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In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity
Microbial Pathogenesis 34 (2003) 297–308 www.elsevier.com/locate/micpath
In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity Margareta Aili, Maxim Telepnev1, Bengt Hallberg2, Hans Wolf-Watz, Roland Rosqvist* Department of Molecular Biology, Umea˚ University, SE-901 87 Umea˚, Sweden Received 20 December 2002; received in revised form 7 March 2003; accepted 7 March 2003
Abstract The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmidencoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166– 183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Yersinia; GAP activity; cytotoxicity; GTPase; YopE
Yersinia pseudotuberculosis, a Gram-negative enteropathogen, causes gastrointestinal infections in humans and animals that are characterized by diarrhea, abdominal pain and fever. Infection occurs mainly through the oral route and the enteropathogenic Yersinia transit to the terminal ileum and initiate infection by penetrating Peyer’s patches. The bacteria can then colonize the mesenteric lymph nodes and may spread to deeper tissues such as the spleen. In rare cases, septicaemia can occur [1]. YopE is an essential virulence determinant during a Y. pseudotuberculosis infection, being secreted and translocated into eukaryotic cells via a contact dependent type III secretion system [2]. The cytosolic localization of YopE induces depolymerisation of the actin cytoskeleton, which results in a rounding up of infected cells. This changed * Corresponding author. Tel.: þ 46-90-785-2529; fax: þ46-90-77-1420. E-mail address: [email protected] (R. Rosqvist). 1 Present address: Department of Clinical Microbiology, Umea˚ University, SE-901 87 Umea˚, Sweden. 2 Present address: Department of Medical Biosciences/Pathology, Umea˚ University, SE-901 87 Umea˚, Sweden.
morphology has been described as a cytotoxic effect [3]. A similar cytotoxic effect is also induced by YopT [4], however YopT is not expressed by the strain used in this study. Utilizing YopE fusions to the Cya reporter indicated that the first 11 amino acids of YopE are sufficient for secretion through the bacterial envelope [5,6], while at least the first 49 amino acids are required for YopE to be translocated into HeLa cells during infection [5]. The YerA/ SycE chaperone requires the first 75 amino acids of YopE to form a stable complex and promote its efficient secretion [5] (Fig. 2). The enzymatic activity of YopE resides in the Cterminus [7]. YopE possesses a GTPase activating protein (GAP) activity towards RhoA, Rac1 and Cdc42 in vitro [7,8]. A conserved arginine finger at position 144 is essential for the GAP activity, indicating the same function as the arginine finger of the eukaryotic GAPs. No other obvious homologies with eukaryotic GAPs can be detected by sequence alignment. On the other hand, YopE shares a high degree of homology with the N-terminal domain of two other bacterial toxins, ExoS of Pseudomonas and SptP of Salmonella,
0882-4010/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0882-4010(03)00063-9
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possessing in vitro GAP activity towards members of the Rho family [9,10]. Thus, all three bacterial GAPs inactivate the Rho family of small GTP-binding proteins. Since small GTP-binding proteins are involved in rearrangement of the actin cytoskeleton, it has been assumed that the cytotoxic phenotype induced by YopE and ExoS, and the effect on actin rearrangement induced by SptP, is caused through inactivation of RhoA, Rac1 and Cdc42 [7,9,10]. However, no in vivo interaction between the bacterial GAPs and the Rho GTPases has so far been demonstrated. The three dimensional structure of the GAP domain of YopE (amino acids 90– 219) was recently solved (Fig. 1) but no structural data of YopE in complex with a Rho GTPase have been published so far [11]. However, the crystal structures of complexes between SptP and Rac1, and
ExoS and Rac1, respectively, have recently been published [12,13] and based on the similarities between YopE and these bacterial GAPs a model of the YopE – Rac1 complex can be made [11]. As observed from SptP and ExoS, the model indicates that interactions between YopE and Rac are limited to three distinct regions of the structure. Residues Ile106, Leu109, Thr138, Gly139, Ser140, and Gln149 of YopE are contacting Switch II region of the GTPase. The arginine finger Arg144 and the bulge residues Thr183, Ile184, and Gly185 are contacting GTP and both of the switch regions, while residues Thr148, Gln151, Pro177, Ser179, and Gln180, are contacting Switch I and the bound nucleotide [11]. By exploiting the known structures of YopE, SptP and ExoS, we can analyze structural effects of mutations in YopE in an effort to define the eukaryotic
Fig. 1. Structure of YopE. A. Structure-based sequence alignment of the bacterial GAP domains of YopE, ExoS and SptP. Residues that are identical in all three sequences are shown in red; residues identical in two out of three sequences, in green. The critical arginine finger is enclosed by a red box. The positions of ahelices and b-strands are indicated above the sequence. B. Overall structure of YopEGAP. The critical arginine residue is shown as a stick model (modified from Ref. [11]). Reproduced with permission from the copyright holder Cold Spring Harbor Laboratory Press, copyright 2002.
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molecular target of YopE and the phenotypical effect of YopE on eukaryotic cells. Herein, we show that mutations flanking the arginine finger are important for the YopE cytotoxic phenotype and in vitro GAP activity towards RhoA, Rac1 and Cdc42. Our results also define regions in YopE that are specific for cytotoxicity and in vitro GAP activity towards RhoA, Rac1 and Cdc42 in isolation, showing these two functions can be separated.
1. Results 1.1. Regions important for the cytotoxic activity of YopE We and others have shown that the arginine residue in position 144 of YopE is essential for YopE-mediated cytotoxicity and in vitro GAP activity towards RhoA, Rac1 and Cdc42 [7,8]. To identify other regions of YopE that are essential to generate the cytotoxic effect, a series of deletions in wild-type yopE were created. The full-length yopE gene harbored on pAF19 was replaced with different variants of yopE (Fig. 2). With this approach, the yopE gene is under the control of its native promoter on a high copy number plasmid and a mutated protein can easily be expressed in any Yersinia strain. Each YopE variant was then analyzed with respect to secretion, translocation and ability to induce a cytotoxic response on HeLa cells. We first examined the requirement of the YopE Cterminus in the cytotoxic process by creating sequential deletions from the C-terminal end of the protein. A FLAGepitope was introduced in the C-terminal end of the fulllength 219 amino acid YopE protein and in five C-terminal deletion mutants creating: YopE219FLAG, YopE215FLAG, YopE202FLAG, YopE191FLAG, YopE179FLAG and YopE134FLAG (Fig. 2). Even in the presence of a Cterminal FLAG-tag, these variants all displayed elements of native folding as implied by their resistance to endogenous proteases (data not shown, [14]). Moreover, the mutant proteins were indistinguishable with respect to secretion from bacteria and translocation into infected HeLa cells (data not shown). Thus, the C-terminal part of YopE from amino acid 135 –219 is not involved in the secretion or translocation process of YopE, consistent with earlier findings [5]. The cytotoxic effect of YopE on HeLa cells after infection can be visualized as a changed morphology using phase contrast microscopy. Infected cells become well rounded up, leaving tail-like retractions that disappear upon prolonged incubation ultimately leading to their surface detachment [3,15,16]. The effect of each YopE variant on the morphology of HeLa cells were analyzed by infecting HeLa cells with Yersinia defective for YopE and YopH (YPIII(pIB251)) expressing in trans wild-type yopE (pAF19) or the respective deletion mutant. Like wild-type YopE a C-terminal deletion of only four amino acids, YopE215FLAG, showed full cytotoxic effect
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on HeLa cells as early as 45 min post-infection. However, the subsequent deletions of 17 (YopE202FLAG), 28 (YopE191FLAG), 40 (YopE179FLAG), and 85 (YopE134FLAG) amino acids, did not induce cytotoxicity on HeLa cells comparable to the arginine finger mutant YopE(R144A) (Fig. 2). Thus, the C-terminus of YopE is essential for cytotoxicity. The N-terminal region of Yop effectors contains secretion and translocation signals needed for transportation of the protein across the bacterial envelope as well as the eukaryotic plasma membrane [5,6]. In order to analyze the importance of the YopE N-terminal region for cytotoxicity, we fused different YopE variants to the N-terminal domain (amino acids 1 –85) of YopH (Fig. 2). With this approach, the secretion and translocation signals were supplied by YopH, providing a tool to discern phenotypes of the YopE N-terminus. Each hybrid was analyzed for secretion and its susceptibility to endogenous proteases to confer protein stability (data not shown, [14]). Furthermore, an analysis of protein secretion and subsequent translocation into infected HeLa cells revealed that these YopH-YopE hybrids were essentially indistinguishable from wild-type YopE (data not shown). Deletion of the first 89 amino acids of YopE (YopH85YopED1-89) showed a full cytotoxic effect on HeLa cells (Fig. 2). However, deletion of additional 10 amino acids (YopH85-YopED1-99) resulted in a significantly reduced cytotoxic effect. Not only was the effect delayed, but only a fraction of the cell population exhibited an altered morphology. The observation that not every cell was cytotoxically affected, might be explained by the fact that a higher amount of the mutant YopE protein must be injected to affect cells than wild-type YopE protein. Given that the infection is a stochastic event, it is possible that only cells with a number of bacteria above the threshold were affected. Nevertheless, this shows that the mutants are able to induce cytotoxicity, although they are less active than wild-type YopE. Furthermore, deletion of additional ten amino acids (YopH85-YopED1-109) totally abolished the cytotoxic effect on the cell monolayer. It follows that further deletions of amino acids 1– 119 (YopH85-YopED1-119) and amino acids 1– 128 (YopH85-YopED1-128) were also unable to induce cytotoxicity (Fig. 2). Collectively, these investigations showed that the minimal region required to induce YopE cytotoxicity is located between amino acids 90 and 215. To further dissect this region, we made in-frame deletions within the amino acids 108 – 215 of YopE (Fig. 2). These mutants were used to infect semi-confluent HeLa cell monolayers to assess their ability to induce morphologically changes. An internal deletion of four amino acids (YopED131-135) did not change the cytotoxic activity of YopE on HeLa cells, whereas larger deletions (YopED108-128, YopED131-149, YopED131-169, YopED131-189) rendered the protein inactive. Therefore,
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Fig. 2. Deletion mutants used in this study. Wild-type yopE, located in pAF19 (a high-copy plasmid), and derivatives containing mutant alleles. All variants were expressed in Y. pseudotuberculosis YPIII(pIB251) (yopEyopH) background to asses cytotoxic effect on HeLa cells and in MYM background for expression of protein used for in vitro GAP activity measurements.
