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Infection and Immunity, September 2007, p. 4423-4431, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00528-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Serogroup-Related Escape of Yersinia enterocolitica YopE from Degradation by the Ubiquitin-Proteasome Pathway

Moritz Hentschke,¹ Konrad Trülzsch,² Jürgen Heesemann,² Martin Aepfelbacher,¹ and Klaus Ruckdeschel^1*

Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany,¹ Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Pettenkoferstrasse 9a, 80336 Munich, Germany²

Received 13 April 2007/ Returned for modification 22 May 2007/ Accepted 18 June 2007

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Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.

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Pathogenic microorganisms have evolved complex tactics to manipulate the immune response of the host. An archetypical pathogen for studying the interaction between bacteria and the host cell is the gram-negative bacterium Yersinia. Three Yersinia spp. can cause diseases in humans and rodents. These are Yersinia pestis, the causative agent of bubonic plague, and Yersinia enterocolitica and Yersinia pseudotuberculosis, which mediate gastrointestinal syndromes, lymphadenitis, and septicemia (9). Although the Yersinia spp. take different routes of infection, they share the characteristic that they survive and multiply extracellularly in the host lymphoid tissue. This ability depends on the presence of a common, plasmid-encoded type III protein secretion system that acts as a Yersinia core virulence determinant (9, 18). The type III protein secretion system is activated upon host cell contact. It mediates the polarized translocation of Yersinia effector proteins (Yersinia outer proteins, or Yops) inside eukaryotic cells, where the Yops interfere with critical signaling processes of the host immune response. The Yops neutralize a sequence of programmed effector functions of host immunity. YopE, YopT, YopH, and YopO/YpkA inhibit rearrangements of the actin cytoskeleton that otherwise mediate internalization of the bacteria by the infected cell (6, 9, 18). This helps Yersinia to prevent its uptake and killing by phagocytes. Interestingly, YopE, YopT, and YopO/YpkA all act on members of the Rho-GTPase family (2, 6). The Rho-GTPases regulate the dynamics of the actin cytoskeleton and a multitude of other cellular functions. YopE is a GTPase-activating protein which inactivates Rho-GTPases by increasing their intrinsic GTPase activity (5, 45). This switches the GTPases into an inactive state. YopT is a cysteine protease that represses Rho-GTPase members by cleaving off their C-terminal isoprenoid moieties (35). The serine/threonine kinase YopO/YpkA possesses a Rac1 binding domain that mimics Rho guanidine nucleotide dissociation inhibitors. YopO/YpkA consequently inhibits the nucleotide exchange in Rac1 and RhoA which locks these GTPases in an inactive state (31). YopH dismantles peripheral focal adhesion complexes by dephosphorylating host cell proteins, such as p130Cas and the focal adhesion kinase (4, 28). Furthermore, Yersinia represses the proinflammatory response of infected cells and triggers apoptosis in macrophages. These effects are mediated by YopP/YopJ, which acetylates members of the mitogen-activated protein kinase kinase superfamily and the NF-B-activating IB kinase-ß. These events deactivate the mitogen-activated protein kinase and NF-B signaling pathways and prevent the production of protective cytokines, such as tumor necrosis factor alpha and interleukin-8 (25-27).

While these immunomodulatory activities of Yops have been intensively studied, little is known about the reaction of the host cell to Yop infection. Our previous studies have shown that the infected cell has developed mechanisms to counteract the Yop effects. It was revealed that YopE is degraded and inactivated through the ubiquitin-proteasome pathway after it has been translocated inside the host cell (32). The proteasome is a self-compartmentalizing protease complex that executes the controlled breakdown of intracellular proteins. It regulates the half-life of the vast majority of the eukaryotic proteins and thereby contributes to maintain cellular homeostasis (12, 29). The proteins that are destined for proteasomal destruction are marked with lysine-48-linked polyubiquitin chains to allow recognition and processing by the proteasome complex in order to generate oligopeptides and recyclable ubiquitin (29, 39). This protein-editing function mediates the processing of intracellular antigens for presentation by major histocompatibility complex class I (21). Additionally, the ubiquitin-proteasome system apparently plays a direct role in the innate immune response against bacterial infection. Our data have demonstrated that the degradation of YopE by the proteasome contributes to reverse the antiphagocytic effect of Yersinia (32). This indicates an immediate function of the proteasome to fight bacterial infection. In line with this conclusion, several other bacterial effector proteins have been demonstrated to be subjected to proteasomal degradation. Accordingly, SopE and SopA from Salmonella enterica and ExoT from Pseudomonas aeruginosa are target molecules of the ubiquitin-proteasome pathway (3, 22, 49). The inactivation of ExoT participates to limit P. aeruginosa-mediated disease (3). This suggests that the modification with polyubiquitin and the subsequent destruction by the proteasome could be a basic strategy of the host cell to restrict the activities of a subset of bacterial proteins translocated via type III protein secretion.

