Yersinia enterocolitica Promotes Deactivation of Macrophage Mitogen-activated Protein Kinases Extracellular Signal-regulated Kinase-1/2, p38, and c-Jun NH2-terminal Kinase
CORRELATION WITH ITS INHIBITORY EFFECT ON TUMOR NECROSIS FACTOR-alpha PRODUCTION*

(Received for publication, November 19, 1996, and in revised form, March 3, 1997)

Klaus Ruckdeschel Dagger , Jan Machold Dagger , Andreas Roggenkamp §, Sören Schubert §, Josiane Pierre , Robert Zumbihl Dagger , Jean-Pierre Liautard Dagger , Jürgen Heesemann § and Bruno Rouot Dagger par

From Dagger  INSERM U431, Université Montpellier II, Place E. Bataillon, CC100, F-34095 Montpellier Cedex 05, France,  INSERM U461, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France, and § Max von Pettenkofer-Institut für Hygiene und Mikrobiologie, Pettenkoferstrasse 9a, D-80336 München, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha ) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.


INTRODUCTION

The enteropathogenic Gram-negative bacterium Yersinia enterocolitica has developed strategies to resist the host immune defense. This enables extracellular survival and multiplication of the bacteria in host lymphoid tissue after infection and invasion of the intestinal mucosa. It is becoming increasingly evident that Yersinia sp. evade host defense mechanisms by disrupting key functions of the host cell. This ability is linked to the expression of a set of released plasmid-encoded proteins, termed Yersinia outer proteins (Yops)1 (1, 2). Export of Yops is triggered by attachment of Yersinia sp. to the host cell (3-5). Eleven Yops have been described so far (2). At least four of them, i.e. YopE, YopH, YopM, and YopO (the homolog of YpkA in Yersinia pseudotuberculosis), are translocated across the host cell membrane to their putative intracellular targets (3, 4, 6-10). YopE disrupts actin filaments (3, 4, 11) and acts synergistically with the protein-tyrosine phosphatase YopH (12) to inhibit phagocytosis and to suppress the oxidative burst of professional phagocytes (11, 13-16). YopH and also YopO, which displays serine/threonine kinase activity (17), share homologies with eukaryotic proteins, and both are supposed to interfere or block host cell signal transduction pathways (12, 17-20).

Y. enterocolitica, like other pathogens (Brucella sp. (21, 22), Bacillus anthracis (23), or Leishmania donovani (24)), also interferes with cytokine production. It suppresses chemokine interleukin-8 secretion of epithelial cells (25) and prevents production of the macrophage proinflammatory cytokine TNFalpha (26-29). Released TNFalpha enhances the activation of cells involved in the immune defense (i.e. macrophages, polymorphonuclear leukocytes, NK cells, and T lymphocytes) and thus contributes in overcoming bacterial infection. Previous studies already demonstrated that TNFalpha also plays an important role in limiting the severity of Y. enterocolitica infection (30). However, the impact of Y. enterocolitica on signaling pathways of mammalian cells, leading to suppression of cytokine release, is still completely unknown. Since LPS itself stimulates macrophage secretion of TNFalpha , it seems reasonable to assume that Y. enterocolitica interferes with LPS-stimulated pathways.

LPS from Gram-negative bacteria was reported to activate the three different MAPK families, i.e. ERK, JNK/SAPK, and p38, in macrophages (31-35). The mechanism of MAPK activation by LPS remains unclear (36). On the one hand, ceramide seems to play an important role (37), since LPS was shown to activate a ceramide-dependent kinase (38) and ceramide itself stimulates the JNK/SAPK pathway (39). Moreover, c-Raf, the upstream kinase activator of MEK1/2 and ERK1/2, was recently shown to be activated by ceramide (40). On the other hand, LPS, through its fatty acid chains, has structural homology with ceramide, and it was suggested that LPS stimulates a ceramide-dependent kinase by mimicking the ceramide molecule (38). However, irrespective of the mechanism of LPS-induced MAPK stimulation, it is well established that among MAPKs, p38 plays an important role in LPS-induced TNFalpha production in macrophages (41-42).

In the present study, we analyzed possible alteration of MAPK activation during infection with Y. enterocolitica. We thus chose the macrophage-like J774A.1 cell line as a well established infection model to study Yersinia sp.-macrophage interactions (13-14, 18-19, 43). Interestingly, there is a nonvirulent Y. enterocolitica strain that is virulence plasmid-cured, thus providing an ideal control for comparison experiments with virulent wild-type or mutated Y. enterocolitica strains (Table I). Here, we report that virulent Y. enterocolitica indeed strongly interferes with macrophage signal transduction, resulting in blockade of ERK, JNK, and p38 MAPK activities. This MAPK inhibition correlates with the suppression of TNFalpha production but is not required for the inhibition of macrophage phagocytosis and oxidative burst.

