell wall integrity pathway from other signalling pathways. We found that co-expression of ROM2, RHO1 and, to a lesser extent, BCK1 overcame the cytostatic effect of YopE on yeast cells (Fig. 3). We could not observe any effect of TOR2, SAC7, PCK1 or ACT1 (data not shown). The weak suppression observed by co-expression of BCK1 was improved when the dominant active allele BCK1-20 (Lee and Levin, 1992) was transformed (Fig. 3), indicating that YopE probably affects processes upstream of Bck1p. Thus, these data strongly suggest that an activated Rho1 pathway can overcome the cytostatic effect mediated by YopE in yeast cells.

YopE specifically affects the Rho-mediated pathway in yeast

The Rho GEF Rom2p has been found to function as a Rho-specific exchange factor (Ozaki et al., 1996). The finding that co-expression of ROM2 suppressed YopE cytotoxicity strongly implies that YopE actually targets the Rho pathway in yeast. To confirm this specificity towards the Rho pathway further, we tested whether other small GTPases would be able to suppress YopE cytotoxicity. We transformed strain FY24 carrying either pRS415MyopE or pRG416MyopE with high-copy-number plasmids carrying CDC42, RAS2 or RHO4. In addition, we also tested a dominant active allele of RAS2 (RAS2V19) on a low-copy-number plasmid. We found that co-expression of CDC42, RAS2 or RAS2V19 did not rescue strains expressing YopE. Co-expression of RHO4, however, did suppress the cytostatic effect of YopE expression on yeast cells (Fig. 4). RHO4 is considered to be functionally related to RHO3 and to be involved in bud growth. However, overexpression of RHO4 also rescues the temperature-sensitive growth phenotype of a Δrom2 mutant, indicating that RHO4 might have overlapping functions with RHO1 (Ozaki et al., 1996).

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Figure 4. Intrinsic and YopE-stimulated GTPase activity of RhoA, Rac and Cdc42. Recombinant Rho proteins were loaded with [γ-32P]-GTP for 10 min at 30°C in loading buffer. MgCl2 and unlabeled GTP were added. For GAP stimulation, YopE (300 nM) was added to RhoA (▪), Rac (●) and Cdc42 (▴) (3 µM) and incubated for the indicated time intervals at 30°C.As a control, GTPases were incubated without YopE (open symbols). GTPase activity was analysed by filter binding assay. The remaining GTP at each time is shown as the mean (± SD) of three independent experiments.

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The MEKK kinase Bck1p also suppressed the cytotoxic effect of YopE in yeast (Fig. 3). We therefore tested whether expression of STE11, a MEKK kinase with 31% identity to BCK1 that functions in the Rho1-independent pheromone response pathway, could also suppress YopE cytotoxicity. We found that expression of STE11 did not rescue strains expressing YopE (Fig. 5). However, co-expression of the PAK kinase homologue STE20, which is involved in both the activation of Ste11p (Herskowitz, 1995) and the activation of Bck1p (Zarzov et al., 1996; Jacoby et al., 1998), did suppress the cytotoxic effect of YopE. Thus, co-expression of genes for components of the Rho1-Pkc1 pathway, or genes implicated in the activation of the Rho1-Pkc1 pathway, suppresses the cytotoxic effect of YopE in yeast.

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Figure 5. Intrinsic and YopE-stimulated GTPase activity of small GTPases. Recombinant RhoA, Rac1, Cdc42, RhoA-Q63E, Ras and GSTRal were loaded with [γ-32P]-GTP for 10 min at 30°C in loading buffer. MgCl2 and unlabelled GTP were added. For GAP stimulation, YopE (3 µM) was added to 3 µM GTPase and incubated for 4 min at 30°C. GTPase activity was analysed by filter binding assay. The remaining GTP at each time point is shown as the mean (± SD) of three independent experiments.

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Effect of mammalian RhoGTPases on YopE cytotoxicity in yeast

We tested whether expression of the mammalian rhoA gene (under control of the Rho1 promoter, Qadota et al., 1994), as well as mammalian rac1 and the human cdc42 homologue G25K (under the MET3 promoter), could rescue yeast cells expressing YopE. We found that expression of RhoA, or activated forms of G25K and Rac1 (G25KV12 and RAC1V12), did suppress the cytotoxic effect of YopE, even though to a lesser extent than Rho1 (Fig. 3 and data not shown). Thus, these data support further the finding that YopE specifically interferes with the activity of small Rho-GTPases in vivo.