In this study, we analyzed several pathogenic Y. enterocolitica serotypes for the susceptibilities of their YopE protein species to degradation by the ubiquitin-proteasome pathway. We report that YopE of Y. enterocolitica serogroups O3 and O9 evades polyubiquitination and proteasomal destabilization. In contrast, YopE from serotype O8 is effectively ubiquitinated and inactivated. The susceptibility of the YopE O8 isotype to polyubiquitination and degradation is determined by two critical lysine residues in the YopE N terminus (K62 and K75). These lysines are absent in the YopE protein species from serogroups O3 and O9. Introduction of these lysine residues in YopE from serogroup O9 induces YopE ubiquitination and destabilization, whereas replacement of the lysines in YopE from serogroup O8 by arginine and glutamine of YopE O9 stabilizes the YopE protein levels and its cytotoxic effect. Thus, the amino acids at positions 62 and 75 critically control the stability and activity of the Yersinia effector protein YopE. The susceptibility of YopE to polyubiquitination could be relevant for differential function and virulence of Y. enterocolitica serotypes.

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Yersinia strains, cell lines, and infection conditions. The Y. enterocolitica strains used in this study are listed in Table 1. Three different Y. enterocolitica wild-type strains (WA-314, E40, and 108-P) and a virulence plasmid-cured derivative of strain WA-314, termed WA-C, were investigated. The Y. enterocolitica serotype O9 strain E40 (38) was kindly provided by G. R. Cornelis (Biozentrum der Universität Basel, Basel, Switzerland). Furthermore, we utilized a number of Yersinia strains that produced diverse isotypes of YopE as the sole effector Yop. These strains harbor one plasmid encoding the Yop secretion and translocation machinery (pTTSS, where TTSS is type III secretion system) and a second plasmid encoding the YopE module (pYopE-SycE) either from strain WA-314 (WA-TTSS/YopE_O8, where YopE_O8 is YopE from the Y. enterocolitica serogroup O8) or E40 (WA-TTSS/YopE_O9), respectively (32, 41). The resulting strains produce only YopE but no other effector Yop. For the mutagenesis of YopE in these strains, point mutations were inserted into the wild-type yopE genes by using a QuikChange site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA).

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TABLE 1. Y. enterocolitica strains used in this study

For infection, overnight cultures grown at 27°C were diluted 1:20 in fresh Luria-Bertani broth and grown for another 2 h at 37°C. Shift of the growth temperature to 37°C initializes activation of the Yersinia type III secretion machinery for efficient translocation of Yops into the host cell upon cellular contact (32). To equalize and synchronize infection, bacteria were seeded on the cells by centrifugation at 400 x g for 5 min at a ratio of 50 to 100 bacteria per cell. For incubation times longer than 90 min, bacteria were killed by the addition of gentamicin (100 µg/ml) after 90 min. The human embryonic kidney 293 (HEK293) cell line was cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum (Invitrogen, Karlsruhe, Germany). Where indicated, the cells were treated with a 5 µM concentration of the proteasome inhibitor MG-132 (Z-Leu-Leu-Leu-CHO; Biomol, Plymouth Meeting, PA) 30 min prior to infection, unless stated otherwise. The application of the proteasome inhibitor did not trigger apoptosis or cytotoxically alter the viability of the cells by another mechanism within the investigated time frames (32).