Table I. Y. enterocolitica strains used in this study


Strain Relevant characteristics Former designation Reference

Virulent Serogroup O8; clinical isolate harboring virulence plasmid pYVO8 WA-314 44
Nonvirulent Plasmidless derivative of the virulent strain WA-C 44
YopH(1)- Mutant strain, deficient in YopH secretion; insertional inactivation of sycH, the gene for the YopH-specific chaperone SycH WA-C(pYV-7146) 16
WA-C(pYV O8::Tn7) 45
YopH(1)-/H+ YopH(1)- strain, complemented with sycH and yopH; YopH secretion-positive WA-C(pYV-7146, pB8-64) 16
YopH(2)- YopH mutant; insertional inactivation of the yopH gene WA-C(pYV 08Delta H) 46
Yop secr.- Mutant strain, deficient in secretion of Yops; insertional inactivation of lcrD, the gene encoding LcrD, which is essential for Yop secretion WA-C(pYV-515) 16
YopD,B,N,V+ Strain harboring plasmid pLCR encoding the secretion apparatus of Y. enterocolitica including the genes for YopD, YopB, YopN, and the V antigen WA-C(pLCR) 46
YopD,B,N,V,H,E,YadA+ Strain YopD,B,N,V+ harboring an additional plasmid encoding the genes for YopH, YopE, and YadA WA-C(pLCR, pB8-23) 46


EXPERIMENTAL PROCEDURES

Bacterial Strains, Growth, and Infection Conditions

The bacterial strains used in this study are listed in Table I. Overnight cultures grown at 26 °C were diluted 1:20 in fresh Luria-Bertani broth and grown for 2 h at 37 °C as described previously (16). Bacteria were then washed once and resuspended in phosphate-buffered saline. Cells were infected at a ratio of 50 bacteria/cell. The desired bacterial concentration was adjusted by measuring the optical density at 600 nm and checked by plating serial dilutions from every sample on agar and counting colony-forming units after incubation at 26 °C for 20 h.

Cell Culture and Stimulation

The murine macrophage-like cell line J774.A1 (ATCC TIB 67) was cultured in RPMI 1640 medium (Life Technologies, Cergy, Pontoise, France) supplemented with 10% heat-inactivated fetal calf serum and 5 mM L-glutamine at 37 °C in a humidified atmosphere (5% CO2). Before treatment with bacteria or 10 µg/ml LPS from Escherichia coli (Sigma), cells were scraped, washed, and resuspended in complete culture medium. Cell stimulation occurred at 37 °C for different periods of time as indicated. Cell viability was more than 90% after 90 min of bacterial infection, as determined by trypan blue exclusion.

Preparation of Affinity-purified Anti-p38 Antibodies

Antibodies directed against the C-terminal end (peptide KPLDQEEMES) of p38 kinase were raised in New Zealand rabbits as described previously (47). Antibodies were purified from immune sera by ammonium sulfate precipitation followed by overnight recycling through Affi-Gel 10 (Bio-Rad) to which the antigen peptide had been linked. After acidic elution, neutralized affinity-purified antibodies were dialyzed against phosphate-buffered saline and thereafter against a glycerol/phosphate-buffered saline solution (1:1) before storage at -20 °C.

Immunoprecipitation, Anti-phosphotyrosine Immunoblotting Assays

5 × 106 cells were treated with bacteria and/or LPS for different periods of time and lysed with radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 20 mM beta -glycerophosphate, 10 mM p-nitrophenyl phosphate, 0.1 mM Na3VO4, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 0.5 mM PMSF). For immunoprecipitation, cell lysates were incubated with polyclonal anti-p38 antibody at 4 °C for 1 h. Immune complexes were then collected with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) and washed three times with radioimmune precipitation buffer. Both whole cell lysates and immunoprecipitates were mixed with 4 × or 2 × Laemmli buffer, respectively. Proteins were separated by 10% SDS-PAGE, electrotransferred to PVDF membrane (Polyscreen, DuPont NEN), blocked with 3% bovine serum albumin, and probed with appropriate antibodies. Phosphotyrosine immunostaining was performed with the monoclonal antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). Immunoblotting for p38 and ERK1/2 was performed using rabbit polyclonal anti-p38 antibodies (1:3000 dilution) or goat polyclonal anti-ERK1/2 antibodies (1:30,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively. Immunoreactive bands were visualized by incubation (1 h) with rabbit anti-mouse (1:10,000 dilution; Sigma), goat anti-rabbit (1:20,000 dilution; Sigma), or rabbit anti-goat (1:10,000; Santa Cruz Biotechnology) antibodies conjugated to horseradish peroxidase using enhanced chemiluminescence reagents (Renaissance; DuPont NEN). When required, membranes were stripped in 62.5 mM Tris, pH 6.7, 0.1 mM 2-mercaptoethanol, and 2% SDS for 30 min at 50 °C after film exposure. Thereafter, membranes were reprobed with appropriate primary and secondary antibodies and developed by chemiluminescence.