YopE is a GAP protein for RhoGTPases

As the activated Rho1 pathway suppressed the cytotoxic effect of YopE, we reasoned that YopE functions through an inactivation of the RhoGTPase switch. This could be accomplished by either an inactivation of RhoGTPase exchange factors or by an inactivation of Rho1-GTP. As RhoGTPase exchange factors are redundant (Rom1p and Rom2p in S. cerevisiae), it was unlikely that YopE would act through an inactivation of these proteins. We therefore investigated whether YopE would possess a GTPase-activating activity in vitro using a cell-free system. To facilitate purification, a his-tagged variant of YopE was constructed and expressed in the Yersinia MYM strain unable to express YopH, M, E, K, B and YpkA (Persson et al., 1997). Secreted YopE was recovered from the culture medium as described by Michiels et al. (1990). The YopE-containing aggregates were dissolved in 3 M guanidium-HCl and purified by affinity chromatography using Ni-NTA agarose in the presence of 3 M guanidium-HCl. The N-terminal part of YopE, including the his-tag, was cleaved off by the enterokinase protease. With the use of a bead-loading technique (Rosqvist et al., 1991), purified YopE (90–219) was introduced into the cytosol of HeLa cells and found to induce a cytotoxic effect similar to that of the full-length YopE, showing that the purified protein was active (data not shown).

The influence of purified YopE(90–219) on GTP hydrolysis by RhoA was studied after loading of RhoA with [γ-32P]-GTP in a membrane filter assay. YopE(90–219) increased the intrinsic rate of GTP hydrolysis by RhoA (Fig. 4). In the presence of YopE(90–219), 50% of GTP was hydrolysed within 4 min, whereas the half-time (t1/2) for the intrinsic rate of GTP hydrolysis by RhoA was about 30 min. The effect of YopE on other members of the Rho family of GTPases was also tested. A similar increase in the rate of GTP hydrolysis could be seen when Rac (t1/2 = 4 min) and Cdc42 (t1/2 = 4 min) were incubated in the presence of YopE(90–219) (Fig. 4). In the absence of YopE(90–219), the t1/2 was 10 min for Rac and 12 min for Cdc42 respectively. The constitutive active RhoA(Q63E) that has a low intrinsic GTPase activity was not stimulated by the addition of as high a concentration as 3 μM YopE(90–219). The activity of YopE was also tested in the presence of the small GTPases, Ras and Ral. Whereas the GTPase activity of Rho, Rac and Cdc42 was stimulated by YopE(90–219) (see above), the rate of GTP hydrolysis of Ras and Ral was not activated by YopE(90–219) (Fig. 5).

Common to all GAP proteins is the so-called arginine finger sequence motif, which is required and essential for GAP activity (Scheffzek et al., 1998). In YopE, a sequence, amino acids 141–144, resembles a potential arginine finger. The arginine residue in position 144 was therefore replaced with alanine, the resulting yopE(90–219)R144A mutant allele was cloned, and the corresponding mutant protein was tested for its ability to stimulate GTP hydrolysis. As expected, the addition of YopE(90–219)R144A had no effect on the stimulation of RhoA-coupled GTP hydrolysis (Fig. 6). Together, these results show that YopE is a Rho-specific GAP protein.

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Figure 6. YopE- and YopE(R144A)-stimulated GTPase activity of RhoA. Recombinant RhoA was loaded with [γ-32P]-GTP for 10 min at 30°C in loading buffer. MgCl2 and unlabelled GTP were added. For GAP stimulation, YopE (3 µM) or YopE(R144A) (3 µM) was added to 3 µM RhoA and incubated for 4 min at 30°C. As a control, RhoA was incubated without YopE. GTPase activity was analysed by filter binding assay. The remaining GTP at each time is shown as the mean (± SD) of three independent experiments.

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The GAP activity of YopE is essential for the cytotoxic effect on HeLa cells

Thus, in a cell-free assay, YopE exhibits GAP activity towards RhoA, Rac1 and Cdc42. However, it should be noted that several GAP proteins have been shown to exhibit a broad activity against several RhoGTPases in vitro, but their substrate specificity in vivo has been found to be more restricted (Ridley et al., 1993; Van Aelst and D'Souza-Schorey, 1997).