Western immunoblotting, immunoprecipitation, and cell transfection. For assessment of the cellular YopE levels, infected cells were solubilized, at the time points indicated in the figure legends, with a buffer containing 10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol, and phosphatase and protease inhibitors (Roche, Mannheim, Germany). The lysates were cleared by centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with polyclonal antibodies directed against YopE (32). Immunoreactive bands were visualized using appropriate secondary antibodies and enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). To overexpress ubiquitin for the YopE immunoprecipitation experiments, HEK293 cells were seeded in six-well cell culture plates and transfected with a human cDNA construct encoding an octameric tandem fusion of hemagglutinin-ubiquitin (40) or empty control vector. Transfections were conducted by the calcium-phosphate transfection method as described previously (13). The ubiquitin expression vector was kindly provided by M. Treier (European Molecular Biology Laboratory, Heidelberg, Germany). Eighteen hours after transfection, the cells were infected with Yersinia strains and processed for immunoprecipitation similarly to nontransfected cells. Accordingly, the cells were lysed 90 min after onset of infection with a lysis buffer containing 50 mM Tris, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µM MG-132, a cocktail of protease inhibitors (Roche), and a 10 µM concentration of the deubiquitinase inhibitor N-ethylmaleimide (22, 32). The lysates were preabsorbed to protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C and then incubated with rabbit polyclonal antibodies directed against YopE or flagellin as a negative control serum for 16 h at 4°C to precipitate YopE from the infected cells (32, 46). The immune complexes were collected with protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA), washed five times with lysis buffer, and subjected to Western immunoblotting as described above. Ubiquitin-YopE conjugates were detected by immunoblotting with the monoclonal mouse anti-ubiquitin antibody FK2 (Biomol International, Plymouth Meeting). When required, the membranes were stripped in 62.5 mM Tris, pH 6.7, 0.1 mM 2-mercaptoethanol, and 2% sodium dodecyl sulfate for 30 min at 50°C and reused for a second immunoblotting procedure. This stripping method was also applied to recycle the membranes for successive labeling with anti-YopE antibodies and for controlling equal protein loading of the gels by detecting ß-tubulin with mouse monoclonal antibody (D-10; Santa Cruz Biotechnology, Santa Cruz, CA) in the cellular lysates. The shown data are from one experiment and are representative for at least three performed.

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YopE from Y. enterocolitica serogroups O9 and O3 evades destabilization by the host cell proteasome. Our previous studies demonstrated that YopE of Y. enterocolitica serogroup O8 is subjected to proteasomal degradation after it is translocated inside the host cell. The destabilization of YopE by the proteasome helps to restrict and to counteract the immunomodulatory activities of the Yop in the infected cell (32). YopE is one of the major Yops that mediate reorganization of the actin cytoskeleton and thereby induce a typical, contracted or rounded morphology of the infected cells. We found that the cytotoxic effect of YopE is enhanced when the degradation of YopE by the proteasome is prevented by the application of proteasome-inhibitory compounds (32). Here, we analyzed the influence of the proteasome inhibitor MG-132 on the appearance of HEK293 cells that were infected with different pathogenic serotypes of Y. enterocolitica. The peptide MG-132 serves as substrate analogue of the proteasome and thereby blocks its protease activity (23). Figure 1 A shows that the serotype O8 strain WA-314 induced a characteristic, contracted cell morphology. This effect was enhanced by MG-132 treatment, with the cells displaying a pronounced rounding phenotype compared to untreated cells. Replacement of MG-132 by the proteasome inhibitor epoxomicin (24) provided comparable results (32; also data not shown). The virulence plasmid-cured strain WA-C was unable to alter the morphology of the infected cells, whether they were treated with MG-132 or not (Fig. 1A). This confirms the idea that cell rounding is mediated by some of the effector Yops, most likely involving YopE (2, 6, 9, 32). Surprisingly, rounding of cells infected with the Y. enterocolitica serotype O9 strain E40 was not intensified by MG-132 (Fig. 1A). E40-infected cells displayed a severely rounded phenotype similar to that of MG-132-treated cells infected with the O8 strain under the same conditions. In the same manner, cytotoxicity conferred by the serogroup O3 strain 108-P was not pronounced upon proteasome inhibition. This suggests that the cytotoxic effects conferred by strains E40 and 108-P, which most likely are brought about by YopE activity, are not influenced by the host cell proteasome.

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FIG. 1. Inhibition of the proteasome augments the cytotoxicity and the protein levels of YopE in HEK293 cells infected with Y. enterocolitica serogroup O8 but not O9 or O3. (A) Proteasome inhibitor-dependent enhancement of the morphological alterations induced by Y. enterocolitica O8 but not serogroup O9 or O3. HEK293 cells were left untreated (–) or were treated (+) with the proteasome inhibitor MG-132 for 30 min prior to infection with virulence plasmid-cured yersiniae (WA-C) or the serogroup O8 (WA-314), O9 (E40), or O3 (108-P) wild-type strains. Ninety minutes after onset of infection, the yersiniae were killed by the addition of gentamicin. The cells were fixed, and cellular morphologies were microscopically analyzed after a total incubation period of 4 h. (B) Selective stabilization of YopE from Y. enterocolitica serogroup O8 by MG-132. HEK293 cells were left untreated (–) or were treated (+) with the proteasome inhibitor MG-132 and infected with the different Yersinia strains as described for panel A. Cellular lysates were prepared 150 min after onset of infection and processed for immunoblotting using anti-YopE antibodies. Equal loading of gels with cellular lysates was controlled by successive immunoblotting against ß-tubulin.