amino acids 108– 128 and 136– 189 are required to generate a cytotoxic effect. The latter region was further investigated by generating seven small in-frame deletion mutants ranging from amino acid 162 to 183. In addition, YopED189-192 and YopED211-215 were created (Fig. 2). These in-frame deletions were stable and secreted normally (data not shown, [14]). The YopE deletions were analyzed for their cytotoxic effect on HeLa cells during infection. One mutant,
YopED189-192 showed cytotoxic activity equivalent to wild-type, while two mutants, YopED162-165 and YopED211-215, did not cause a complete rounding up of infected cells although the cells were clearly cytotoxically affected (Fig. 3). The remaining six deletion mutants (YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180 and YopED180-183) had lost the ability to cause morphological changes on HeLa cells (Figs. 2 and 3). This indicates that the region between
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Fig. 3. Cytotoxic effect on HeLa cells after 2 h bacterial infection. HeLa cells were seeded at 2 £ 105 cells/ml on cover slips placed in 24 well tissue culture plates. The effect on the cell morphology was assessed by phase-contrast microscopy after 2 h infection with no bacteria or Y. pseudotuberculosis YPIII(pIB251) containing pAF19 and the nine small deletion constructs described in Fig. 2. The images were captured by a Hamamatzu CCD camera through a 40 £ magnification objective. Shown are images from HeLa cells infected with four different deletion mutants.
amino acid 165 and 183 contained amino acids essential to induce a cytotoxic response on HeLa cells. The lack of a cytotoxic response could either be caused by an inactive protein or through defective translocation into the HeLa cells. To distinguish between these two possibilities, infected cells were assayed for protease protection of YopE after digitonin fractionation of infected HeLa cells [17]. To discriminate between YopE located outside of the HeLa cells from translocated YopE, the infected HeLa cell monolayers were treated with proteinase K, which has previously been shown to degrade secreted YopE [17]. The amount of translocated protein was assessed by comparing supernatants obtained from non-lysed cells (containing only proteins released to the culture medium) with the soluble contents derived from lysed cells. The eukaryotic protein Erk was used as an internal control for efficient lysis and to ensure that an equivalent level of protein was loaded onto gels. The translocation defective yopB null mutant [YPIII(pIB604)] showed the similar low level of protein in both lysed and non-lysed samples, confirming its inability to translocate YopE (Fig. 4). In some cases, we observed that a small amount of YopE was recovered from infected protease-treated non-lysed cells. A likely explanation is that during infection of HeLa cells with a Yersinia strain overexpressing YopE, secreted YopE is trapped between the bacteria and the eukaryotic cell in a protease protected environment, which is released into
the media when the cells are scraped off the culture dish. However, this is not the case when the wild-type protein is expressed at wild-type levels from the virulence plasmid, and not in trans. The different YopE deletions were generally translocated into HeLa cells to similar or higher levels as wild-type YopE. However, we noted that YopED162-165 and YopED165-168 were translocated less efficiently (Fig. 4), although YopED162-165 was still able to induce a cytotoxic response, whereas YopED165-168 was not able to induce a cytotoxic response (Fig. 3). 1.2. In vitro GAP activity YopE posses an in vitro GAP activity towards members of the small Rho GTPase subfamily [7,8]. Therefore, we wanted to examine the relationship between cytotoxic effect of YopE on HeLa cells and its in vitro GAP activity. We measured the in vitro GAP activity of secreted protein deletions (YopED162-165, YopED165-168, YopED168171, YopED171-174, YopED174-177, YopED177-180, YopED180-183, YopED189-192 and YopED211-215) expressed in trans in a Multiple Yop Mutant background, MYM [YPIII(pIB29MEKBA)], to avoid possible adverse effects of other effector proteins of Yersinia. As this MYM strain is devoid of YopH, YopM, YopE, YopK, YopB, and YpkA, measurable GAP activity could only be attributed to YopE variants produced in trans. To ensure an equal amount
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Fig. 4. Translocation of YopE wild type and mutant proteins after 3 h infection of HeLa cells. Infections of HeLa cells with strains of Y. pseudotuberculosis expressing mutant alleles of yopE in trans were carried out in duplicate. Three hours postinfection all the HeLa cells monolayers were treated with proteinase K for 20 min, prior to the addition of phenylmethylsulfonyl flouride (4 mM in PBS) to block the remaining protease activity. To one set of the HeLa cells cultures digitonin (þ ) were added to lyse the cells, while to the other set no digitonin were added (2). The HeLa cells were harvested and collected in eppendorf tubes and centrifuged to clear the sample from cell debris and bacteria. The supernatant was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using antiserum raised against YopE (upper panel) or commercially available pan-Erk antibodies (lower panel). The estimated molecular weights are for YopE 24 kD and Erk 42 kD. The translocation defective YPIII(pIB604) (yopB) was included as a negative control [32].
of each protein was used for analysis of the in vitro GAP activity supernatant samples were initially characterized by immunoblot using anti-YopE antiserum. The GTPase assay was performed as described [18]. As expected, all of the deletion mutants unable to induce a cytotoxic effect on HeLa cells (YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180 and YopED180-183) were also unable to inactivate RhoA, Rac1 and Cdc42 in vitro (Fig. 5a). Surprisingly, three deletion mutants causing cytotoxic effect on HeLa cells (YopED162-165, YopED189-192 and YopED211-215) were still unable to inactivate RhoA, Rac1 and Cdc42 in vitro. We interpret this finding to indicate that induction of a cytotoxic effect on HeLa cells is not absolutely linked to the ability of YopE to inactivate RhoA, Rac1 and Cdc42 in vitro. Deletion of four amino acids might cause an adverse effect on the overall structure of the protein, leading to loss of function. To minimize these possible effects, we made nine substitution mutants where three consecutive amino acids were exchanged for alanine (Fig. 6). We detected no difference in stability or secretion of these variants compared to wild-type YopE (data not shown). In contrast to the corresponding deletion mutants however, eight of the nine substitution mutants, induced a cytotoxic effect on semi-confluent monolayers of HeLa cells after infection (Table 1). This result suggests that the spacing of the amino acids in this region is important for the cytotoxic phenotype. In addition, the fact that either a deletion of amino acids 180 –183 or substitution of amino acids 181– 183 rendered the protein inactive in the cytotoxic assay reflects a direct functional role for this site. We further investigated the in vitro GAP activity of the alanine-substitution mutants towards RhoA, Rac1 and Cdc42. One substitution mutant (YopE210-12A) inactivated all of the three GTPases, although not as effectively as the wild-type YopE (Fig. 5b, Table 1). The relative in vitro GAP activity of YopE210-12A towards RhoA, Rac1 and Cdc42 was 4.5 ^ 1.3, 2.8 ^ 0.9 and 4.3 ^ 2.1, respectively, compared to 6.7 ^ 1.7, 21.9 ^ 10.3 and 7.9 ^ 1.4 for
wild-type YopE. The cytotoxic negative mutant (YopE18183A) had no measurable in vitro GAP activity. Interestingly, five of the substitution mutants (166-68A, 169-71A, 17577A, 178-80A and 206-08A) showed similar low levels of in vitro GAP activity against RhoA, Rac1 and Cdc42 as the MYM control strain. Of these mutants YopE175-77A showed the highest relative in vitro GAP activity towards RhoA, 1.2 ^ 0.2, Rac1, 1.2 ^ 0.1 and Cdc42, 1.3 ^ 0.2 compared to the MYM control strain values of RhoA, 1.0 ^ 0.1, Rac1, 1.2 ^ 0.0 and Cdc42, 1.0 ^ 0.1. Thus, all these five mutants show in vitro GAP activity of the same level as the MYM control strain. Interestingly, all five mutants were able to induce a cytotoxic response on HeLa cells. As for the other two mutants (172-74A and 213-15A), somewhat higher activities towards the GTPases tested were detected, but none to the same level as wild-type YopE (Table 1). 1.3. Structure modelling of YopE Recently the three dimensional structure of the GAP ˚ domain of YopE (amino acids 90 – 219) was solved at 2.2 A resolution [11]. The structure is almost entirely composed of a-helices and shows no obvious structural similarity to eukaryotic RhoGAP domains (Fig. 1). However, the structure of YopE is very similar to the GAP domains of ExoS of Pseudomonas aeruginosa [12] and SptP of Salmonella typhimurium [13]. We have used the SWISSMODEL Protein Modeling Server [19 – 21] to create structural models of the nine deletion mutants (YopED162-165, YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180, YopED180-183, YopED189-192 and YopED211-215) and the nine corresponding alanine-substitution mutants. With the help of the structural viewing program MOLMOL [22], we have compared the models of the deletion mutants with wild-type YopE. In four cases (YopED162-165, YopED165-168, YopED168-171 and YopED189-192), a change in the position of the arginine finger was observed (data not shown). This may explain the observed lack of
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Fig. 5. YopE in vitro GAP activity towards RhoA, Rac1 and Cdc42. Recombinant RhoA, Rac1 and Cdc42 were loaded with g-32P GTP for 5 minutes at 37 8C. For analysis of GTP hydrolysis, supernatants from Y. pseudotuberculosis MYM strains producing in trans YopE deletions (panel A) or substitutions (panel B) were added to 11 nM Rac1, 11 nM Cdc42, or 11 nM RhoA, respectively, and incubated for 20 minutes at 16 8C (Rac1 and Cdc42) or 20 8C (RhoA). To establish the intrinsic level of hydrolysis, loaded GTPases were incubated at the reaction temperature for 20 minutes without addition of GAP protein. Shown is the remaining g-32P GTP bound to RhoA, Rac1 and Cdc42 as a percentage of the loaded GTPases. Each point represents the mean of three independent experiments in duplicate samples with standard deviations.
GAP activity towards the Rho proteins. In the other five deletion mutants, the arginine finger occupies the same position as in the wild type protein and no visible change in the Rac1 contact area was observed. Nevertheless, large differences in the structure of the deletion mutants were
generally observed in the local area of the deletion. In line with this, these deletions severely affected the GAP activity of YopE. However, deletion mutants YopED162-165 and YopED189-192 were still able to induce cytotoxicity on HeLa cells although they lacked in vitro GAP activity.
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2. Discussion
Fig. 6. YopE triple-alanine substitution mutants used in this study. Wildtype yopE, located in pAF19 (a high-copy plasmid), and derivatives containing mutant alleles. All variants were expressed in Y. pseudotuberculosis YPIII(pIB251) (yopE, yopH) background to asses cytotoxic effect on HeLa cells and in MYM background for expression of protein used for in vitro GAP activity measurements.