We therefore investigated whether the GAP activity of YopE was essential for the in vivo cytotoxic effect mediated by YopE on S. cerevisiae cells and on HeLa cell monolayers. S. cerevisiae strains expressing a YopE(R144A) mutant protein did not mediate a cytotoxic effect on yeast cells, even though growth appeared to be somewhat slower than in the plasmid control (Fig. 7). The mutant YopE(R144A) protein was expressed at the same level as the wild-type YopE protein, as determined by Western blotting using anti-YopE antibodies (data not shown). Thus, the GAP activity of YopE was essential for mediating the cytotoxic effect in yeast cells.

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Figure 7. The arginine finger motif is essential for YopE cytotoxicity. Strain FY24 carrying pRS415MYopE or pRS415MyopER144A was streaked onto SC plates lacking leucine and onto SC plates lacking methionine, cysteine and leucine. Strains expressing the YopE(R144A) mutant protein (SC-met,cys,leu plate) do not mediate a cytotoxic effect on yeast cells.

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The cytotoxic effect induced by Yersinia infections on cultured HeLa cells can be visualized as a changed morphology. The cells are rounded up, leaving a retraction, tail-formed, cytoplasmic membrane rest that disappears upon prolonged incubation (16 h) and leads to detachment of the HeLa cells from the surface (Rosqvist and Wolf-Watz, 1986). This change in morphology is dependent on the presence of translocated YopE in the cytosol of the eukaryotic cell (Rosqvist et al., 1990; 1991; 1994). The effect of YopE is characterized by disruption and condensation of the actin microfilament structure of the cells (Rosqvist et al., 1991; Frithz-Lindsten et al., 1997).

To analyse the importance of the GAP activity of YopE in the cytotoxic process, HeLa cells were infected with a strain carrying either the wild-type yopE gene (pYopE219) or the mutated yopE allele (pYopE(R144A)). The latter construct is completely devoid of any GAP activity (Fig. 6). One hour after infection, the cells infected with the wild-type strain showed changed morphology, and the actin microfilaments were disrupted and appeared in condensed aggregates. In contrast, cells infected with the pYopE(R144A) mutant strain showed normal morphology, and an intact actin microfilament structure was seen (Fig. 8). Even after prolonged incubation (6 h), no change in morphology could be detected when the mutant was studied. Both wild-type YopE and the mutated variant were translocated into the HeLa cell cytosol to the same extent, as determined by both immunofluorescence staining and biochemical fractionation of the infected cells (data not shown). Thus, the GAP activity of YopE is essential for the cytotoxic effect of YopE on eukaryotic cells.

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Figure 8. The YopE GAP activity is essential to induce disruption of the actin microfilament structure in HeLa cells. Phalloidin staining of the actin microfilament structure of cells infected with Yersinia strains expressing wild-type YopE (A and D), mutated YopE(R144A) (B and E) or non-infected cells (C and F). Cytotoxic necrotizing factor 1 (CNF-1) counteracts the cytotoxic effect of YopE. CNF-1 was added to the infected cells 1 h after infection. Three hours after infection, the cells were fixed and processed for visualization of the actin microfilament structure (D–F). Note the recovery of the microfilament structure in the wild-type YopE-infected cells (D) and the numerous thick actin stress fibres induced by CNF-1 in (E) and (F). A, B and C analysed 1 h after infection.

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CNF-1 rescues HeLa cells from the YopE cytotoxic effect

The Escherichia coli cytotoxic necrotizing factor 1 (CNF-1) acts on eukaryotic cells by activating the small GTP-binding proteins Rho, Rac and Cdc42 via deamidation of Gln-61 or 63 to Glu, which results in a constitutive active protein (Flatau et al., 1997; Schmidt G. et al., 1997; Lerm et al., 1999). The activation of the Rho proteins leads to the production of enlarged, multinucleated cells, which possess numerous actin stress fibres (Fiorentini et al., 1997).

As YopE acts as a Rho-GAP, we investigated whether CNF-1 could counteract the cytotoxic effect of YopE. Five minutes before infection of HeLa cells, CNF-1 (900 ng ml−1) was added to the cells. One hour after infection, the cells were fixed, and the actin filament structure was stained with rhodamine-conjugated phalloidin. In cells infected with the wild-type YopE strain not treated with CNF-1, the filament structure was completely disrupted, whereas in cells treated with CNF-1, almost all actin filament structures were still intact (data not shown). Thus, CNF-1 rescued the cells from the YopE intoxication.