MAPK Assays

5 × 106 cells treated with bacteria or LPS for different periods of time were lysed in 200 µl of cell extract buffer (25 mM Hepes, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM beta -glycerophosphate, 0.1 mM Na3VO4, 2 µg/ml leupeptin, 10 µg/ml benzamidin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 100 µg/ml PMSF). After centrifugation, the supernatant was diluted with 600 µl of dilution buffer (20 mM Hepes, pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 20 mM beta -glycerophosphate, 0.1 mM Na3VO4, 2 µg/ml leupeptin, 10 µg/ml benzamidin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 100 µg/ml PMSF), incubated on ice for 10 min, and centrifuged again. Lysates were then mixed with GST fusion protein kinase substrates (8 µg of each, as indicated) and glutathione-agarose (20 µl, Sigma) and incubated overnight at 4 °C. Experiments were performed as described (48) with four different GST fusion protein kinase substrates obtained from M. Karin (GST-c-Jun-(1-222)), B. Dérijard (GST-c-Jun-(1-79) and GST-ATF2), and A. Nordheim (GST-Elk-1). The substrate-agarose complexes were washed four times with binding buffer (20 mM Hepes, pH 7.7, 50 mM NaCl, 25 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100), and in vitro phosphorylation was carried out for 20 min at 30 °C in the presence of 20 mM Hepes, pH 7.6, 20 mM MgCl2, 2 mM dithiothreitol, 20 mM beta -glycerophosphate, 0.1 mM Na3VO4, 20 mM p-nitrophenyl phosphate, 20 µM ATP (4 µCi of [gamma -32P]ATP) (total volume, 30 µl). The reaction was stopped by a single wash with binding buffer and by adding 30 µl of 2 × Laemmli buffer. Proteins were fractionated by 10% SDS-PAGE, electrotransferred to PVDF membrane, and subjected to autoradiography or quantified with a PhosphorImager (Molecular Dynamics, Inc.).

Raf-1 Kinase Assay

Raf-1 kinase activity was assayed, after immunoprecipitation, by phosphorylation of exogenously added MEK1 and ERK2 (49). 1 × 107 cells, treated with bacteria or LPS for 90 min, were lysed with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM beta -glycerophosphate, 1% Triton X-100, 50 mM NaF, 2 mM dithiothreitol, 100 µM Na3VO4, 5 mM benzamidin, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Cell lysates were precleared with an irrelevant polyclonal rabbit antibody and protein A-Sepharose (Pharmacia) before incubation with a rabbit anti-Raf-1 antibody (Sc133; Santa Cruz Biotechnology). Immune complexes were collected with protein A-Sepharose and washed twice with lysis buffer and twice with washing buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2). Immunoprecipitated Raf-1 was incubated with 1 µg of purified histidyl-tagged recombinant MEK1 in the presence of 100 µM ATP (5 µCi of [gamma -32P]ATP), 20 mM Hepes, pH 7.4, 10 mM MgCl2, 10 mM p-nitrophenyl phosphate, and 1 mM dithiothreitol at 30 °C for 30 min in a total volume of 50 µl. Thereafter, 1 µg of histidyl-tagged recombinant kinase-inactive ERK2 was added to each sample, and incubation was carried out for another 10 min at 30 °C. The reaction was stopped by adding 15 µl of 4 × Laemmli buffer. Proteins were separated by 10% SDS-PAGE, electrotransferred to PVDF, subjected to autoradiography, and analyzed with a PhosphorImager (Molecular Dynamics, Inc.). Purification of recombinant histidyl-tagged wild-type MEK1 and kinase-inactive ERK2 was performed as described (49).