Modeling of the substitution mutants showed no significant change in their structure compared to wild-type YopE. In particular, the position of the essential argininefinger was maintained. As expected, this confirms that the substitution mutants have less effect on the structure compared to the deletion mutants. Thus, the lack of in vitro GAP activity might be due to loss of specific interaction with RhoA, Rac1 and Cdc42.
YopE of Yersinia is an essential virulence determinant, which is translocated into the eukaryotic target cell through a contact-dependent mechanism. The bacteria remains bound to the surface of the target cell while YopE is translocated into the target cell cytosol via the type III secretion system [2,16]. YopE causes disruption of the actin microfilament stress fibers [15] and causes the eukaryotic cell to round up, leaving a tail-like cytoplasmic membrane retraction. This characteristic morphological change of the cell shape can easily be visualized by phase contrast microscopy and has been denoted as a cytotoxic effect on the HeLa cell [3,15]. It has recently been shown that YopE possess a GTPaseactivating protein (GAP) activity that down-regulates RhoA, Rac1 and Cdc42 in vitro [7,8,23]. Mutation of a critical arginine residue at position 144 completely abolishes the GAP activity of YopE and renders this mutant avirulent [34] and unable to induce a cytotoxic effect on HeLa cells [7,8]. This has led to the conclusion that the YopE GAP activity is essential for induction of the HeLa cell cytotoxic phenotype [7]. This is supported by the fact that HeLa cells pre-treated with CNF1 (cytotoxic necrotizing factor 1) results in constitutive active Rho proteins that rescue the HeLa cells from the YopE cytotoxic effect [7]. In parallel, introduction of a
Table 1 Ability of wild-type YopE and different YopE mutants to induce a cytotoxic response on cultured HeLa cells, and their relative in vitro GAP activity towards RhoA, Rac1 and Cdc42 Construct
PIB251(pAF19) YPIII(pIB29)MYM PIB251(pYopED162-165) PIB251(pYopED165-168) PIB251(pYopED168-171) PIB251(pYopED171-174) PIB251(pYopED174-177) PIB251(pYopED177-180) PIB251(pYopED180-183) PIB251(pYopED189-192) PIB251(pYopED211-215) PIB251(pYopED166-8A) PIB251(pYopED169-71A) PIB251(pYopE172-4A) PIB251(pYopE175-7A) PIB251(pYopE178-80A) PIB251(pYopE181-3A) PIB251(pYopE206-8A) PIB251(pYopE210-12A) PIB251(pYopE213-15A) RhoGAP
Mutation
Wt YopE, yopH, yopM, ypkA, yopB, yopK D162–165 D165–168 D168–171 D171–174 D174–177 D177–180 D180–183 D189–192 D211–215 166-8A 169-71A 172-4A 175-7A 178-80A 181-3A 206-8A 210-12A 213-15A 2
Cytotoxicity on HeLe-cells
þ þþ 2 þþ 2 2 2 2 2 2 þ þþ þþ þ þþ þ þþ þ þþ þ þþ þ þþ 2 þ þþ þ þþ þ þþ n.d.
In vitro GAPactivity RhoA
Rac1
Cdc42
6.7 ^ 1.7 1.0 ^ 0.1 1.0 ^ 0.2 1.2 ^ 0.1 1.1 ^ 0.1 1.0 ^ 0.1 1.2 ^ 0.1 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.2 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.1 1.4 ^ 0.1 1.2 ^ 0.2 1.1 ^ 0.1 0.9 ^ 0.0 0.8 ^ 0.0 4.5 ^ 1.3 1.4 ^ 0.1 18.4 ^ 3.8
21.9 ^ 10.3 1.2 ^ 0.1 1.1 ^ 0.3 0.9 ^ 0.3 1.4 ^ 0.3 1.6 ^ 0.6 1.6 ^ 0.8 1.5 ^ 0.3 1.5 ^ 0.3 1.4 ^ 0.3 1.4 ^ 0.2 1.2 ^ 0.2 1.1 ^ 0.0 1.6 ^ 0.3 1.2 ^ 0.1 1.1 ^ 0.1 0.9 ^ 0.1 1.1 ^ 0.3 2.8 ^ 0.9 1.4 ^ 0.3 12.7 ^ 4.6
7.9 ^ 1.4 1.0 ^ 0.1 1.0 ^ 0.1 1.2 ^ 0.3 1.1 ^ 0.2 1.2 ^ 0.3 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.1 1.2 ^ 0.1 1.0 ^ 0.1 1.0 ^ 0.3 1.0 ^ 0.0 1.4 ^ 0.1 1.3 ^ 0.2 1.2 ^ 0.2 1.0 ^ 0.0 1.2 ^ 0.2 4.3 ^ 2.1 1.4 ^ 0.4 12.9 ^ 1.5
Cytotoxic effect after infection of HeLa cells evaluated 2 h post-infection. A sliding scale of þþ þ indicates complete rounding up of the HeLa cells, while 2 indicates no visible effect on the cell morphology. The relative GAP-activity has been calculated as intrinsic hydrolysis set to 100% divided by the hydrolysis after addition of the YopE variant. The mean from three independent experiments together with the standard deviation (^) has been calculated.
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constitutive active RhoA (RhoA-V14) into HeLa cells rescued infected cells from the cytotoxic response [8]. While these studies collectively support the view that the GAP activity is necessary for the cytotoxic phenotype of YopE on HeLa cells, evidence for a direct interaction between YopE and the Rho proteins during infection is lacking. To better understand the biological role of YopE, we investigated what regions of YopE are important for the induction of cytotoxicity on HeLa cells. Our strategy was to create deletion and substitution mutations along the YopE protein and analyze their effect on secretion, translocation and the ability to cause a cytotoxic effect on HeLa cells. In addition, to discern the relationship between cytotoxicity and the enzymatic activity of YopE, mutants were also analyzed for GAP activity towards the Rho proteins RhoA, Rac1 and Cdc42 in vitro. Sequential removal of the N- and C-terminus of YopE identified a region between amino acids 90 and 215 that is necessary to induce cytotoxicity. As expected, internal deletions containing the essential arginine at position 144 resulted in a total loss of the cytotoxic response. Smaller inframe deletions flanking the arginine finger defined an additional region important for the cytotoxic effect to amino acids 166– 183. To further analyze this region, alaninesubstitution of three consecutive amino acids were made to reduce impact on the protein structure. We tested nine alanine-substitution mutants for in vitro GAP activity towards RhoA, Rac1 and Cdc42 and for ability to induce cytotoxicity. Out of the nine ala-substitution mutants, one (YopE210-12A) showed activity towards RhoA, Rac1 and Cdc42, albeit not as high as wild-type YopE. This mutation is located in the a8 helix which is not making a direct contact with the GTPase (Fig. 1). Two mutants (YopE17274A and YopE213-15A) showed a slightly higher in vitro GAP activity than background levels, while the other six substitution mutants showed an in vitro GAP activity comparable to background activity. Interestingly, five out of these six substitution-mutants were still able to induce a cytotoxic response on infected HeLa cells. These five mutations are located in a region spanning the a5, b1-2, and the a6 helices. YopE175-7A and YopE178-80A are located in the b1 –2 and the a6 region affecting amino acids YopE177 and YopE179 that most likely are contacting the Switch 1 region and the bound nucleotide in Rac1 [11], explaining the loss of activity in these mutants. Interestingly, we have only been able to identify one substitution mutant (YopE181-3A) that is completely devoid of any measurable in vitro GAP activity as well as unable to induce any cytotoxic response on HeLa cells. This mutation is located in the bulge region that connects helices a6 and a7, and the residues within and around this bulge are strictly conserved in the bacterial GAPs. This region is contacting the two switch regions as well as the bound GTP in Rac1. Single amino acid substitutions of the corresponding residues in SptP (Q246 and T249 (Q180 and T183 in
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YopE)) render the SptP mutants to loose its GAP activity, similar to what we have found here. Thus, this region in YopE is essential for both the GTPase activity and the cytotoxic response. These results reinforce the close relationship between SptP and YopE and provide additional evidences for the importance of this region in YopE. Thus, we have identified five substitution mutants located in different regions of YopE with a similar phenotype: able to induce cytotoxicity on HeLa cells but showed no in vitro GAP activity towards the tested Rho GTPases. We have been unable to identify mutants that are unable to induce cytotoxicity but still possess in vitro GAP activity, reinforcing the earlier notation that GAP activity of YopE is important for the cytotoxic response. Assuming that the folding properties of YopE are the same during in vivo and in vitro conditions these results suggest that YopE might exhibit additional unidentified target(s) within the eukaryotic cell. Some of the mutants causing a cytotoxic response in HeLa cells have an in vitro GAP activity towards one or more of the tested GTPases just above background levels (Table 1). This low activity might be enough to explain the observed cytotoxic phenotype, but since there are mutants having similar low in vitro GAP activity that is completely non-cytotoxic, it seems unlikely that this residual activity is the cause of the cytotoxicity. In addition, other mutants show a full cytotoxic effect, but have no measurable in vitro GAP activity suggesting that YopE has additional eukaryotic target(s) other than RhoA, Rac1 and Cdc42. However, this remains to be shown. Black and Bliska showed that constitutive active RhoA is able to rescue cells from YopE induced cytotoxicity [8], arguing the fact that RhoA is the target of YopE. However, transfection of active RhoA may alter the balance of other substrates involved in the actin homeostasis. Thus, the effect of YopE on its eukaryotic target could be masked by the dominant effect of RhoA on the actin cytoskeleton dynamics, especially if YopE’s target is upstream of RhoA or in a parallel pathway. The fact that overexpression of mammalian RhoA, Rac1 and Cdc42 in yeast suppressed the toxic effect of YopE, although to a lower extent then Rho1, the yeast homologue to RhoA [7], also supports the notion that the use of transfection to produce higher intracellular amounts of the respective GTPase may sequester YopE from its bona vide cellular target. In addition it was recently published that YopE selectively targets Rac1 mediated signalling pathways [23], since YopE was able to prevent Cdc42 dependent Rac1 activated ruffling after stimulation with bradykinin, but YopE was unable to affect actin structures regulated by Cdc42 (filopodia formation) and RhoA (stress fiber assembly). Thus, contradicting the results presented by Black and Bliska [8]. However, since these studies utilize drugs that may have broader specificity than only towards RhoA, Rac1 and Cdc42, it is possible that other targets of YopE were overlooked. In addition, cell type-specific responses may also explain the observation that YopE
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specifically targeted Rac1 pathways in vivo [23], while in other studies the RhoA and Cdc42 pathway was targeted [7, 8]. Thus, although several studies suggest that YopE targets RhoA, Rac1 and Cdc42, it is not at all clear that the YopE GAP activity is specifically directed towards these three proteins. The family of small Rho GTPases to date consists of around 20 members in humans, and of these RhoA, Rac1 and Cdc42 are the most extensively studied. RhoA has two highly related homologues, RhoB and RhoC. Given that RhoB is involved in actin organization and vesicle transport [24] and shares high homology with RhoA, might make it an interesting target candidate of YopE. Other possible candidates include the Cdc42 relatives TC10 and TCL, which are implicated in actin-based reorganization [25,26]. Thus, from the results presented here we find support for the hypothesis that YopE has additional targets within the eukaryotic cell and we are currently looking for the bona vide eukaryotic molecular target of YopE.