We subsequently investigated whether the different cell-rounding activities of the Y. enterocolitica serotypes could be related to differences in the regulation of YopE stability. HEK293 cells were infected with the diverse Yersinia strains in the absence or presence of the proteasome inhibitor, and the protein levels of translocated YopE were examined in cellular lysates by immunoblotting with antibodies directed against YopE. Figure 1B shows that addition of the proteasome inhibitor markedly enhanced the protein levels of YopE from Y. enterocolitica serogroup O8. In contrast, the YopE levels from serogroups O9 and O3, appearing as double protein bands, were not affected by MG-132 and instead remained constant. This suggests that the proteasome pathway governs the stability of YopE_O8, but not from the serogroups O9 and O3 (YopE_O9 and YopE_O3, respectively). This finding could explain why the cytotoxic activity of only YopE_O8 increases in proteasome inhibitor-treated cells. The proteasome apparently degrades and inactivates YopE_O8 but not YopE_O9 or YopE_O3. YopE_O9 and YopE_O3, instead, appear to be subjected to another form of modification. The double protein band detected for YopE_O9 and YopE_O3 (Fig. 1B) could be consistent with intracellular processing of YopE_O9 and YopE_O3. The nature and significance of this modification, however, are not yet clear.

We next attempted to analyze the relationship between the stabilities of the different YopE isotypes and their primary structures. For that reason, we sequenced the yopE genes of the investigated strains and compared the deduced YopE amino acid sequences (Fig. 2). The sequence of the serogroup O8 strain WA-314 corresponds to the published sequences of YopE_O8 from strain A127/90 (accession no. NP_783702), whereas YopE from strain E40 was homologous to YopE_O9 from strain W22703 (accession no. NP_052427). Finally, YopE from the serogroup O3 strain 108-P, which behaved like YopE_O9 in our experiments (Fig. 1B), was identical to serogroup O9 YopE (data not shown). Figure 2 shows that the amino acid sequences of YopE_O8 and YopE_O9 are highly homologous (93.6% identity). Single amino acids that differ between the two serogroups are marked in boldface. Interestingly, at positions 62 and 75 YopE_O9 lacks two lysine residues that are present in YopE_O8. Conjugation of lysine residues with ubiquitin moieties is the best-characterized posttranslational modification event that targets cellular proteins to the proteasome for degradation (29, 39). In fact, one of the two lysines (lysine-75) was already identified to be involved in mediating the destabilization of YopE_O8 (32). On this basis, we explored the possibility that translocated YopE_O9 could escape the modification with polyubiquitin inside the host cell because it lacks the two lysines (Fig. 2). This could prevent its degradation through the proteasome pathway.

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FIG. 2. Comparison of the YopE sequences from Y. enterocolitica serogroups O8 and O9. Comparison of the deduced amino acid sequences of YopEO8 (NP_783702) and YopEO9 (NP_052427) reveals two lysines in YopEO8 at amino acid positions 62 and 75 that are not conserved in YopEO9 (boxed and shaded). The residues found in YopEO9 at these positions are arginine and glutamine, respectively. Additional amino acids that differ between YopEO8 and YopEO9 are underlined and shown in boldface.