We also analysed whether the YopE cytotoxic effect was reversible or not. One hour after infection, the infected HeLa cells were washed, and CNF-1 (1500 ng ml−1) and gentamicin (50 μg ml−1, to kill extracellular bacteria) were added. Three hours after infection, the HeLa cell morphology appeared normal, and the actin stress fibre structures were intact (Fig. 8D). In cells infected with the mutated YopE variant [YopE(R144A)], the addition of CNF-1 resulted in the production of numerous actin stress fibres 3 h after infection (Fig. 8E). A similar effect on stress fibre formation could be seen in uninfected control cells (Fig. 8F). Thus, the effect of YopE is reversible and can be counteracted by the effect of CNF-1.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The YopE protein of Yersinia is an essential virulence determinant, which is injected into the eukaryotic target cell through a contact-dependent type III secretion system (Rosqvist et al., 1994). The effect of YopE on cultured cells is manifested by rapid depolymerization of the actin cytoskeleton. YopE has no effect in vitro on the polymerization/depolymerization of actin; neither has YopE any effect on DNA, RNA or protein synthesis (Rosqvist et al., 1991). The cytotoxic effect of YopE on eukaryotic cells is strikingly similar to the effect seen with other bacterial toxins known to modify Rho and Rho-like proteins.

The family of Rho-GTPases, Rho, Rac and Cdc42, are all important regulatory proteins involved in the control of a range of biological functions, including cytoskeleton formation and cell proliferation. Several different bacterial toxins have evolved that specifically target and modify this protein family of regulators (Aktories, 1997), indicating that they are specifically suited to the attack of bacterial toxins. The Clostridium botulinum C3 toxin ADP-ribosylate RhoA, B and C (but not Rac1 and Cdc42) (Aktories and Koch, 1997) and the C. difficile toxins A and B UDP-glucosylate and inactivate all members of the Rho protein family (Just et al., 1995a, b). These toxins induce a similar cytotoxic effect on cultured cells to YopE. Interestingly, the C. sordellii lethal toxin, which glucosylates Rac1, Cdc42 and Ras, but not Rho (Just et al., 1996; Popoff et al., 1996), gives a different cytotoxic phenotype to YopE. This suggests that downregulation of Rho causes the typical morphology that can be seen in YopE-intoxicated cells. Based on this and other similar observations, it has been suggested that YopE also inactivates Rho proteins (Cornelis and Wolf-Watz, 1997).

In line with this hypothesis is our present observation that YopE exhibits a Rho-coupled GAP activity in vitro towards RhoA, Rac and Cdc42, but not towards Ras. This activity was abolished when a critical arginine residue in position 144 was replaced with alanine. This arginine residue is located in a region of YopE (142–144) that shows homology with the so-called arginine finger motif (Scheffzek et al., 1998). These motifs are present in all Rho-GAP proteins, and they are essential for stimulation of the GTP-hydrolysing activity of the GTPases, resulting in their inactivation.

Interestingly, in contrast to the wild-type strain, the yopE(R144A) mutant did not induce any cytotoxic effect on HeLa cells, although the mutant protein was secreted and translocated at the same level as the corresponding wild-type protein. Thus, the GAP activity of YopE is crucial for the YopE-promoted biological activity, leading to depolymerization of the actin stress fibres and cytotoxicity. This was also evident when YopE and YopE(R144A) were expressed in yeast cells from an inducible yeast promoter. Expression of wild-type YopE prevented growth, while expression of YopE(R144A) had no obvious effect on growth and proliferation of the yeast cells. We conclude from this that the GAP activity of YopE is important for the inactivation of RhoGTPases in yeast as well as in mammalian cells, and that this activity is essential for the YopE-associated biological effect during in vivo conditions.