Quantitation of TNFalpha and Analysis of TNFalpha Expression by Reverse Transcriptase-PCR

Cells dispatched in plastic culture plates (1 × 106 cells/sample for TNFalpha quantitation and 1 × 107 cells/sample for analysis of TNFalpha mRNA expression) were treated with bacteria or LPS at 37 °C in a humidified atmosphere (5% CO2). After 60 min, extracellular bacteria were killed by the addition of 100 µg/ml gentamicin. In some experiments, cells were treated with both LPS and gentamicin after 60 min of bacterial infection. For TNFalpha quantitation, the cell culture supernatants were removed after a final 120-min incubation. The TNFalpha cytokine level in the culture supernatant was evaluated by a cytotoxic assay performed with the TNFalpha -sensitive murine fibroblast cell line L929 as described (21, 22). L929 viability was colorimetrically determined using the CellTiter 96 AQ Assay (Promega, Madison, WI) according to the manufacturer's instructions. For analysis of TNFalpha expression, total RNA was extracted with Trizol (Life Technologies), as described by the manufacturer, after a final 120-min incubation. The reverse transcription reaction was performed at 42 °C for 50 min on 5 µg of total RNA, using the murine Moloney leukemia virus reverse transcriptase (Life Technologies) and oligo(dT)(12-18) (Life Technologies). 1 µl of each cDNA was amplified using 1 unit of Gold Star polymerase (Eurogentec, Seraing, Belgium) and 0.5 µM specific primers. Primer pairs specific for TNFalpha (sense, 5'-TCT CAT CAG TTC TAT GGC CC-3'; antisense, 5'-GGG AGT AGA CAA GGT ACA AC-3'; PCR product, 212 base pairs) and for beta 2m (sense, 5'-TGA CCG GCT TGT ATG CTA TC-3'; antisense, 5'-CAG TGT GAC CCA GGA TAT AG-3'; PCR product, 222 base pairs) were designed and purchased from Eurobio (Les Ulis, France). PCR was performed with 20 cycles. Amplification of beta 2m was used as a control. The PCR products were run on a 1.5% agarose gel supplemented with ethidium bromide.

Oxidative Burst Assays

We analyzed the oxidative burst of J774.A1 cells in response to opsonized zymosan after pretreatment of the cells with different Y. enterocolitica strains. The oxidative burst was measured as luminol-enhanced chemiluminescence in an automatic luminescence analyzer (Lumicon, Hamilton, Bonaduz, Switzerland) as described (50). 5 × 105 cells were infected with bacteria for 1.5 h. Thereafter, cells were resuspended in 0.95 ml of phosphate-buffered saline containing 5 µg/ml luminol (Boehringer, Mannheim, Germany). Stimulation was started by the addition of 50 µl of opsonized zymosan (Sigma), and chemiluminescence was recorded for a total of 30 min. Assays were repeated at least three times.

Phagocytosis Assays

5 × 105 cells/well were infected with bacteria in 24-well culture plates for 1 h. To discriminate between intra- and extracellularly located bacteria, cells were then stained using a double-immunofluorescence technique as described (16, 51). This technique allows determination of the numbers of both cell-associated (red and green fluorescence) and phagocytosed (exclusively green fluorescence) bacteria. For every strain investigated, three separate experiments were performed, and 100 cells from each experiment were analyzed under a fluorescence microscope. Mean percentages of phagocytosed versus total numbers of bacteria per cell were determined.


RESULTS

Y. enterocolitica Reduces p38 and ERK1/2 Tyrosine Phosphorylation and MAPK Activities

To determine possible differences between the virulent and nonvirulent Y. enterocolitica strain during infection, we compared the patterns of tyrosine-phosphorylated proteins in cells stimulated with LPS from E. coli or with the two Y. enterocolitica strains. Cell lysates prepared after different stimulation times were immunostained with the monoclonal anti-phosphotyrosine antibody 4G10. After 15 min of stimulation with LPS, nonvirulent and virulent yersiniae, there was a dramatic increase in tyrosine phosphorylation of two proteins at the 38- and 42-kDa level (Fig. 1A). The phosphorylated 38-kDa protein was shown, by stripping and reprobing the membrane with the anti-p38 peptide antibody, to have the same electrophoretical mobility as p38, also known as RK, reactivating kinase (52, 53), or for cytokine-suppressive anti-inflammatory drug binding proteins (41) (data not shown). Reprobing the membrane in the same manner with a pan-anti-ERK antibody demonstrated that the tyrosine-phosphorylated protein at 42 kDa was ERK2 (Fig. 1B). This immunoblot also allowed us to distinguish between the unphosphorylated and phosphorylated forms of ERK2 and ERK1, since the phosphorylated forms exhibited slower electrophoretical mobilities. In unstimulated cells (Fig. 1B, lane 4), ERK labeling corresponded to unphosphorylated forms of ERK2 (lower band) and ERK1 (upper band). Cell treatment with LPS and Y. enterocolitica strains induced a total upward shift in the ERK proteins, in accordance with the strong tyrosine phosphorylation of ERK2 at about 42 kDa in panel A. It was not clear from panel A whether or not ERK1 (44 kDa) was also phosphorylated, because of intense phosphotyrosine labeling at about 44 kDa that was not regulated by LPS or bacterial stimulation. Nevertheless, Fig. 1B revealed a electrophoretical shift that also occurred at the ERK1 level, demonstrating that ERK1/p44MAPK was phosphorylated over the same time course as ERK2. We thus referred to these proteins as ERK1/2, since both ERK proteins behaved similarly. Interestingly, in the 46-55-kDa region, where the JNK subtypes migrate, no substantial tyrosine phosphorylation change could be detected by immunoblotting with the anti-phosphotyrosine antibody 4G10 (Fig. 1A).