To be able to characterize the ubiquitination of YopE_O8 and YopE_O9 in parallel under otherwise identical conditions, we constructed two Yersinia strains that translocate either YopE_O8 or YopE_O9 as the sole effector Yop. These strains harbor two plasmids (32, 41), one encoding the structural and regulatory components of the Y. enterocolitica type III protein secretion system and the other encoding YopE either from the serogroup O8 strain WA-314 (resulting in strain WA-TTSS/ YopEO8) or the serogroup O9 strain E40 (resulting in WA-TTSS/YopEO9). These two strains were then used to infect HEK293 cells, and the virulence plasmid-cured strain WA-C served as a negative control. A Yersinia strain that expresses the secretion-translocation module without YopE is not an adequate control because this strain causes necrotic cell death (44; also data not shown). This nonspecific effect is thought to result from the formation of pores in the host cell membrane by the Yersinia type III protein secretion apparatus in the absence of YopE. YopE likely inhibits the pore-forming activity by reducing F-actin that somehow is required for keeping the pores open (44). The cells were treated with the proteasome inhibitor MG-132 prior to infection, a procedure that causes the accumulation of proteins which are destined for proteasomal degradation. The YopE proteins were then immunoprecipitated from cellular lysates with anti-YopE antibodies and immunoblotted with anti-ubiquitin antibody to detect ubiquitin-modified YopE protein species. Figure 3 shows that a number of anti-ubiquitin antibody-immunoreactive bands specifically precipitated with YopE_O8. These ubiquitin-modified protein species migrated with slower electrophoretic mobilities than unmodified YopE_O8 (23 kDa) and were separated from original YopE by increasing distances, starting from about 8 kDa. These higher molecular weights are consistent with the modification of YopE_O8 by mono- and polyubiquitination. In fact, the majority of the ubiquitinated proteins were detected also by the anti-YopE antibody (Fig. 3, right panel), which gives evidence that the ubiquitin-modified proteins represent ubiquitinated YopE_O8 protein species. These YopE-ubiquitin bands were not found in either the precipitates prepared from WA-C-infected cells or in control samples using an anti-flagellin antibody. Furthermore, no substantial ubiquitination was detected in the immunoprecipitates of YopE_O9. The elevated protein levels in the YopE_O8 immunoprecipitates compared to those of YopE_O9 probably result from the accumulation of YopE_O8 upon proteasome inhibition, which is not observed for YopE_O9 (Fig. 1B). These results indicate that YopE_O8 is subjected to ubiquitination inside the Yersinia-infected host cell, whereas YopE_O9 evades ubiquitination. In agreement with the role of polyubiquitin moieties in targeting a selected protein for proteasomal destruction (12, 29, 39), the failure of YopE_O9 to be modified by polyubiquitination apparently prevents its degradation by the host proteasome.

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FIG. 3. YopEO8 but not YopEO9 is subjected to ubiquitination. HEK293 cells were infected with virulence plasmid-cured yersiniae (WA-C) or the Yersinia strains that overproduce either YopEO8 or YopEO9 as the sole effector Yop (WA-TTSS/YopEO8 and WA-TTSS/YopEO9) in the presence of the proteasome inhibitor MG-132. Cellular extracts were prepared 90 min after onset of infection and immunoprecipitated using anti-YopE or anti-flagellin antibodies as negative controls, as indicated at the top of the panels. The immunoprecipitates were first immunoblotted with the anti-ubiquitin antibody for the detection of ubiquitin-modified proteins (left panel). Subsequently, to control successful and specific precipitation of YopE, the membrane was stripped and reprobed with anti-YopE antibodies (right panel). The apparent ubiquitin-modified forms of YopE are indicated. YopE Ubpoly, polyubiquitinated YopE; YopE Ubmono, monoubiquitinated YopE; IP, immunoprecipitation; WB, Western blotting.

Loss of the lysines-62 and -75 protects YopE against polyubiquitination and proteasomal destabilization. To specify the impact of lysine-62 and -75 on the ubiquitination and stability of translocated YopE, we mutated the two lysines of YopE_O8 either individually or together. They were replaced by arginine or glutamine, which are located at the respective amino acid positions in YopE_O9 [resulting in plasmids YopE_O8(K62R), YopE_O8(K75Q), and YopE_O8(K62R K75Q)]. In the same manner, we replaced arginine-62 and glutamine-75 in YopE_O9 with lysine residues [resulting in plasmids YopE_O9(R62K), YopE_O9(Q75K), and YopE_O9(R62K Q75K)]. These YopE mutant proteins were then used to replace the wild-type YopE isotypes in strains WA-TTSS/YopE_O8 [resulting in strains WA-TTSS/YopE_O8(K62R), WA-TTSS/YopE_O8(K75Q), and WA-TTSS/YopE_O8(K62R, K75Q)] and WA-TTSS/YopEO9 [resulting in strains WA-TTSS/YopE_O9(R62K), WA-TTSS/YopE_O9(Q75K), and WA-TTSS/YopE_O9(R62K Q75K)], respectively. The cellular effects of the different strains were compared (Fig. 4). These experiments revealed that the mutation of only one lysine in YopE_O8, either at position 62 or 75, could not significantly diminish the polyubiquitination of YopE_O8 [Fig. 4A, strains WA-TTSS/YopE_O8(K62R) and WA-TTSS/YopE_O8(K75Q)]. In contrast, the ubiquitination of YopE_O8 was abolished when both lysines were mutagenized [Fig. 4A, strain WA-TTSS/YopE_O8(K62R K75Q)]. The negative controls applying anti-flagellin instead of anti-YopE antibodies in the immunoprecipitations in Fig. 4 did not show significant ubiquitination patterns (data not shown). This indicates that lysine-62 as well as lysine-75 can serve as ubiquitin acceptor sites and that only the loss of both lysines efficiently prevents the polyubiquitination of YopE_O8. The importance of these lysine residues in enabling YopE ubiquitination was confirmed by studies on YopE_O9. Accordingly, the YopE_O9 double point mutant that harbors both lysines at positions 62 and 75 was efficiently ubiquitinated [Fig. 4B, strain WA-TTSS/YopE_O9(R62K Q75K)]. In contrast, the YopE_O9 mutants with only one of these lysine residues did not exhibit remarkable polyubiquitination in this set of experiments [Fig. 4B, strains WA-TTSS/YopE_O9(R62K) and WA-TTSS/YopE_O9(Q75K)]. A faint band that appeared in the anti-YopE immunoblot could be consistent with a weak modification of these YopE_O9 mutant proteins by monoubiquitination (Fig. 4B, right panel). However, when ubiquitin was overexpressed in the infected HEK293 cells, both single lysine mutants of YopE_O9 became efficiently mono- and polyubiquitinated (Fig. 4C). This confirms that either lysine can principally ensure the ubiquitination of YopE. The effective and proper ubiquitination of YopE under more restricted conditions with physiological ubiquitin levels may, however, be influenced by the secondary structure of YopE_O9 and require the presence of both lysine residues.