3. Material and methods 3.1. Construction of yopE mutants PCR-amplified DNA fragments used for constructing the in-frame deletion mutants of yopE were generated by overlap PCR [27] using the pAF19 plasmid [28] as a template for the deletions within the yopE gene. Isolation of plasmid DNA from E. coli strains was performed using the Quantum Prep Plasmid Miniprep kit (Bio-RAD) as described by the manufacturer. Oligonucleotides were synthesized with an Applied Biosystems DNA/RNA synthesizer model 392. Oligonucleotide sequences can be obtained by request. Standard DNA manipulation techniques were essentially used as described [29]. Recovery of DNA fragments from agarose was achieved by spin-column purification (Amicon) as detailed by the manufacturer. The PCR-generated DNA-fragments were gel purified, digested with restriction enzymes and cloned back into the pAF19 plasmid. The clones were screened by PCR and plasmid DNA from one positive clone was isolated, and the sequence was confirmed using the T7 sequencing kit (Pharmacia/Amersham). For the mutants containing the N-terminal part of yopH, the first 89 amino acids of YopE were substituted with the N-terminal 85 amino acids of YopH prior to the introduction of the yopE deletion variants. Plasmids carrying the different YopE variants were introduced into the Yersinia strains by electroporation [30] using a Gene Pulsar apparatus (BioRad Laboratories, Richmond, CA). 3.2. Bacterial strains and growth conditions The Y. pseudotuberculosis strain YPIII(pIB251), unable to express YopE and YopH [3], were used to assess the
cytotoxicity of YopE variants in trans after infection of HeLa cells. The Yersinia Multiple Yop Mutant (MYM [YPIII(pIB29MEKBA)]) strain [31] is unable to express YopM, YopE, YopK, YpkA, YopH, and YopB, respectively. This MYM strain was used to produce YopE variant protein in trans for analysis of in vitro GAP activity. YPIII(pIB604) (yopB) is unable to translocate Yop proteins into HeLa cells during infection [32] and was used as a translocation negative control in the protease protection assay. Bacterial strains used to infect HeLa cell cultures were grown overnight in Luria Broth (LB) medium containing the appropriate antibiotic on a rotary shaker at 26 8C. Bacterial strains used to produce protein for in vitro GAP activity measurements were grown overnight at 26 8C in Ca2þ-depleted BHI (Brain Heart Infusion) media containing the appropriate antibiotic and supplemented with 5 mM EGTA (ethylene glycol-bis(b-aminoehtyl ether)-N0 ,N0 ,N0 ,N0 -tetraacetic acid) and 20 mM MgCl2. The overnight culture was sub-cultured to 0.1 OD600 in 2 ml fresh Ca2þ-depleted BHI media and grown for 30 min at 26 8C prior to 4 hours incubation at 37 8C. These conditions produced an optimal amount of soluble YopE protein, and no aggregated protein filaments were detected. The bacteria were separated from the secreted YopE by centrifugation at 13,000g for 10 min. The amount of YopE was characterized after 2-fold serial dilution by immunoblotting with YopE antiserum. 3.3. Cultivation of HeLa cells and the cytotoxicity assay HeLa cell cultures were routinely maintained as previously described [3]. For the cytotoxic assay, cells were plated on cover slips (12 mm diameter) placed in 24 well tissue culture plates. HeLa cells were seeded on cover slips and incubated overnight at 37 8C. Bacteria grown overnight were diluted in fresh cell culture media and incubated for 30 min at 26 8C, followed by one hour at 37 8C prior to infection of HeLa cells were carried out with a multiplicity of infection (MOI) of 10. The HeLa cells were observed over a 2-hour period. Cover slips were fixed in 2% paraformaldehyde at 45 min and two hours post infection, respectively, and the cytotoxicity was assessed by phase contrast microscopy. Infection of HeLa cells and analysis of the cytotoxic effect (altered morphology of cultured cells) were performed as described previously [3]. Cytotoxicity is characterized by a destruction of the actin cytoskeleton of the target cell, resulting in rounding up of the cell [15]. 3.4. Protease protection assay To investigate the translocation of the YopE variants, a modification of the protocol of Nordfelth and Wolf-Watz [17] was followed. Overnight cultures of bacteria were subcultured and grown for 30 min at 26 8C and 1 h at 37 8C before they were added to duplicates of HeLa cells monolayers to give a MOI of 10 as described previously
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[17]. Infection was carried out for 3 h, after which all monolayers were treated with 1 ml of proteinase K (PK) (500 mg/ml in PBS). The PK solution was removed after 60 s to leave only a thin film of liquid on the cells. After 20 min at room temperature, 500 ml of freshly prepared phenylmethylsulfonyl flouride (4 mM in PBS) was added to all monolayers to block protease activity. One set of the monolayers was lysed with 400 ml of digitonin (1% in PBS) before the cells were collected in eppendorf tubes. The incubation continued at room temperature for 10 min. Centrifugation at 4 8C for 10 min at 13,000 rpm cleared the lysate of cell debris and bacteria. The supernatant was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using antiserum raised against YopE or commercially available pan-Erk antibodies (BD Transduction Laboratories, Mississauga, Canada). The other set of the monolayers were not lysed by digitonin, but incubated with 400 ml PBS before collection. The collected cells were cleared of bacteria, cell debris and intact HeLa cells by centrifugation at 5000 rpm for 5 min at 4 8C. The supernatant was analyzed by immunoblotting as described above. 3.5. The GTPase assay Recombinant wild-type Rho GTPases were produced and purified as GST-fusion proteins as described [18,33]. Rho GTPases were loaded with [g-32P]-GTP for 5 min at 37 8C in loading buffer (50 mM Hepes, pH 7.3, 5 mM EDTA, 5 mg/ml BSA). Cold GTP to a final concentration of 0.1 mM was added together with hydrolysis buffer (50 mM Hepes, pH 7.3, 10 mM MgCl2, 1 mM DTT, 100 mM KCl, 0.1 mg/ml BSA). Supernatant from YPIII(pIB29 MEKBA) strains expressing wild-type YopE and the different variants was added to 25 ml loading reaction with a final concentration of Rho GTPases of 11 nM and incubated at 16 8C for Cdc42 and Rac1, and 20 8C for RhoA. The hydrolysis reaction was stopped after 20 min by adding 1 ml of ice cold stop solution (50 mM Hepes pH 7.3, 20 mM MgCl2, 1 mM DTT, 10 mg/ml BSA, 0.1 mM cold GTP) and the GTPase activity was analyzed by a filter binding assay [18,33]. To facilitate comparison between the different strains the relative GAP activity in Ref. Table 1 was calculated as intrinsic hydrolysis set to 100% divided by the % hydrolysis obtained after addition of a given YopE variant.
Acknowledgements We thank PhD Matthew Francis for critical review of the manuscript and PhD Magnus Wolf-Watz for help with the structure modelling programs. This work was supported by grants from the Swedish Medical Research Council, the Swedish Foundation of Strategic Research, the Swedish Cancer Society and the Kempe foundation.
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References [1] Cover TL, Aber RC. Yersinia enterocolitica. N Engl J Med 1989;1: 16–24. [2] Cornelis GR, Wolf-Watz H. The Yersinia Yop virulon: A bacterial system for subverting eukaryotic cells. Mol Microbiol 1997;5: 861–7. [3] Rosqvist R, Forsberg A, Rimpilainen M, Bergman T, Wolf-Watz H. The cytotoxic protein YopE of Yersinia obstructs the primary host defence. Mol Microbiol 1990;4:657–67. [4] Iriarte M, Cornelis GR. YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells. Mol Microbiol 1998;3:915– 29. [5] Schesser K, Frithz-Lindsten E, Wolf-Watz H. Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes. J Bacteriol 1996;24:7227 –33. [6] Sory MP, Boland A, Lambermont I, Cornelis GR. Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci USA 1995;26:11998–2002. [7] Von Pawel-Rammingen U, Telepnev MV, Schmidt G, Aktories K, Wolf-Watz H, Rosqvist R. GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: A mechanism for disruption of actin microfilament structure. Mol Microbiol 2000;3: 737–48. [8] Black DS, Bliska JB. The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol Microbiol 2000;3:515–27. [9] Fu Y, Galan JE. A salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. Nature 1999;6750: 293–7. [10] Goehring UM, Schmidt G, Pederson KJ, Aktories K, Barbieri JT. The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases. J Biol Chem 1999;51: 36369– 72. [11] Evdokimov AG, Tropea JE, Routzahn KM, Waugh DS. Crystal structure of the Yersinia pestis GTPase activator YopE. Protein Sci 2002;2:401 –8. [12] Wurtele M, Wolf E, Pederson KJ, et al. How the Pseudomonas aeruginosa ExoS toxin downregulates Rac. Nat Struct Biol 2001;1: 23–6. [13] Stebbins CE, Galan JE. Modulation of host signaling by a bacterial mimic: Structure of the Salmonella effector SptP bound to Rac1. Mol Cell 2000;6:1449–60. [14] Francis MS, Aili M, Wiklund ML, Wolf-Watz H. A study of the YopD–lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD. Mol Microbiol 2000;1:85–102. [15] Rosqvist R, Forsberg A, Wolf-Watz H. Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun 1991;12:4562–9. [16] Rosqvist R, Wolf-Watz KE, Magnusson H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. Embo J 1994;4:964–72. [17] Nordfelth R, Wolf-Watz H. YopB of Yersinia enterocolitica is essential for YopE translocation. Infect Immun 2001;5:3516–8. [18] Self AJ, Hall A. Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol 1995;67–76. [19] Guex N, Diemand A, Peitsch MC. Protein modelling for all. Trends Biochem Sci 1999;9:364–7. [20] Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 1997; 15:2714–23. [21] Peitsch MC, Wells TN, Stampf DR, Sussman JL. The Swiss-3DImage collection and PDB-Browser on the World-Wide Web. Trends Biochem Sci 1995;2:82–4.