Fig. 1. Time course of tyrosine phosphorylation of J774A.1 cells treated with Y. enterocolitica or LPS. Cells were stimulated for the indicated time with the nonvirulent (lane 1) or the virulent (lane 2) Y. enterocolitica strain, stimulated with LPS from E. coli (lane 3), or remained nonstimulated (lane 4). After centrifugation, the cellular pellets were subjected to SDS-PAGE. A, the membrane was immunoblotted with the anti-phosphotyrosine antibody 4G10 as described under "Experimental Procedures." B, the same membrane was stripped and reprobed to assess the relative positions of the phosphorylated and unphosphorylated forms of ERK. In this figure, ERK refers to the unphosphorylated forms of ERK1 (panel B, upper line) and ERK2 (panel B, lower line), while ERK* indicates the positions of their respective phosphorylated forms in panels A and B.
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Phosphorylation of p38 and ERK1/2 tyrosine residues remained unchanged for at least 30 min in each stimulating condition. Thereafter, a slow decrease in phosphorylation was observed under stimulation with LPS and the nonvirulent strain (Fig. 1, lanes 1 and 3), whereas almost complete dephosphorylation of p38 and ERK1/2 occurred after a 90-min infection with the virulent Y. enterocolitica strain (Fig. 1, lane 2). The decrease in tyrosine phosphorylation of p38 might have been faster than that of ERK1/2, since a preferential decrease in p38 tyrosine phosphorylation was already visible after only 60 min of infection. After a 90-min infection, inhibition of tyrosine phosphorylation induced by the virulent Y. enterocolitica strain affected p38 and ERK1/2 and also some other proteins. This phenomenon, i.e. interference of Yersinia sp. with macrophage tyrosine phosphorylation, has already been described (18, 19, 54, 55) and can at least partially be attributed to the tyrosine phosphatase of Yersinia sp., named YopH. In any case, the virulent Y. enterocolitica strain selectively decreased p38 and ERK1/2 tyrosine phosphorylation levels, indicating that their kinase activities, conferred by dual phosphorylation of the Thr-X-Tyr motif, should be concomitantly reduced.

To directly measure the activities of ERK and p38 and to determine whether JNK activity was also affected by the inhibitory effect of Y. enterocolitica, we analyzed the ability of cytosolic extracts to phosphorylate the transcription factors Elk-1, ATF2 and c-Jun (Fig. 2). Although Elk-1 and ATF2 cannot be considered as selective substrates for the kinases ERK and p38, respectively (48), c-Jun appears to be specifically phosphorylated by JNK. A 60-min incubation of J774A.1 cells with LPS (Fig. 2, lanes 2) or the nonvirulent Y. enterocolitica strain (Fig. 2, lanes 3) induced a substantial (3-10-fold) increase in phosphotransferase activities toward the different GST fusion proteins as compared with basal levels. For example, GST-c-Jun-(1-79) phosphorylation was increased 6-fold with LPS and 11-fold with nonvirulent yersiniae. This clearly indicated that Y. enterocolitica also stimulated kinase activity of the JNK protein. However, after infection with the virulent Y. enterocolitica strain (Fig. 2, lanes 4), phosphorylation of all substrates was markedly reduced as compared with the nonvirulent strain, since the substrate phosphorylation was only 1.5-3-fold that of the control level. The parallel alteration of the different GST fusion proteins, including the two GST-c-Jun substrates, indicated that, in addition to the reduction in ERK and p38 kinase activities, the virulent Y. enterocolitica strain also inhibited JNK activity, as revealed by the weaker phosphorylation of both GST-c-Jun-(1-79) and GST-c-Jun-(1-222) substrates.