In S. cerevisiae, the Rho homologue Rho1p has been shown to be important for several different processes. Rho1p is essential for the activity of β(1[RIGHTWARDS ARROW]3) glucan synthase, which catalyses the synthesis of β(1[RIGHTWARDS ARROW]3) glucan, the main structural component of the cell wall in yeast (Drgonováet al., 1996, Qadota et al., 1996). Rho1p is also required for cell cycle progression and polarization of the yeast cell (Drgonováet al., 1999). Importantly, Rho1p has been shown to regulate the activity of protein kinase C (Pkc1p), which mediates Rho1p signalling to the cytoskeleton (Kamada et al., 1996; Kohno et al., 1996; Helliwell et al., 1998; Bickle et al., 1998). RhoGTPases interact with phosphatidylinositol (PI) kinases, and the PI-kinase homologue Tor2p has been demonstrated to be required for the organization of the actin cytoskeleton in S. cerevisiae (Schmidt et al., 1996). Signalling from Tor2p to the Rho1p effector Pkc1p and subsequently to the actin cytoskeleton is mediated by the Rho1 guanine nucleotide exchange factor (GEF) Rom2p (Schmidt A. et al., 1997).

Rho1p also targets Bni1p, a protein with formin homology (FH) domains that interacts with profilin and is therefore suggested to be involved in the regulation of the actin cytoskeleton (Kohno et al., 1996; Imamura et al., 1997). However, Bni1p is a non-essential protein and also interacts with other small GTPases and might function as a general target of yeast Rho proteins in response to various stimuli (Evangelista et al., 1997). The Rho-Pkc pathway is highly conserved between species. Mammalian RhoA is highly homologous to Rho1p, and the RhoA gene can complement a rho1 null mutant in S. cerevisiae (Qadota et al., 1994). Human PKC-type η is functionally related to Pkc1p, as PKC-η can activate the Pkc1p-mediated pathway through an activation of the Bck1p kinase. Finally, mammalian MEKK kinase can functionally replace Bck1p in the Pkc pathway in yeast (Blumer et al., 1994).

Interestingly, the expression of Rho1p, Rom2p, Bck1p and Ste20p in yeast suppressed the YopE-mediated inhibitory effect on cell growth. These proteins are all involved in different steps of the Rho1 regulatory pathway (see above and Fig. 2), indicating that YopE interferes with this pathway. As Rom2p is a Rho-specific guanine nucleotide exchange factor and as Bck1p acts downstream of Rho1p, these results strongly support the results obtained from in vitro experiments showing that YopE exhibits GAP activity towards Rho. Thus, both in vivo and in vitro experiments suggest that YopE is a GAP of RhoGTPases.

Interestingly, expression of RhoA and constitutive activated forms of Rac and human Cdc42 in yeast also rescued the yeast cell from the YopE-mediated growth inhibition, showing that YopE can interact with pathways controlled by these proteins. Human G25K has been shown to complement lethal CDC42 mutant alleles in S. cerevisiae functionally (Munemitsu et al., 1990), and human G25K shows ≈ 80% similarity to S. cerevisiae CDC42. However, co-expression of wild-type yeast CDC42 did not rescue the yeast strain from the cytotoxic effect mediated by YopE. These data also imply that YopE interferes with the activation of small GTPases and suggest that YopE recognizes targets from mammalian cells better than the corresponding proteins in yeast. Moreover, treatment of HeLa cells with CNF-1 toxin, which specifically modifies the Rho family of proteins, making them constitutively active, suppressed the YopE-mediated effect on eukaryotic cells. This result provided further support for the involvement of YopE in the Rho pathway in vivo and indicated that YopE targets only the Rho family of proteins. However, whether one or several of these proteins are in vivo targets remains to be evaluated.

It was shown recently that SptP, a translocated effector protein of Salmonella, also acts as a GAP of RhoGTPases (Fu and Galán, 1999). The N-terminal half of SptP shows a high degree of homology with YopE. However, it was found that the activity of SptP was directed towards Rac1 and Cdc42, but not towards RhoA, suggesting that SptP and YopE have different functions during infection. This idea is also supported from in vivo experiments showing that, while YopE is involved in antiphagocytosis and depolymerization of actin, the GAP-mediated activity of SptP reverses cytoskeletal changes triggered by Salmonella during the induced entry process. By analogy with SptP, the N-terminal half of exoenzyme S of Pseudomonas aeruginosa also shows a high degree of homology with YopE, and ExoS is also a GAP for RhoA, Rac and Cdc42 (Goehring et al., 1999). The effect of the N-terminal part of ExoS on HeLa cells is indistinguishable from that of YopE (Frithz-Lindsten et al., 1997), and this effect can also be blocked and reversed by CNF-1 (Pederson et al., 1999). Thus, these two effectors may serve the same function during infection. Whether SptP or ExoS target one or several proteins of the Rho family is not known at present. All three bacterial GAP proteins, YopE, SptP and ExoS, have a crucial arginine residue located in homologous arginine finger motifs, and removal of this arginine results in the inactivation of the G-coupled GAP activity of these proteins (Fu and Galán, 1999; Goehring et al. 2000). Interestingly, the three different bacterial Rho-GAP proteins show high homology with each other, but they show no obvious similarity with their eukaryotic counterparts, indicating that YopE, SptP and ExoS have evolved from a common ancestor and gained their activities by convergent evolution.