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[22] Koradi R, Billeter M, Wuthrich K. MOLMOL: A program for display and analysis of macromolecular structures. J Mol Graph 1996;1:51– 5. see also p. 29–32. [23] Andor A, Trulzsch K, Essler M, Roggenkamp A, Wiedemann A, Heesemann J, Aepfelbacher M. YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells. Cell Microbiol 2001;5:301– 10. [24] Ridley AJ. Rho proteins: Linking signaling with membrane trafficking. Traffic 2001;5:303–10. [25] Murphy GA, Solski PA, Jillian SA, et al. Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth. Oncogene 1999;26:3831 –45. [26] Vignal E, De Toledo M, Comunale F, Ladopoulou A, GauthierRouvieve C, Blangy A, Fort P. Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42. J Biol Chem 2000;46:36457–64. [27] Horton RM, Pease LR. Recombination and mutagenesis of DNA sequences using PCR. In: McPherson MJ, editor. Directed mutagenesis: A practical approach. New York: Oxford University Press; 1991. p. 217 –47. [28] Rosqvist R, Hakansson S, Forsberg A, Wolf-Watz H. Functional conservation of the secretion and translocation machinery for
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virulence proteins of yersiniae, salmonellae and shigellae. Embo J 1995;17:4187–95. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Press; 1989. Conchas RF, Carniel E. A highly efficient electroporation system for transformation of Yersinia. Gene 1990;1:133–7. Hakansson S, Galyov EE, Rosqvist R, Wolf-Watz H. The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol 1996;3:593–603. Ha˚kansson S, Schesser K, Persson C, Galyov EE, Rosqvist R, Homble F, Wolf-Watz H. The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity. Embo J 1996;21: 5812–23. Self AJ, Hall A. Purification of recombinant Rho/Rac/G25K from Escherichia coli. Methods Enzymol 1995;3–10. Aili M, Hallberg B, Wolf-Watz H, Rosqvist R. Gap activity of Yersinia YopE. Methods Enzymol 2002;358:359 –70.
In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity Margareta Aili, Maxim Telepnev1, Bengt Hallberg2, Hans Wolf-Watz, Roland Rosqvist* Department of Molecular Biology, Umea˚ University, SE-901 87 Umea˚, Sweden Received 20 December 2002; received in revised form 7 March 2003; accepted 7 March 2003
Abstract The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmidencoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166– 183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Yersinia; GAP activity; cytotoxicity; GTPase; YopE
Yersinia pseudotuberculosis, a Gram-negative enteropathogen, causes gastrointestinal infections in humans and animals that are characterized by diarrhea, abdominal pain and fever. Infection occurs mainly through the oral route and the enteropathogenic Yersinia transit to the terminal ileum and initiate infection by penetrating Peyer’s patches. The bacteria can then colonize the mesenteric lymph nodes and may spread to deeper tissues such as the spleen. In rare cases, septicaemia can occur [1]. YopE is an essential virulence determinant during a Y. pseudotuberculosis infection, being secreted and translocated into eukaryotic cells via a contact dependent type III secretion system [2]. The cytosolic localization of YopE induces depolymerisation of the actin cytoskeleton, which results in a rounding up of infected cells. This changed * Corresponding author. Tel.: þ 46-90-785-2529; fax: þ46-90-77-1420. E-mail address: [email protected] (R. Rosqvist). 1 Present address: Department of Clinical Microbiology, Umea˚ University, SE-901 87 Umea˚, Sweden. 2 Present address: Department of Medical Biosciences/Pathology, Umea˚ University, SE-901 87 Umea˚, Sweden.
morphology has been described as a cytotoxic effect [3]. A similar cytotoxic effect is also induced by YopT [4], however YopT is not expressed by the strain used in this study. Utilizing YopE fusions to the Cya reporter indicated that the first 11 amino acids of YopE are sufficient for secretion through the bacterial envelope [5,6], while at least the first 49 amino acids are required for YopE to be translocated into HeLa cells during infection [5]. The YerA/ SycE chaperone requires the first 75 amino acids of YopE to form a stable complex and promote its efficient secretion [5] (Fig. 2). The enzymatic activity of YopE resides in the Cterminus [7]. YopE possesses a GTPase activating protein (GAP) activity towards RhoA, Rac1 and Cdc42 in vitro [7,8]. A conserved arginine finger at position 144 is essential for the GAP activity, indicating the same function as the arginine finger of the eukaryotic GAPs. No other obvious homologies with eukaryotic GAPs can be detected by sequence alignment. On the other hand, YopE shares a high degree of homology with the N-terminal domain of two other bacterial toxins, ExoS of Pseudomonas and SptP of Salmonella,
0882-4010/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0882-4010(03)00063-9
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possessing in vitro GAP activity towards members of the Rho family [9,10]. Thus, all three bacterial GAPs inactivate the Rho family of small GTP-binding proteins. Since small GTP-binding proteins are involved in rearrangement of the actin cytoskeleton, it has been assumed that the cytotoxic phenotype induced by YopE and ExoS, and the effect on actin rearrangement induced by SptP, is caused through inactivation of RhoA, Rac1 and Cdc42 [7,9,10]. However, no in vivo interaction between the bacterial GAPs and the Rho GTPases has so far been demonstrated. The three dimensional structure of the GAP domain of YopE (amino acids 90– 219) was recently solved (Fig. 1) but no structural data of YopE in complex with a Rho GTPase have been published so far [11]. However, the crystal structures of complexes between SptP and Rac1, and
ExoS and Rac1, respectively, have recently been published [12,13] and based on the similarities between YopE and these bacterial GAPs a model of the YopE – Rac1 complex can be made [11]. As observed from SptP and ExoS, the model indicates that interactions between YopE and Rac are limited to three distinct regions of the structure. Residues Ile106, Leu109, Thr138, Gly139, Ser140, and Gln149 of YopE are contacting Switch II region of the GTPase. The arginine finger Arg144 and the bulge residues Thr183, Ile184, and Gly185 are contacting GTP and both of the switch regions, while residues Thr148, Gln151, Pro177, Ser179, and Gln180, are contacting Switch I and the bound nucleotide [11]. By exploiting the known structures of YopE, SptP and ExoS, we can analyze structural effects of mutations in YopE in an effort to define the eukaryotic
Fig. 1. Structure of YopE. A. Structure-based sequence alignment of the bacterial GAP domains of YopE, ExoS and SptP. Residues that are identical in all three sequences are shown in red; residues identical in two out of three sequences, in green. The critical arginine finger is enclosed by a red box. The positions of ahelices and b-strands are indicated above the sequence. B. Overall structure of YopEGAP. The critical arginine residue is shown as a stick model (modified from Ref. [11]). Reproduced with permission from the copyright holder Cold Spring Harbor Laboratory Press, copyright 2002.
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molecular target of YopE and the phenotypical effect of YopE on eukaryotic cells. Herein, we show that mutations flanking the arginine finger are important for the YopE cytotoxic phenotype and in vitro GAP activity towards RhoA, Rac1 and Cdc42. Our results also define regions in YopE that are specific for cytotoxicity and in vitro GAP activity towards RhoA, Rac1 and Cdc42 in isolation, showing these two functions can be separated.
1. Results 1.1. Regions important for the cytotoxic activity of YopE We and others have shown that the arginine residue in position 144 of YopE is essential for YopE-mediated cytotoxicity and in vitro GAP activity towards RhoA, Rac1 and Cdc42 [7,8]. To identify other regions of YopE that are essential to generate the cytotoxic effect, a series of deletions in wild-type yopE were created. The full-length yopE gene harbored on pAF19 was replaced with different variants of yopE (Fig. 2). With this approach, the yopE gene is under the control of its native promoter on a high copy number plasmid and a mutated protein can easily be expressed in any Yersinia strain. Each YopE variant was then analyzed with respect to secretion, translocation and ability to induce a cytotoxic response on HeLa cells. We first examined the requirement of the YopE Cterminus in the cytotoxic process by creating sequential deletions from the C-terminal end of the protein. A FLAGepitope was introduced in the C-terminal end of the fulllength 219 amino acid YopE protein and in five C-terminal deletion mutants creating: YopE219FLAG, YopE215FLAG, YopE202FLAG, YopE191FLAG, YopE179FLAG and YopE134FLAG (Fig. 2). Even in the presence of a Cterminal FLAG-tag, these variants all displayed elements of native folding as implied by their resistance to endogenous proteases (data not shown, [14]). Moreover, the mutant proteins were indistinguishable with respect to secretion from bacteria and translocation into infected HeLa cells (data not shown). Thus, the C-terminal part of YopE from amino acid 135 –219 is not involved in the secretion or translocation process of YopE, consistent with earlier findings [5]. The cytotoxic effect of YopE on HeLa cells after infection can be visualized as a changed morphology using phase contrast microscopy. Infected cells become well rounded up, leaving tail-like retractions that disappear upon prolonged incubation ultimately leading to their surface detachment [3,15,16]. The effect of each YopE variant on the morphology of HeLa cells were analyzed by infecting HeLa cells with Yersinia defective for YopE and YopH (YPIII(pIB251)) expressing in trans wild-type yopE (pAF19) or the respective deletion mutant. Like wild-type YopE a C-terminal deletion of only four amino acids, YopE215FLAG, showed full cytotoxic effect
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on HeLa cells as early as 45 min post-infection. However, the subsequent deletions of 17 (YopE202FLAG), 28 (YopE191FLAG), 40 (YopE179FLAG), and 85 (YopE134FLAG) amino acids, did not induce cytotoxicity on HeLa cells comparable to the arginine finger mutant YopE(R144A) (Fig. 2). Thus, the C-terminus of YopE is essential for cytotoxicity. The N-terminal region of Yop effectors contains secretion and translocation signals needed for transportation of the protein across the bacterial envelope as well as the eukaryotic plasma membrane [5,6]. In order to analyze the importance of the YopE N-terminal region for cytotoxicity, we fused different YopE variants to the N-terminal domain (amino acids 1 –85) of YopH (Fig. 2). With this approach, the secretion and translocation signals were supplied by YopH, providing a tool to discern phenotypes of the YopE N-terminus. Each hybrid was analyzed for secretion and its susceptibility to endogenous proteases to confer protein stability (data not shown, [14]). Furthermore, an analysis of protein secretion and subsequent translocation into infected HeLa cells revealed that these YopH-YopE hybrids were essentially indistinguishable from wild-type YopE (data not shown). Deletion of the first 89 amino acids of YopE (YopH85YopED1-89) showed a full cytotoxic effect on HeLa cells (Fig. 2). However, deletion of additional 10 amino acids (YopH85-YopED1-99) resulted in a significantly reduced cytotoxic effect. Not only was the effect delayed, but only a fraction of the cell population exhibited an altered morphology. The observation that not every cell was cytotoxically affected, might be explained by the fact that a higher amount of the mutant YopE protein must be injected to affect cells than wild-type YopE protein. Given that the infection is a stochastic event, it is possible that only cells with a number of bacteria above the threshold were affected. Nevertheless, this shows that the mutants are able to induce cytotoxicity, although they are less active than wild-type YopE. Furthermore, deletion of additional ten amino acids (YopH85-YopED1-109) totally abolished the cytotoxic effect on the cell monolayer. It follows that further deletions of amino acids 1– 119 (YopH85-YopED1-119) and amino acids 1– 128 (YopH85-YopED1-128) were also unable to induce cytotoxicity (Fig. 2). Collectively, these investigations showed that the minimal region required to induce YopE cytotoxicity is located between amino acids 90 and 215. To further dissect this region, we made in-frame deletions within the amino acids 108 – 215 of YopE (Fig. 2). These mutants were used to infect semi-confluent HeLa cell monolayers to assess their ability to induce morphologically changes. An internal deletion of four amino acids (YopED131-135) did not change the cytotoxic activity of YopE on HeLa cells, whereas larger deletions (YopED108-128, YopED131-149, YopED131-169, YopED131-189) rendered the protein inactive. Therefore,
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Fig. 2. Deletion mutants used in this study. Wild-type yopE, located in pAF19 (a high-copy plasmid), and derivatives containing mutant alleles. All variants were expressed in Y. pseudotuberculosis YPIII(pIB251) (yopEyopH) background to asses cytotoxic effect on HeLa cells and in MYM background for expression of protein used for in vitro GAP activity measurements.