Fig. 2. Effects of the virulent Y. enterocolitica strain on the ability of J774A.1 cells to phosphorylate c-Jun, ATF2, and Elk-1 transcription factors. Extracts from untreated cells (lane 1) or cells treated with LPS (lane 2) or with the nonvirulent (lane 3) or the virulent Y. enterocolitica strain (lane 4) for 60 min were incubated with recombinant GST-c-Jun-(1-79), GST-c-Jun-(1-222), GST-ATF2, and GST-Elk-1 and isolated with glutathione-agarose. Kinase activities were assessed in the washed pellets by incorporation of [gamma -32P]ATP in the GST fusion proteins, followed by SDS-PAGE, transfer to PVDF membrane, and autoradiography detection.
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Inhibition of TNFalpha Production Is Associated with a Reduction in MAPK Activities

Since the virulent Y. enterocolitica strain abolished MAPK activation, we wondered whether MAPK deactivation was related to the inhibition of macrophage TNFalpha secretion. Fig. 3 (lanes 2-4) confirms that LPS and nonvirulent yersiniae induced a strong TNFalpha -response in J774A.1 cells, while the virulent Y. enterocolitica strain, which prevented p38 and ERK1/2 tyrosine phosphorylation, completely blocked TNFalpha secretion (Fig. 3A) as well as TNFalpha mRNA expression (Fig. 3B). Furthermore, when cells were first infected with the virulent Y. enterocolitica strain, further stimulation with LPS from E. coli could trigger neither TNFalpha production (data not shown) nor tyrosine rephosphorylation of p38 and ERK2 (Fig. 3C, bottom panel). To gain further insight into a possible relation between the lack of TNFalpha production and the decreased p38 and ERK1/2 tyrosine phosphorylation, we analyzed defined Y. enterocolitica mutants. The YopH(1)- strain, a mutant with selectively impaired secretion of the protein-tyrosine phosphatase YopH, prevented tyrosine phosphorylation and TNF production to a similar extent as the virulent wild-type strain (Fig. 3, lanes 4 and 5). On the contrary, the Yop secretion-negative LcrD- mutant with defective secretion of all Yops (Yop secr.-; Fig. 3, lane 6), did not decrease p38 and ERK2 tyrosine phosphorylation and induced strong TNFalpha release, similar to the nonvirulent strain. Fig. 3 also shows the results obtained with two other mutants expressing a restricted repertoire of yop genes. The YopD,B,N,V+ strain harbors the fragment of the Y. enterocolitica virulence plasmid encoding the Yop secretion machinery, including the genes coding for YopD, YopB, YopN, and the V antigen, which are necessary for Yop expression, secretion, and translocation. The second strain, referred to as YopD,B,N,V,H,E,YadA+ expresses, in addition to yopD, yopB, yopN, and lcrV (encoding the V antigen), yopH and yopE, which encode the translocated proteins YopH and YopE, and yadA, encoding the cell adhesin YadA. Analysis of these two mutants indicated that they were able neither to reduce p38 and ERK1/2 phosphorylation nor to block TNFalpha -production of J774A.1 cells (Fig. 3, lanes 7 and 8).


Fig. 3. Correlation between inhibition of TNFalpha production and reduction of p38/ERK1/2 tyrosine phosphorylation. These two properties were compared with untreated cells (lane 1) or cells treated with LPS (lane 2), the nonvirulent Y. enterocolitica strain (lane 3), the virulent Y. enterocolitica strain (lane 4), the YopH(1)- strain (lane 5), the Yop secr.- strain (lane 6), the YopD,B,N,V+ strain (lane 7), or the YopD,B,N,V,H,E,YadA+ strain (lane 8) (for description of the strains, see Table I). A, TNFalpha -production; cells were untreated or treated with bacteria or LPS. After 60 min of infection, extracellular bacteria were killed with gentamicin, and the TNFalpha activity of the cell culture supernatant was measured after a final incubation time of 120 min, using a cytotoxic assay performed with the TNFalpha -sensitive fibroblast cell line L929. B, reverse transcriptase-PCR detection of TNFalpha mRNA. Total RNA was isolated from cells treated as described above. RNA was reverse transcribed. The PCR products for TNFalpha and beta 2m obtained after 20 cycles were analyzed on agarose gel. Results shown are representative of two independent experiments. C, p38/ERK2 tyrosine phosphorylation; cells were treated with bacteria and/or LPS and lysed at the times indicated. In the bottom panel, cells treated for 90 min, as indicated, were challenged with LPS for another 30 min. Lysates were subjected to SDS-PAGE and immunoblotted with the anti-phosphotyrosine antibody 4G10. Only the relevant part of each immunoblot displaying the levels of tyrosine phosphorylation of ERK2 and p38 is shown.
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To compare the action of various mutants on MAPK activities, a time course study was performed with GST-Elk-1, GST-ATF2, and GST-c-Jun-(1-79) as in Fig. 2, except that the three substrates were added together in the kinase assay. In agreement with the phosphorylation of p38 and ERK1/2 seen in Fig. 3B, LPS of E. coli and all Y. enterocolitica strains induced strong phosphorylation of the three substrates within 30 min of stimulation (Fig. 4). Thereafter, only cells infected with the virulent Y. enterocolitica strain and with the YopH(1)- mutant exhibited almost complete disappearance of kinase activities within 60-90 min. The reduction in phosphorylation occurred over a similar time course for the three substrates, suggesting that the virulent and the YopH(1)- strain decreased the activities of MAPK cascades simultaneously. MAPK activities were also inhibited within 90 min, when virulent yersiniae were killed after 30 min of infection by the addition of 100 µg/ml gentamicin (data not shown). Taken together, these results indicate the existence of a relation among blockade of p38/ERK1/2 tyrosine phosphorylation, inhibition of p38/ERK1/2/JNK kinase activities, and suppression of TNFalpha -production.