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FIG. 4. Lysines at positions 62 and 75 are polyubiquitin acceptor sites of YopE. (A) Mutation of lysine-62 and -75 abolishes polyubiquitination of YopEO8. HEK293 cells were infected in the presence of the proteasome inhibitor MG-132. The investigated Yersinia strains produced either wild-type YopEO8 (strain WA-TTSS/YopEO8), wild-type YopEO9 (WA-TTSS/YopEO9), or YopEO8 with mutations in lysine-62 [WA-TTSS/YopEO8(K62R)], lysine-75 [WA-TTSS/YopEO8(K75Q)], or both lysine residues [WA-TTSS/YopEO8(K62R K75Q)]. Cellular extracts were prepared 90 min after onset of infection, and immunoprecipitations were performed using anti-YopE or anti-flagellin antibodies as negative controls (immunoblots not shown). The immunoprecipitates were first immunoblotted with the anti-ubiquitin antibody to detect ubiquitin-modified proteins (Western blotting, left) and then stripped and reprobed with anti-YopE antibodies to detect YopE protein species (Western blotting, right). The apparent ubiquitin-modified forms of YopE are indicated. (B) Mutation of arginine-62 and glutamine-75 to lysines subjects YopEO9 to polyubiquitination. Experiments were performed as in described for panel A except that different strains were used. The investigated Yersinia strains produced either wild-type YopEO8 (WA-TTSS/YopEO8), wild-type YopEO9 (WA-TTSS/YopEO9), or YopEO9 with the replacement of arginine-62 [WA-TTSS/YopEO9(R62K)], glutamine-75 [WA-TTSS/YopEO9(Q75K)], or both amino acids [WA-TTSS/YopEO9(R62K Q75K)] by lysine(s). (C) Overexpression of ubiquitin reveals polyubiquitination of YopEO9 harboring either lysine-62 or lysine-75. HEK293 cells were transfected with a ubiquitin expression plasmid and 20 h later were infected in the presence of MG-132. Experiments were performed as described for panel A, except that different strains were used. The investigated Yersinia strains produced either wild-type YopEO9 (WA-TTSS/YopEO9) or YopEO9 with the replacement of arginine-62 [WA-TTSS/YopEO9(R62K)], glutamine-75 [WA-TTSS/YopEO9(Q75K)], or both amino acids [WA-TTSS/YopEO9(R62K Q75K)] by lysine(s). YopE Ubmono, monoubiquitinated YopE; YopE Ubpoly, polyubiquitinated YopE; IP, immunoprecipitation; WB, Western blotting.

Having characterized the ubiquitination pattern of the different YopE point mutants, we next studied their intracellular stability in response to treatment with the proteasome inhibitor (Fig. 5). The results showed that the levels of the YopE protein species that harbor at least one lysine at either position 62 or 75 [YopE_O8, YopE_O8(K62R), YopE_O8(K75Q), YopE_O9(R62K), YopE_O9(Q75K), and YopE_O9(R62K Q75K)] increased upon proteasome inhibitor treatment. This effect correlated with the ability of these YopE proteins to become polyubiquitinated (Fig. 4). The insertion of alanines instead of the lysines in YopE_O9 [Fig. 5, compare strains WA-TTSS/YopE_O9(R62A Q75A) and WA-TTSS/YopE_O9(R62K Q75K)] did not increase the quantity of YopE upon proteasome inhibition, suggesting that the accumulation of YopE_O9 in proteasome-inhibited cells is specifically mediated by lysine insertion. YopE_O9 and YopE_O8(K62R K75Q), which do not undergo polyubiquitination, were not stabilized by treatment with the proteasome inhibitor. These results show that lysine-62 and -75 determine the predisposition of YopE to become subjected to ubiquitination and proteasomal degradation.