amino acids 108– 128 and 136– 189 are required to generate a cytotoxic effect. The latter region was further investigated by generating seven small in-frame deletion mutants ranging from amino acid 162 to 183. In addition, YopED189-192 and YopED211-215 were created (Fig. 2). These in-frame deletions were stable and secreted normally (data not shown, [14]). The YopE deletions were analyzed for their cytotoxic effect on HeLa cells during infection. One mutant,
YopED189-192 showed cytotoxic activity equivalent to wild-type, while two mutants, YopED162-165 and YopED211-215, did not cause a complete rounding up of infected cells although the cells were clearly cytotoxically affected (Fig. 3). The remaining six deletion mutants (YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180 and YopED180-183) had lost the ability to cause morphological changes on HeLa cells (Figs. 2 and 3). This indicates that the region between
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Fig. 3. Cytotoxic effect on HeLa cells after 2 h bacterial infection. HeLa cells were seeded at 2 £ 105 cells/ml on cover slips placed in 24 well tissue culture plates. The effect on the cell morphology was assessed by phase-contrast microscopy after 2 h infection with no bacteria or Y. pseudotuberculosis YPIII(pIB251) containing pAF19 and the nine small deletion constructs described in Fig. 2. The images were captured by a Hamamatzu CCD camera through a 40 £ magnification objective. Shown are images from HeLa cells infected with four different deletion mutants.
amino acid 165 and 183 contained amino acids essential to induce a cytotoxic response on HeLa cells. The lack of a cytotoxic response could either be caused by an inactive protein or through defective translocation into the HeLa cells. To distinguish between these two possibilities, infected cells were assayed for protease protection of YopE after digitonin fractionation of infected HeLa cells [17]. To discriminate between YopE located outside of the HeLa cells from translocated YopE, the infected HeLa cell monolayers were treated with proteinase K, which has previously been shown to degrade secreted YopE [17]. The amount of translocated protein was assessed by comparing supernatants obtained from non-lysed cells (containing only proteins released to the culture medium) with the soluble contents derived from lysed cells. The eukaryotic protein Erk was used as an internal control for efficient lysis and to ensure that an equivalent level of protein was loaded onto gels. The translocation defective yopB null mutant [YPIII(pIB604)] showed the similar low level of protein in both lysed and non-lysed samples, confirming its inability to translocate YopE (Fig. 4). In some cases, we observed that a small amount of YopE was recovered from infected protease-treated non-lysed cells. A likely explanation is that during infection of HeLa cells with a Yersinia strain overexpressing YopE, secreted YopE is trapped between the bacteria and the eukaryotic cell in a protease protected environment, which is released into
the media when the cells are scraped off the culture dish. However, this is not the case when the wild-type protein is expressed at wild-type levels from the virulence plasmid, and not in trans. The different YopE deletions were generally translocated into HeLa cells to similar or higher levels as wild-type YopE. However, we noted that YopED162-165 and YopED165-168 were translocated less efficiently (Fig. 4), although YopED162-165 was still able to induce a cytotoxic response, whereas YopED165-168 was not able to induce a cytotoxic response (Fig. 3). 1.2. In vitro GAP activity YopE posses an in vitro GAP activity towards members of the small Rho GTPase subfamily [7,8]. Therefore, we wanted to examine the relationship between cytotoxic effect of YopE on HeLa cells and its in vitro GAP activity. We measured the in vitro GAP activity of secreted protein deletions (YopED162-165, YopED165-168, YopED168171, YopED171-174, YopED174-177, YopED177-180, YopED180-183, YopED189-192 and YopED211-215) expressed in trans in a Multiple Yop Mutant background, MYM [YPIII(pIB29MEKBA)], to avoid possible adverse effects of other effector proteins of Yersinia. As this MYM strain is devoid of YopH, YopM, YopE, YopK, YopB, and YpkA, measurable GAP activity could only be attributed to YopE variants produced in trans. To ensure an equal amount
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Fig. 4. Translocation of YopE wild type and mutant proteins after 3 h infection of HeLa cells. Infections of HeLa cells with strains of Y. pseudotuberculosis expressing mutant alleles of yopE in trans were carried out in duplicate. Three hours postinfection all the HeLa cells monolayers were treated with proteinase K for 20 min, prior to the addition of phenylmethylsulfonyl flouride (4 mM in PBS) to block the remaining protease activity. To one set of the HeLa cells cultures digitonin (þ ) were added to lyse the cells, while to the other set no digitonin were added (2). The HeLa cells were harvested and collected in eppendorf tubes and centrifuged to clear the sample from cell debris and bacteria. The supernatant was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using antiserum raised against YopE (upper panel) or commercially available pan-Erk antibodies (lower panel). The estimated molecular weights are for YopE 24 kD and Erk 42 kD. The translocation defective YPIII(pIB604) (yopB) was included as a negative control [32].
of each protein was used for analysis of the in vitro GAP activity supernatant samples were initially characterized by immunoblot using anti-YopE antiserum. The GTPase assay was performed as described [18]. As expected, all of the deletion mutants unable to induce a cytotoxic effect on HeLa cells (YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180 and YopED180-183) were also unable to inactivate RhoA, Rac1 and Cdc42 in vitro (Fig. 5a). Surprisingly, three deletion mutants causing cytotoxic effect on HeLa cells (YopED162-165, YopED189-192 and YopED211-215) were still unable to inactivate RhoA, Rac1 and Cdc42 in vitro. We interpret this finding to indicate that induction of a cytotoxic effect on HeLa cells is not absolutely linked to the ability of YopE to inactivate RhoA, Rac1 and Cdc42 in vitro. Deletion of four amino acids might cause an adverse effect on the overall structure of the protein, leading to loss of function. To minimize these possible effects, we made nine substitution mutants where three consecutive amino acids were exchanged for alanine (Fig. 6). We detected no difference in stability or secretion of these variants compared to wild-type YopE (data not shown). In contrast to the corresponding deletion mutants however, eight of the nine substitution mutants, induced a cytotoxic effect on semi-confluent monolayers of HeLa cells after infection (Table 1). This result suggests that the spacing of the amino acids in this region is important for the cytotoxic phenotype. In addition, the fact that either a deletion of amino acids 180 –183 or substitution of amino acids 181– 183 rendered the protein inactive in the cytotoxic assay reflects a direct functional role for this site. We further investigated the in vitro GAP activity of the alanine-substitution mutants towards RhoA, Rac1 and Cdc42. One substitution mutant (YopE210-12A) inactivated all of the three GTPases, although not as effectively as the wild-type YopE (Fig. 5b, Table 1). The relative in vitro GAP activity of YopE210-12A towards RhoA, Rac1 and Cdc42 was 4.5 ^ 1.3, 2.8 ^ 0.9 and 4.3 ^ 2.1, respectively, compared to 6.7 ^ 1.7, 21.9 ^ 10.3 and 7.9 ^ 1.4 for
wild-type YopE. The cytotoxic negative mutant (YopE18183A) had no measurable in vitro GAP activity. Interestingly, five of the substitution mutants (166-68A, 169-71A, 17577A, 178-80A and 206-08A) showed similar low levels of in vitro GAP activity against RhoA, Rac1 and Cdc42 as the MYM control strain. Of these mutants YopE175-77A showed the highest relative in vitro GAP activity towards RhoA, 1.2 ^ 0.2, Rac1, 1.2 ^ 0.1 and Cdc42, 1.3 ^ 0.2 compared to the MYM control strain values of RhoA, 1.0 ^ 0.1, Rac1, 1.2 ^ 0.0 and Cdc42, 1.0 ^ 0.1. Thus, all these five mutants show in vitro GAP activity of the same level as the MYM control strain. Interestingly, all five mutants were able to induce a cytotoxic response on HeLa cells. As for the other two mutants (172-74A and 213-15A), somewhat higher activities towards the GTPases tested were detected, but none to the same level as wild-type YopE (Table 1). 1.3. Structure modelling of YopE Recently the three dimensional structure of the GAP ˚ domain of YopE (amino acids 90 – 219) was solved at 2.2 A resolution [11]. The structure is almost entirely composed of a-helices and shows no obvious structural similarity to eukaryotic RhoGAP domains (Fig. 1). However, the structure of YopE is very similar to the GAP domains of ExoS of Pseudomonas aeruginosa [12] and SptP of Salmonella typhimurium [13]. We have used the SWISSMODEL Protein Modeling Server [19 – 21] to create structural models of the nine deletion mutants (YopED162-165, YopED165-168, YopED168-171, YopED171-174, YopED174-177, YopED177-180, YopED180-183, YopED189-192 and YopED211-215) and the nine corresponding alanine-substitution mutants. With the help of the structural viewing program MOLMOL [22], we have compared the models of the deletion mutants with wild-type YopE. In four cases (YopED162-165, YopED165-168, YopED168-171 and YopED189-192), a change in the position of the arginine finger was observed (data not shown). This may explain the observed lack of
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Fig. 5. YopE in vitro GAP activity towards RhoA, Rac1 and Cdc42. Recombinant RhoA, Rac1 and Cdc42 were loaded with g-32P GTP for 5 minutes at 37 8C. For analysis of GTP hydrolysis, supernatants from Y. pseudotuberculosis MYM strains producing in trans YopE deletions (panel A) or substitutions (panel B) were added to 11 nM Rac1, 11 nM Cdc42, or 11 nM RhoA, respectively, and incubated for 20 minutes at 16 8C (Rac1 and Cdc42) or 20 8C (RhoA). To establish the intrinsic level of hydrolysis, loaded GTPases were incubated at the reaction temperature for 20 minutes without addition of GAP protein. Shown is the remaining g-32P GTP bound to RhoA, Rac1 and Cdc42 as a percentage of the loaded GTPases. Each point represents the mean of three independent experiments in duplicate samples with standard deviations.