Fig. 4. Time course of kinase activations in response to different Y. enterocolitica strains. Cells were untreated (lane 1) or treated with LPS (lane 2), the nonvirulent Y. enterocolitica strain (lane 3), the virulent Y. enterocolitica strain (lane 4), the YopH(1)- strain (lane 5), or the YopD,B,N,V,H,E,YadA+ strain (6) for 30, 60, or 90 min, respectively. Cell extracts prepared as described under "Experimental Procedures" were incubated with a mixture of equal amounts (8 µg) of recombinant GST-c-Jun-(1-79), GST-ATF2, and GST-Elk-1. Protein kinase activities were measured on the precipitated complexes by phosphorylation of the transcription factors using [gamma -32P]ATP. After SDS-PAGE and electrotransfer, the incorporation of radioactive phosphate was detected by autoradiography.
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Inhibition of TNFalpha Production Is Distinct from Other Y. enterocolitica Virulence Properties

Resistance of Yersinia sp. to phagocytosis and inhibition of the oxidative burst of professional phagocytes is known to be conferred by YopH and by YopE (11, 13-16). This was confirmed by strain YopD,B,N,V,H,E,YadA+, which effectively resisted phagocytosis and inhibited the J774A.1 cell oxidative burst, in contrast to strain YopD,B,N,V+ lacking YopH and YopE (Table II). However, since these two strains induced strong TNFalpha release (Fig. 3 and Table II), the ability of Y. enterocolitica to resist phagocytosis and to suppress the oxidative burst is obviously completely independent of its ability to block p38/ERK1/2 tyrosine phosphorylation and TNFalpha production. Interestingly, the YopH protein, which possesses tyrosine phosphatase activity (12), seems not to be involved in p38 and ERK1/2 dephosphorylation and TNFalpha inhibition (Fig. 3, lane 5). In the YopH(1)- strain, the mutation actually involves the YopH-specific chaperone sycH, and thus residual secretion of YopH by leaky cells cannot be excluded. To definitely rule out a possible inhibitory role of YopH, we analyzed another YopH- strain affected in YopH expression (YopH(2)-) and the complemented YopH(1)- strain, secreting YopH (YopH(1)-/H+). Cells were infected with bacteria for 60 min and thereafter were restimulated by LPS treatment. The p38 protein was immunoprecipitated and immunoblotted with the anti-phosphotyrosine antibody 4G10. As expected, the nonvirulent strain and the LcrD- mutant, impaired in Yop secretion (Yop secr.-), did not prevent p38 tyrosine phosphorylation (Fig. 5, lanes 3 and 8). In contrast, the virulent wild-type strain, the two YopH- mutants, and the complemented mutant (YopH(1)-/H+), which all inhibited TNFalpha production (data not shown), blocked tyrosine phosphorylation of p38. These results demonstrate that the inhibition of tyrosine phosphorylation of p38 (Fig. 5) ERK1/2 (Fig. 3) and probably that of JNK occur independently of the presence of YopH but depend on a functional Yop secretion/translocation apparatus (e.g. LcrD). This suggests that one or several secreted Y. enterocolitica virulence proteins are involved.

Table II. Comparison of the effects of four different Y. enterocolitica strains on J774A.1 cell TNFalpha production, phagocytosis, and oxidative burst


Y. enterocolitica strain TNFalpha releasea Phagocytosisb Oxidative burstc

Nonvirulent 87  ± 8 91  ± 1 65  ± 2
Virulent 8  ± 1 5  ± 1 5  ± 4
YopD,B,N,V+ 64  ± 6 87  ± 3 58  ± 16
YopD,B,N,V,H,E,YadA+ 97  ± 10 5  ± 2 5  ± 5

a Results for TNFalpha release are the values from the experiment depicted in Fig. 3, expressed as percentages of released TNFalpha induced by 10 µg/ml LPS (551 ± 20 pg/ml = 100%).
b Cells were incubated with bacteria for 1 h and then stained by a double-immunofluorescence technique to discriminate between intra- and extracellularly located bacteria. Mean percentages of ingested bacteria with respect to the total number of bacteria per cell were determined by counting 100 cells from each experiment.
c Cells were preexposed to Y. enterocolitica for 90 min and then treated with opsonized zymosan. Chemiluminescence responses were recorded for a total of 30 min. Mean percentages of the zymosan-induced chemiluminescence response of cells not preexposed to bacteria (100%) are shown. Values ± S.E. shown in columns three and four are from three independent experiments.