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FIG. 5. The lysines at position 62 and 75 determine the intracellular stability of YopE. HEK293 cells were infected with the indicated yersiniae strains in the absence (–) or presence (+) of MG-132. Two hours after onset of infection, cellular lysates were prepared and subjected to immunoblotting using anti-YopE antibody. Equal loading of the gel with cellular lysates was controlled by stripping the membrane and subsequent immunoblotting against ß-tubulin.

Microscopic evaluations of the infected cells further revealed that lysine-62 and -75 also control the cytotoxic activity of YopE on the host cell. Only YopE_O9, YopE_O8(K62R K75Q), and YopE_O9(R62A Q75QA), which resist polyubiquitination because of the absence of lysine-62 and -75, induced a pronounced rounded phenotype in both the presence and absence of MG-132 (Fig. 6). In contrast, the morphological cell alterations induced by the other investigated YopE protein species, which harbor at least either lysine-62 or -75, were enforced by MG-132 treatment [Fig. 6, YopE_O8 and YopE_O9(R62K Q75K)] (data not shown). The cellular effects of these Yops are thus enhanced by MG-132 exposure according to their protein levels. Together, these data indicate that loss of the two lysine residues 62 and 75 provides YopE with the ability to escape polyubiquitination. This increases its intracellular stability and concomitantly enhances its cytotoxic potential on the host cell. It may be concluded from these observations that the protection of YopE_O9 and YopE_O3 against ubiquitination may aid yersiniae of these serotypes to intensify their immunomodulatory activities.

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FIG. 6. The lysines at positions 62 and 75 determine the cytotoxic activity of YopE on the host cell. HEK293 cells were infected with the indicated Yersinia strains in the absence (–) or presence (+) of MG-132. Ninety minutes after infection, the bacteria were killed by the addition of gentamicin. The cells were fixed, and the cellular morphologies were microscopically evaluated after a total incubation period of 4 h.

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This study demonstrates that YopE isotypes from diverse pathogenic Y. enterocolitica serogroups differ in their dispositions to become ubiquitinated and degraded by the host cell proteasome. The susceptibility to ubiquitination and degradation concomitantly determines the effect of YopE on the host cell. YopE of Y. enterocolitica serotypes O3 and O9 resisted ubiquitination and exerted an enhanced cytotoxic effect on infected host cells, whereas cytotoxicity conferred by serogroup O8 YopE was substantially less pronounced. The reduced activity of YopE_O8 resulted from polyubiquitination and efficient degradation of YopE_O8 through the proteasome pathway. To relate the effects of the different YopE isotypes to single amino acids, we compared the amino acid sequences of YopE_O8, YopE_O9, and YopE_O3. We identified two lysine residues (K62 and K75) as polyubiquitin acceptor sites of YopE_O8 that are absent in YopE_O9 and YopE_O3. Exchange of the lysine residues of YopE_O8 for arginine and glutamine of YopE_O9 and vice versa revealed that either lysine can be modified by polyubiquitination and mediate YopE degradation. These data show that functional differences exist between genetically highly homologous members of YopE proteins from diverse Y. enterocolitica serotypes. These differences are due to distinct stabilities of the respective YopE isotypes inside the host cell. Interestingly, a search in the published protein sequence databases showed that lysine-62 and lysine-75 are unique to YopE from Y. enterocolitica serogroup O8 and are not found in any other YopE proteins such as those from Y. pestis or Y. pseudotuberculosis. This indicates that ubiquitination and destabilization of YopE by the host cell proteasome is a specific feature of Y. enterocolitica serogroup O8.