GAP activity towards the Rho proteins. In the other five deletion mutants, the arginine finger occupies the same position as in the wild type protein and no visible change in the Rac1 contact area was observed. Nevertheless, large differences in the structure of the deletion mutants were
generally observed in the local area of the deletion. In line with this, these deletions severely affected the GAP activity of YopE. However, deletion mutants YopED162-165 and YopED189-192 were still able to induce cytotoxicity on HeLa cells although they lacked in vitro GAP activity.
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2. Discussion
Fig. 6. YopE triple-alanine substitution mutants used in this study. Wildtype yopE, located in pAF19 (a high-copy plasmid), and derivatives containing mutant alleles. All variants were expressed in Y. pseudotuberculosis YPIII(pIB251) (yopE, yopH) background to asses cytotoxic effect on HeLa cells and in MYM background for expression of protein used for in vitro GAP activity measurements.
Modeling of the substitution mutants showed no significant change in their structure compared to wild-type YopE. In particular, the position of the essential argininefinger was maintained. As expected, this confirms that the substitution mutants have less effect on the structure compared to the deletion mutants. Thus, the lack of in vitro GAP activity might be due to loss of specific interaction with RhoA, Rac1 and Cdc42.
YopE of Yersinia is an essential virulence determinant, which is translocated into the eukaryotic target cell through a contact-dependent mechanism. The bacteria remains bound to the surface of the target cell while YopE is translocated into the target cell cytosol via the type III secretion system [2,16]. YopE causes disruption of the actin microfilament stress fibers [15] and causes the eukaryotic cell to round up, leaving a tail-like cytoplasmic membrane retraction. This characteristic morphological change of the cell shape can easily be visualized by phase contrast microscopy and has been denoted as a cytotoxic effect on the HeLa cell [3,15]. It has recently been shown that YopE possess a GTPaseactivating protein (GAP) activity that down-regulates RhoA, Rac1 and Cdc42 in vitro [7,8,23]. Mutation of a critical arginine residue at position 144 completely abolishes the GAP activity of YopE and renders this mutant avirulent [34] and unable to induce a cytotoxic effect on HeLa cells [7,8]. This has led to the conclusion that the YopE GAP activity is essential for induction of the HeLa cell cytotoxic phenotype [7]. This is supported by the fact that HeLa cells pre-treated with CNF1 (cytotoxic necrotizing factor 1) results in constitutive active Rho proteins that rescue the HeLa cells from the YopE cytotoxic effect [7]. In parallel, introduction of a
Table 1 Ability of wild-type YopE and different YopE mutants to induce a cytotoxic response on cultured HeLa cells, and their relative in vitro GAP activity towards RhoA, Rac1 and Cdc42 Construct
PIB251(pAF19) YPIII(pIB29)MYM PIB251(pYopED162-165) PIB251(pYopED165-168) PIB251(pYopED168-171) PIB251(pYopED171-174) PIB251(pYopED174-177) PIB251(pYopED177-180) PIB251(pYopED180-183) PIB251(pYopED189-192) PIB251(pYopED211-215) PIB251(pYopED166-8A) PIB251(pYopED169-71A) PIB251(pYopE172-4A) PIB251(pYopE175-7A) PIB251(pYopE178-80A) PIB251(pYopE181-3A) PIB251(pYopE206-8A) PIB251(pYopE210-12A) PIB251(pYopE213-15A) RhoGAP
Mutation
Wt YopE, yopH, yopM, ypkA, yopB, yopK D162–165 D165–168 D168–171 D171–174 D174–177 D177–180 D180–183 D189–192 D211–215 166-8A 169-71A 172-4A 175-7A 178-80A 181-3A 206-8A 210-12A 213-15A 2
Cytotoxicity on HeLe-cells
þ þþ 2 þþ 2 2 2 2 2 2 þ þþ þþ þ þþ þ þþ þ þþ þ þþ þ þþ 2 þ þþ þ þþ þ þþ n.d.
In vitro GAPactivity RhoA
Rac1
Cdc42
6.7 ^ 1.7 1.0 ^ 0.1 1.0 ^ 0.2 1.2 ^ 0.1 1.1 ^ 0.1 1.0 ^ 0.1 1.2 ^ 0.1 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.2 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.1 1.4 ^ 0.1 1.2 ^ 0.2 1.1 ^ 0.1 0.9 ^ 0.0 0.8 ^ 0.0 4.5 ^ 1.3 1.4 ^ 0.1 18.4 ^ 3.8
21.9 ^ 10.3 1.2 ^ 0.1 1.1 ^ 0.3 0.9 ^ 0.3 1.4 ^ 0.3 1.6 ^ 0.6 1.6 ^ 0.8 1.5 ^ 0.3 1.5 ^ 0.3 1.4 ^ 0.3 1.4 ^ 0.2 1.2 ^ 0.2 1.1 ^ 0.0 1.6 ^ 0.3 1.2 ^ 0.1 1.1 ^ 0.1 0.9 ^ 0.1 1.1 ^ 0.3 2.8 ^ 0.9 1.4 ^ 0.3 12.7 ^ 4.6
7.9 ^ 1.4 1.0 ^ 0.1 1.0 ^ 0.1 1.2 ^ 0.3 1.1 ^ 0.2 1.2 ^ 0.3 1.1 ^ 0.2 1.1 ^ 0.1 1.1 ^ 0.1 1.2 ^ 0.1 1.0 ^ 0.1 1.0 ^ 0.3 1.0 ^ 0.0 1.4 ^ 0.1 1.3 ^ 0.2 1.2 ^ 0.2 1.0 ^ 0.0 1.2 ^ 0.2 4.3 ^ 2.1 1.4 ^ 0.4 12.9 ^ 1.5
Cytotoxic effect after infection of HeLa cells evaluated 2 h post-infection. A sliding scale of þþ þ indicates complete rounding up of the HeLa cells, while 2 indicates no visible effect on the cell morphology. The relative GAP-activity has been calculated as intrinsic hydrolysis set to 100% divided by the hydrolysis after addition of the YopE variant. The mean from three independent experiments together with the standard deviation (^) has been calculated.