In conclusion, we have shown here that YopE possesses a GAP activity directed towards RhoA, Rac and Cdc42. This activity is essential for the biological effect of YopE. It remains to be shown whether YopE has one preferred target or whether YopE targets several proteins. These possibilities are currently under investigation in our laboratory.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Materials

All restriction enzymes and DNA modification enzymes were purchased from Roche Boehringer Biochemicals, New England Biolabs or Gibco BRL. Pwo DNA polymerase for polymerase chain reaction (PCR) amplifications was purchased from Roche Boehringer Biochemicals. Enterokinase EKMax was purchased from Invitrogen.

Strains, plasmids, media and genetic procedures

Saccharomyces cerevisiae strains used in this study were FY24 (MATα ura3-52 trp1Δ63 leu2Δ1) and FY23 (MATa ura3-52 trp1Δ63 leu2Δ1) (both F. Winston). Plasmid constructs for expressing YopE in S. cerevisiae are described below. Additional yeast plasmids used in this study are listed in Table 1. Standard yeast media and yeast genetic procedures have been described previously (Rose et al., 1990). All yeast media are synthetic complete and lack cysteine as well as the designated amino acids. Yeast transformations were performed as described by Agatep et al. (1998), and bacterial transformations according to Hanahan (1983). Isolation of plasmid DNA, restriction enzyme analysis and agarose gel electrophoresis were performed according to Sambrook et al. (1989). Plasmid preparations were done using a commercial plasmid mini-prep kit (Roche Boehringer Biochemicals) according to the manufacturer's instructions.

Table 1. Yeast plasmids used in this study.
PlasmidsCharacteristicsReference or source
pRS414 TRP1 CEN similar to pRS314 Sikorski and Hieter (1989)
pRS415MyopE LEU2 CEN, YopE from Y. pseudotuberculosis under MET3 promoterThis study
pRG416MyopE URA3 CEN YopE from Y. pseudotuberculosis under MET3 promoter,This study
pRHO1pYO714, pRS314-RHO1 TRP1 CEN Qadota et al. (1994)
pRhoApYO702, pRS314-RhoA, TRP1, CEN RhoA under RHO1 promoter Qadota et al. (1994)
pROM2 ROM2, TRP1, 2 μm, isolated from a genomic library in pRS314 M. Johansson
pSAC7pMB7, SAC7 in YEplac195 URA3, 2 μm Schmidt et al. (1997)
pBEM2pPB415, BEM2, LEU2, ADE3, 2 μm Schmidt et al. (1997)
pTOR2pJK6, TOR2 in YEplac181, LEU2, 2 μm Schmidt et al. (1997)
pPKC1YCp50PKC1, URA3, CEN PKC1 isolated from a genomic library Levin et al. (1990)
pGAL-PKC1 PKC1 (lacking the N-terminal 363 amino acids) under control of the GAL1 promoter  URA3, CEN Levin et al. (1990)
pBCK1 BCK1 in pRS314 Lee and Levin (1992)
pBCK1–20Genomic fragment containing dominant active allele BCK1–20 in pRS314 TRP1, CEN Lee and Levin, (1992)
pACT1pRB668 ACT1 in YCp50 URA3, CEN Doyle and Botstein (1996)
pCDC42YEp13M4CDC42, LEU2, 2 μm Ozaki et al. (1996)
pRHO4 RHO4 in YEp352 URA3, 2 μm M. Johansson
pRAS2YEplac112RAS2, TRP1, 2 μm M. Wigler
pRAS2V19YCpRAS2V19, URA3, CEN M. Wigler
pSTE11pNC192, STE11, TRP1 CENB. Errede
pSTE20pSTE20-5, pRS316-STE20, URA3, CEN E. Leberer
pRS415MG25KV12pRS415MG25KV12, activated human Cdc42 under MET3 promoterThis study
pRS415MRac1V12pRS415MRac1V12, activated mammalian Rac1 under MET3 promoterThis study