The species Y. enterocolitica comprises a biochemically, serologically, and genetically heterogenous group of organisms. While the serotypes O3 and O9 are most frequently isolated in Europe and Japan, serotype O8 causes infections predominantly in North America. This reflects the independent evolution of the different Y. enterocolitica lineages (19, 47). Y. pestis and Y. pseudotuberculosis are, in contrast, closely related. They display gene homology of nearly 97%, and it was proposed that Y. pestis separated from Y. pseudotuberculosis just 1,500 to 20,000 years ago (1, 47). However, the three pathogenic Yersinia spp. share a related virulence plasmid, termed pYV, which encodes the Yersinia Yop virulon including YopE. It is believed that the pYV plasmids of the diverse Yersinia spp. arose from a common predecessor plasmid that was brought in an environmental, nonpathogenic Yersinia strain to form the ancestor of pathogenic yersiniae (47). Comparison of the virulence plasmids of Y. enterocolitica serogroups O8 and O9 and of Y. pestis reveals deletions, insertions, and rearrangements of DNA sequences (37). This indicates that the virulence plasmids have evolved separately along with the genomes after divergence of the different clades (19, 37, 47). DNA cross-hybridization experiments have shown that the virulence plasmids of the Y. enterocolitica serogroups O3 and O9 display 90% nucleotide identity with one another but only 75% identity with the plasmid from serogroup O8 (16, 37). Y. enterocolitica serotype O8, furthermore, shares only 55% nucleotide identity with the virulence plasmids from Y. pestis and Y. pseudotuberculosis (30, 37). These observations support the concept that Y. enterocolitica serotype O8 arose by a specific evolutionary path, distant from serogroups O3 and O9 (37, 47). This evolutionary aspect can explain the genetic background that determines the specificity in the stability and function of YopE_O8.

The order of evolution of the two lysines in YopE_O8 and their significance for Yersinia virulence are, however, still unclear. Our results show that the absence of the two lysines increases the stability and the activity of YopE inside the host cell. It is tempting to speculate that the lysines were lost in the majority of the Yersinia lineages during evolution to enhance their virulence. On the other hand, because only YopE_O8 carries lysine-62 and -75, it appears more reasonable to assume that these lysines specifically evolved in Y. enterocolitica serogroup O8 from a predecessor strain originally lacking these residues. The lysines may now fulfill a specific function in the pathogenesis of Y. enterocolitica serogroup O8 infection. The formation of the lysines and the concomitant destabilization of YopE_O8 may help to mitigate the virulence of Y. enterocolitica O8. In fact, this could be advantageous for Y. enterocolitica O8 infection. Serotype O8 Y. enterocolitica possesses a number of virulence factors that are absent in the other Y. enterocolitica serogroups. These include the yersiniabactin siderophore system, which is encoded by the high-pathogenicity island (7, 34), the chromosomally encoded type III protein secretion system Ysa (11, 14, 43), and the type II secretion system Yts1 (20). Furthermore, a number of virulence proteins encoded by the pYV plasmid from Y. enterocolitica serogroup O8 are more active than the respective proteins from the other Yersinia clades, i.e., YopP/YopJ and LcrV (10, 33, 36, 48). Y. enterocolitica O8 is consequently characterized by enhanced pathogenicity in the mouse infection model. Y. enterocolitica O8 may therefore have established strategies to counteract excessive bacterial virulence in order to prevent premature consumption of the infected host. The ubiquitination and concomitant reduction of the half-life of YopE could be a mechanism that dampens Y. enterocolitica O8 virulence. This may then enable prolonged, productive bacterial infection. Alternatively, since YopE also possesses a regulatory role in the translocation of the Yops by destabilizing the translocation channel (44), excessive activity of YopE_O8 could be counterproductive for efficient Yop translocation. The specific regulation of the stability of YopE_O8 by the host cell proteasome may consequently help Y. enterocolitica serogroup O8 to fine-tune its immunomodulatory effects on the host cell. Thus, by balancing the activity of YopE_O8, the proteasome could affect the course of Y. enterocolitica serotype O8 infection in the compromised host. It has been shown that YopE_O8 supports the colonization of liver and spleen with yersiniae in infected mice (42). The activity of the proteasome could therefore influence the bacterial counts in the peripheral lymphoid tissue along with the YopE levels. The quantitative effect of YopE_O8 ubiquitination and inactivation on the dissemination of the bacteria in the host will be specified in detail in future studies.

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (grants DFG Ru788/1 and -2).

We are grateful to Nicole Czymmeck for expert technical assistance. We thank Gudrun Pfaffinger for support in establishing the immunoprecipitation assays. We also thank G. R. Cornelis and Mathias Treier for providing us with the E40 Yersinia strain and the ubiquitin expression vector, respectively.

 
* Corresponding author. Mailing address: Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Phone: 49 40 42803 7035. Fax: 49 40 42803 3250. E-mail: k.ruckdeschel{at}uke.uni-hamburg.de

Published ahead of print on 2 July 2007.

Editor: J. B. Bliska

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Infection and Immunity, September 2007, p. 4423-4431, Vol. 75, No. 9
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