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constitutive active RhoA (RhoA-V14) into HeLa cells rescued infected cells from the cytotoxic response [8]. While these studies collectively support the view that the GAP activity is necessary for the cytotoxic phenotype of YopE on HeLa cells, evidence for a direct interaction between YopE and the Rho proteins during infection is lacking. To better understand the biological role of YopE, we investigated what regions of YopE are important for the induction of cytotoxicity on HeLa cells. Our strategy was to create deletion and substitution mutations along the YopE protein and analyze their effect on secretion, translocation and the ability to cause a cytotoxic effect on HeLa cells. In addition, to discern the relationship between cytotoxicity and the enzymatic activity of YopE, mutants were also analyzed for GAP activity towards the Rho proteins RhoA, Rac1 and Cdc42 in vitro. Sequential removal of the N- and C-terminus of YopE identified a region between amino acids 90 and 215 that is necessary to induce cytotoxicity. As expected, internal deletions containing the essential arginine at position 144 resulted in a total loss of the cytotoxic response. Smaller inframe deletions flanking the arginine finger defined an additional region important for the cytotoxic effect to amino acids 166– 183. To further analyze this region, alaninesubstitution of three consecutive amino acids were made to reduce impact on the protein structure. We tested nine alanine-substitution mutants for in vitro GAP activity towards RhoA, Rac1 and Cdc42 and for ability to induce cytotoxicity. Out of the nine ala-substitution mutants, one (YopE210-12A) showed activity towards RhoA, Rac1 and Cdc42, albeit not as high as wild-type YopE. This mutation is located in the a8 helix which is not making a direct contact with the GTPase (Fig. 1). Two mutants (YopE17274A and YopE213-15A) showed a slightly higher in vitro GAP activity than background levels, while the other six substitution mutants showed an in vitro GAP activity comparable to background activity. Interestingly, five out of these six substitution-mutants were still able to induce a cytotoxic response on infected HeLa cells. These five mutations are located in a region spanning the a5, b1-2, and the a6 helices. YopE175-7A and YopE178-80A are located in the b1 –2 and the a6 region affecting amino acids YopE177 and YopE179 that most likely are contacting the Switch 1 region and the bound nucleotide in Rac1 [11], explaining the loss of activity in these mutants. Interestingly, we have only been able to identify one substitution mutant (YopE181-3A) that is completely devoid of any measurable in vitro GAP activity as well as unable to induce any cytotoxic response on HeLa cells. This mutation is located in the bulge region that connects helices a6 and a7, and the residues within and around this bulge are strictly conserved in the bacterial GAPs. This region is contacting the two switch regions as well as the bound GTP in Rac1. Single amino acid substitutions of the corresponding residues in SptP (Q246 and T249 (Q180 and T183 in
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YopE)) render the SptP mutants to loose its GAP activity, similar to what we have found here. Thus, this region in YopE is essential for both the GTPase activity and the cytotoxic response. These results reinforce the close relationship between SptP and YopE and provide additional evidences for the importance of this region in YopE. Thus, we have identified five substitution mutants located in different regions of YopE with a similar phenotype: able to induce cytotoxicity on HeLa cells but showed no in vitro GAP activity towards the tested Rho GTPases. We have been unable to identify mutants that are unable to induce cytotoxicity but still possess in vitro GAP activity, reinforcing the earlier notation that GAP activity of YopE is important for the cytotoxic response. Assuming that the folding properties of YopE are the same during in vivo and in vitro conditions these results suggest that YopE might exhibit additional unidentified target(s) within the eukaryotic cell. Some of the mutants causing a cytotoxic response in HeLa cells have an in vitro GAP activity towards one or more of the tested GTPases just above background levels (Table 1). This low activity might be enough to explain the observed cytotoxic phenotype, but since there are mutants having similar low in vitro GAP activity that is completely non-cytotoxic, it seems unlikely that this residual activity is the cause of the cytotoxicity. In addition, other mutants show a full cytotoxic effect, but have no measurable in vitro GAP activity suggesting that YopE has additional eukaryotic target(s) other than RhoA, Rac1 and Cdc42. However, this remains to be shown. Black and Bliska showed that constitutive active RhoA is able to rescue cells from YopE induced cytotoxicity [8], arguing the fact that RhoA is the target of YopE. However, transfection of active RhoA may alter the balance of other substrates involved in the actin homeostasis. Thus, the effect of YopE on its eukaryotic target could be masked by the dominant effect of RhoA on the actin cytoskeleton dynamics, especially if YopE’s target is upstream of RhoA or in a parallel pathway. The fact that overexpression of mammalian RhoA, Rac1 and Cdc42 in yeast suppressed the toxic effect of YopE, although to a lower extent then Rho1, the yeast homologue to RhoA [7], also supports the notion that the use of transfection to produce higher intracellular amounts of the respective GTPase may sequester YopE from its bona vide cellular target. In addition it was recently published that YopE selectively targets Rac1 mediated signalling pathways [23], since YopE was able to prevent Cdc42 dependent Rac1 activated ruffling after stimulation with bradykinin, but YopE was unable to affect actin structures regulated by Cdc42 (filopodia formation) and RhoA (stress fiber assembly). Thus, contradicting the results presented by Black and Bliska [8]. However, since these studies utilize drugs that may have broader specificity than only towards RhoA, Rac1 and Cdc42, it is possible that other targets of YopE were overlooked. In addition, cell type-specific responses may also explain the observation that YopE
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specifically targeted Rac1 pathways in vivo [23], while in other studies the RhoA and Cdc42 pathway was targeted [7, 8]. Thus, although several studies suggest that YopE targets RhoA, Rac1 and Cdc42, it is not at all clear that the YopE GAP activity is specifically directed towards these three proteins. The family of small Rho GTPases to date consists of around 20 members in humans, and of these RhoA, Rac1 and Cdc42 are the most extensively studied. RhoA has two highly related homologues, RhoB and RhoC. Given that RhoB is involved in actin organization and vesicle transport [24] and shares high homology with RhoA, might make it an interesting target candidate of YopE. Other possible candidates include the Cdc42 relatives TC10 and TCL, which are implicated in actin-based reorganization [25,26]. Thus, from the results presented here we find support for the hypothesis that YopE has additional targets within the eukaryotic cell and we are currently looking for the bona vide eukaryotic molecular target of YopE.
3. Material and methods 3.1. Construction of yopE mutants PCR-amplified DNA fragments used for constructing the in-frame deletion mutants of yopE were generated by overlap PCR [27] using the pAF19 plasmid [28] as a template for the deletions within the yopE gene. Isolation of plasmid DNA from E. coli strains was performed using the Quantum Prep Plasmid Miniprep kit (Bio-RAD) as described by the manufacturer. Oligonucleotides were synthesized with an Applied Biosystems DNA/RNA synthesizer model 392. Oligonucleotide sequences can be obtained by request. Standard DNA manipulation techniques were essentially used as described [29]. Recovery of DNA fragments from agarose was achieved by spin-column purification (Amicon) as detailed by the manufacturer. The PCR-generated DNA-fragments were gel purified, digested with restriction enzymes and cloned back into the pAF19 plasmid. The clones were screened by PCR and plasmid DNA from one positive clone was isolated, and the sequence was confirmed using the T7 sequencing kit (Pharmacia/Amersham). For the mutants containing the N-terminal part of yopH, the first 89 amino acids of YopE were substituted with the N-terminal 85 amino acids of YopH prior to the introduction of the yopE deletion variants. Plasmids carrying the different YopE variants were introduced into the Yersinia strains by electroporation [30] using a Gene Pulsar apparatus (BioRad Laboratories, Richmond, CA). 3.2. Bacterial strains and growth conditions The Y. pseudotuberculosis strain YPIII(pIB251), unable to express YopE and YopH [3], were used to assess the
cytotoxicity of YopE variants in trans after infection of HeLa cells. The Yersinia Multiple Yop Mutant (MYM [YPIII(pIB29MEKBA)]) strain [31] is unable to express YopM, YopE, YopK, YpkA, YopH, and YopB, respectively. This MYM strain was used to produce YopE variant protein in trans for analysis of in vitro GAP activity. YPIII(pIB604) (yopB) is unable to translocate Yop proteins into HeLa cells during infection [32] and was used as a translocation negative control in the protease protection assay. Bacterial strains used to infect HeLa cell cultures were grown overnight in Luria Broth (LB) medium containing the appropriate antibiotic on a rotary shaker at 26 8C. Bacterial strains used to produce protein for in vitro GAP activity measurements were grown overnight at 26 8C in Ca2þ-depleted BHI (Brain Heart Infusion) media containing the appropriate antibiotic and supplemented with 5 mM EGTA (ethylene glycol-bis(b-aminoehtyl ether)-N0 ,N0 ,N0 ,N0 -tetraacetic acid) and 20 mM MgCl2. The overnight culture was sub-cultured to 0.1 OD600 in 2 ml fresh Ca2þ-depleted BHI media and grown for 30 min at 26 8C prior to 4 hours incubation at 37 8C. These conditions produced an optimal amount of soluble YopE protein, and no aggregated protein filaments were detected. The bacteria were separated from the secreted YopE by centrifugation at 13,000g for 10 min. The amount of YopE was characterized after 2-fold serial dilution by immunoblotting with YopE antiserum. 3.3. Cultivation of HeLa cells and the cytotoxicity assay HeLa cell cultures were routinely maintained as previously described [3]. For the cytotoxic assay, cells were plated on cover slips (12 mm diameter) placed in 24 well tissue culture plates. HeLa cells were seeded on cover slips and incubated overnight at 37 8C. Bacteria grown overnight were diluted in fresh cell culture media and incubated for 30 min at 26 8C, followed by one hour at 37 8C prior to infection of HeLa cells were carried out with a multiplicity of infection (MOI) of 10. The HeLa cells were observed over a 2-hour period. Cover slips were fixed in 2% paraformaldehyde at 45 min and two hours post infection, respectively, and the cytotoxicity was assessed by phase contrast microscopy. Infection of HeLa cells and analysis of the cytotoxic effect (altered morphology of cultured cells) were performed as described previously [3]. Cytotoxicity is characterized by a destruction of the actin cytoskeleton of the target cell, resulting in rounding up of the cell [15]. 3.4. Protease protection assay To investigate the translocation of the YopE variants, a modification of the protocol of Nordfelth and Wolf-Watz [17] was followed. Overnight cultures of bacteria were subcultured and grown for 30 min at 26 8C and 1 h at 37 8C before they were added to duplicates of HeLa cells monolayers to give a MOI of 10 as described previously
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[17]. Infection was carried out for 3 h, after which all monolayers were treated with 1 ml of proteinase K (PK) (500 mg/ml in PBS). The PK solution was removed after 60 s to leave only a thin film of liquid on the cells. After 20 min at room temperature, 500 ml of freshly prepared phenylmethylsulfonyl flouride (4 mM in PBS) was added to all monolayers to block protease activity. One set of the monolayers was lysed with 400 ml of digitonin (1% in PBS) before the cells were collected in eppendorf tubes. The incubation continued at room temperature for 10 min. Centrifugation at 4 8C for 10 min at 13,000 rpm cleared the lysate of cell debris and bacteria. The supernatant was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using antiserum raised against YopE or commercially available pan-Erk antibodies (BD Transduction Laboratories, Mississauga, Canada). The other set of the monolayers were not lysed by digitonin, but incubated with 400 ml PBS before collection. The collected cells were cleared of bacteria, cell debris and intact HeLa cells by centrifugation at 5000 rpm for 5 min at 4 8C. The supernatant was analyzed by immunoblotting as described above. 3.5. The GTPase assay Recombinant wild-type Rho GTPases were produced and purified as GST-fusion proteins as described [18,33]. Rho GTPases were loaded with [g-32P]-GTP for 5 min at 37 8C in loading buffer (50 mM Hepes, pH 7.3, 5 mM EDTA, 5 mg/ml BSA). Cold GTP to a final concentration of 0.1 mM was added together with hydrolysis buffer (50 mM Hepes, pH 7.3, 10 mM MgCl2, 1 mM DTT, 100 mM KCl, 0.1 mg/ml BSA). Supernatant from YPIII(pIB29 MEKBA) strains expressing wild-type YopE and the different variants was added to 25 ml loading reaction with a final concentration of Rho GTPases of 11 nM and incubated at 16 8C for Cdc42 and Rac1, and 20 8C for RhoA. The hydrolysis reaction was stopped after 20 min by adding 1 ml of ice cold stop solution (50 mM Hepes pH 7.3, 20 mM MgCl2, 1 mM DTT, 10 mg/ml BSA, 0.1 mM cold GTP) and the GTPase activity was analyzed by a filter binding assay [18,33]. To facilitate comparison between the different strains the relative GAP activity in Ref. Table 1 was calculated as intrinsic hydrolysis set to 100% divided by the % hydrolysis obtained after addition of a given YopE variant.
Acknowledgements We thank PhD Matthew Francis for critical review of the manuscript and PhD Magnus Wolf-Watz for help with the structure modelling programs. This work was supported by grants from the Swedish Medical Research Council, the Swedish Foundation of Strategic Research, the Swedish Cancer Society and the Kempe foundation.
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