Plasmid constructions

A 0.7 kb PCR fragment containing the promoter region of the S. cerevisiae gene MET3 was cloned as a BglII fragment into the BamHI site of plasmid pRS415 (Christianson et al., 1992), creating plasmid pRS415M. A yopE gene lacking almost the entire promoter region was isolated as a 0.9 kb BamHI–HindIII DNA fragment from plasmid pTB1 (Rosqvist et al., 1990) and cloned into the corresponding sites of pUC18 (Yanisch-Perron et al., 1985). The yopE gene was subsequently cloned as a 0.9 kb SmaI–HindIII DNA fragment into the corresponding sites of plasmid pRS415M, creating plasmid pRS415MyopE.

Plasmid pRG416MyopE was created by cloning a 1.6 kb SacI–ClaI fragment from pRS415MyopE into the corresponding sites of plasmid pRG416 (R. Gaber).

Plasmids carrying the yopE mutant alleles pRS415M–yopE191 and pRS415MyopE(R144A) were cloned by replacing a PstI–HindIII fragment containing the N-terminal region of yopE with the same fragment from plasmid pYopE191 and pYopE(R144A)-His, respectively, carrying the indicated mutations. The expression and stability of the different yopE constructs in yeast were confirmed by Western blotting using anti-YopE antibody.

To express the higher eukaryotic GTPases G25K and Rac1 in yeast, the genes were PCR amplified from plasmids pGEX2T-G25KV12 and pGEX2T-Rac1V12, respectively, and cloned into BamHI–HindIII sites of plasmid pRS415M to give plasmids pRS415MG25KV12 and pRS415MRac1V12.

Cloning of His-tagged YopE proteins

The yerA gene and the yopE(1–89) gene, encoding amino acids 1–89, were PCR amplified from the pAF19 (Forsberg and Wolf-Watz, 1988) plasmid, introducing BamHI and NsiI sites with the following primers: 5′-AGT GAA TTC GAG CTC GG-3′ and 5′-AGG TGT ATG CAT TGG TGT CAC CAC CGG-3′.

HindIII sites were introduced into the yopE(90–219) DNA fragment (encoding amino acids 90–219) by PCR amplification of the pAF19 vector with the primers 5′-GTG GTG AAG CTT GCA CCC ACA CCT GCA-3′ and 5′-ATG ACC ATG ATT ACG CCA AGC TTG C-3′. DNA fragments were gel purified (GeneElute spin columns, Supelco) and digested by BamHI–NsiI or HindIII restriction endonucleases.

The His-tag insert, containing six histidine residues, Xpress epitope and enterokinase recognition site (Qiagen) flanked by NsiI–HindIII sites were synthesized using the oligonucleotides: His-tag 5′-T CAT CAT CAT CAT CAT GAT CTG TAC GAC GAT GAC GAT A-3′ and His-tag 5′-AG CTT ATC GTC ATC GTC GTA CAG ATC ATG ATG ATG ATG ATG ATG CA-3′. The His-tag oligonucleotides were annealed and used for cloning without any additional procedures.

The PCR-made yerA-yopE1–89 fragment and the His-tag fragment were cloned into BamHI–HindIII sites of the pUC19 (Yanisch-Perron et al., 1985) vector plasmid. The clones obtained were screened by PCR, and one positive clone was chosen for future work and named pYopE89-His. The yopE90–219 fragment was inserted into the HindIII site of pYopE89-His, creating plasmid pYopE-His. The clones were screened by PCR. Plasmid DNA from one positive clone was isolated, and the sequence was confirmed using the T7 sequencing kit (Pharmacia/Amersham).

Construction of mutated yopE(R144A) DNA. The fragment of the yopE gene encoding amino acids 90–219 was PCR amplified from the pYopE-His plasmid, creating a single amino acid substitution at position 144, arginine to alanine. The DNA fragment was gel purified, digested by HindIII and cloned into HindIII sites of the pYopE-His plasmid. Several clones were sequenced; one positive clone was selected for future work, and the plasmid was named pYopE(R144A)-His.

Construction of truncated yopE191. The fragment of the yopE gene encoding amino acids 130–191 was PCR amplified from the pAF19 plasmid. The DNA fragment was gel purified and recloned back into plasmid pAF19. Clones were screened by PCR, and one positive clone was selected for future work. The sequence of the clone was confirmed, and the plasmid was named pYopE191.

The pYopE-His, pYopE(R144A)-His and pYopE191 plasmids were transformed into the Y. pseudotuberculosis strain MYM [YPIII(pIB29 MEKBA)] for purification of YopE(90–219) and YopE(90–219)(R144A), respectively, and into the strain MYM [YPIII(pIB29 MEKA)] (Persson et al., 1997) for analysis of cytotoxic activity.

Purification of the active YopE(90–219) and the mutant YopE(90–219)(R144A) proteins

The His-tagged YopE variants were expressed in the Y. pseudotuberculosis strain MYM [YPIII(pIB29 MEKBA)], which is unable to produce and secrete YopH, M, E, K, B and YpkA (Persson et al., 1997). The strains were grown at 37°C in TMH media (Straley and Bowmer, 1986), and the secreted YopE was found in aggregated filament-like structures that were collected with an inoculating loop, as described by Michiels et al. (1990). The aggregates were washed briefly in water and then completely dissolved in 3 M guanidium-HCl. At this stage, the dissolved fraction consisted of more than 90% pure YopE. The protein fraction was bound to Ni-NTA agarose-resin (Qiagen) in the presence of 3 M guanidium-HCl in a batch procedure according to the manufacturer's instructions. The agarose-resin was washed with decreasing concentrations of guanidium-HCl (3, 2 and 1 M guanidium-HCl), and finally with 10 volumes of enterokinase cleavage buffer (50 mM Tris, 1 mM CaCl2, pH 8.0). The N-terminal part of the YopE protein containing the His-tag was removed by digestion with the enterokinase EKMax (Invitrogen) for 16 h at 37°C. The enterokinase was washed away, and then the cleaved C-terminal portion of the YopE protein (YopE90–219) was eluted with 3 M guanidium-HCl. Fractions containing YopE(90–219) were pooled and dialysed against PBS-A buffer and then concentrated, using an Amicon ultrafiltration device equipped with a PM10 filter. The protein concentration was determined with Bradford reagent using BSA as the protein standard. The purity of eluted peptides was analysed by SDS–PAGE with Coomassie brilliant blue staining, silver staining and by ECL-Western blots analysed with anti-YopE antiserum, anti-total Yops antiserum and anti-His antibodies (data not shown). No detectable contaminants were observed.

Infection of HeLa cells, cytotoxic assay and visualization of the actin microfilament structure

Cultivation and infection of HeLa cells have been described in detail elsewhere (Rosqvist et al., 1990; Frithz-Lindsten et al., 1997). The morphology of infected cells was observed every hour by phase-contrast microscopy. A changed morphology, as visualized by rounding up of the HeLa cells, indicated a cytotoxic response. E. coli cytotoxic necrotizing factor 1 (CNF-1; 900 ng ml−1 for blockage and 1500 ng ml−1 for reversal of the YopE effect) was added directly to the cell culture media at the indicated times. The actin microfilament structure of the HeLa cells was visualized at the times indicated, after staining with rhodamine-conjugated phalloidin according to the manufacture's instructions (Molecular Probes). Images were obtained by a confocal laser-scanning microscope (Multiprobe 2001, Molecular Dynamics) and scanned with image size 512 × 512 and 0.14 mm pixel size. The pinhole setting was 100 μm.

GTPase assay

Recombinant Rho proteins were loaded with [γ-32P]-GTP for 10 min at 30°C in loading buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 2 mM dithiothreitol). MgCl2 (12 mM, final concentration) and unlabelled GTP (2 mM, final concentration) were added. For GAP stimulation, different concentrations of YopE or YopE(R144A) were added to RhoA (3 µM, final concentration) or other preparations of small GTPases and incubated at 30°C for 4 min or as indicated in the figure legends. GTPase activity was analysed by filter binding assay (Goehring et al., 1999).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Drs D. Botstein, A. S. Byström, M. Hall, B. Hallberg, D. Levin, P. Ljungdahl and Y. Takai for the generous gift of plasmids. This work was supported by grants from the Swedish Medical Research Council, the Natural Science Research Council and the Foundation of Strategic Research. K.A. and G.S. were supported by the DFG, grant 388 SFB.

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Footnotes
  1. Present address: Department of Cell and Molecular Biology, Molecular Pathogenesis, Lund University, SE-22100 Lund, Sweden.