Fig. 5. Role of the Y. enterocolitica protein-tyrosine phosphatase YopH in preventing p38 tyrosine phosphorylation. Cells were untreated (lanes 1 and 2) or treated for 60 min with the nonvirulent (lane 3) or the virulent Y. enterocolitica strain (lane 4), the YopH(1)- strain with defective secretion of YopH (lane 5), the YopH(1)-/H+ strain complemented and secreting YopH (lane 6), the YopH(2)- strain with defective expression of YopH (lane 7), or the Yop secr.- strain with defective secretion of all Yops (lane 8). Thereafter, cells were restimulated with LPS for 30 min (lanes 2-8). After cell lysis, p38 was immunoprecipitated with polyclonal anti-p38 antibodies, subjected to SDS-PAGE, and immunoblotted with the anti-phosphotyrosine antibody 4G10, as described under "Experimental Procedures." The bands stained above the p38 protein correspond to nonspecific labeling of chains of rabbit anti-p38 antibodies used in the immunoprecipitation procedure.
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The Virulent Y. enterocolitica Strain Reduces Raf-1 Kinase Activity

Since the reduction in MAPK tyrosine phosphorylation and activation cannot be attributed to the phosphatase YopH, we analyzed Raf-1 kinase activities to determine whether the virulent Y. enterocolitica strain modulates signaling pathways upstream of MAPKs. Fig. 6 demonstrates that the ability of Raf-1 to activate MEK1, which then in turn phosphorylated ERK2, was markedly reduced when macrophages were infected with the virulent Y. enterocolitica strain, in contrast to treatment of macrophages with the nonvirulent strain or with LPS. Suppression of the Raf-1 kinase activity by the virulent Y. enterocolitica strain may indicate that inhibition of the MAPK signaling cascades occurs at least partially via reduction of upstream kinase activities already at the level of Raf-1.


Fig. 6. Effect of the virulent Y. enterocolitica strain on Raf-1 kinase activity of J774A.1 cells. Raf-1 was immunoprecipitated in precleared extracts from untreated cells (lane 1) or cells treated for 90 min with LPS (lane 2), the nonvirulent (lane 3), or the virulent Y. enterocolitica strain (lane 4). Raf-1 kinase activities were indirectly assayed by measuring Raf-1-dependent activation of MEK1, leading to phosphorylation of ERK2. Washed Raf-1 immune complexes were first incubated with purified recombinant MEK1, and purified recombinant kinase-inactive ERK2 was then added. After SDS-PAGE and electrotransfer, incorporation of [gamma -32P]ATP in ERK2, carried out as described under "Experimental Procedures," was analyzed by autoradiography (upper panel) and quantified with a PhosphorImager (lower panel). Data, expressed as the percentage of radioactivity incorporated in untreated cells (100%), are from one experiment representative of three performed.
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DISCUSSION

Yersinia sp., like a number of other microbial pathogens, are supposed to modulate eukaryotic signaling pathways for their own benefit (20). In this study, we analyzed the impact of Y. enterocolitica on macrophage MAPK signaling pathways using J774A.1 cells as an infection model. Infection with Y. enterocolitica was found to stimulate p38 and ERK1/2 MAPK pathways, as detected by tyrosine phosphorylation. By contrast, direct tyrosine phosphorylation of JNK was not obvious, but this has been attributed to the fact that JNK phosphotyrosine cannot be easily detected by immunoblotting using an anti-phosphotyrosine antibody (56, 57). The patterns of tyrosine-phosphorylated proteins (Figs. 1 and 3) and kinase activities (Figs. 2 and 4) obtained with LPS and virulent and nonvirulent Y. enterocolitica strains were very similar over the first 30 min. Thereafter, the virulent strain harboring the Y. enterocolitica virulence plasmid induced a substantial reduction in kinase activities, as revealed by a decrease in the phosphorylation of ERK1/2 and p38 kinases along with their substrates, the transcription factors Elk-1 and ATF2, as well as that of the JNK-specific substrate c-Jun. The fact that the reduction in kinase activities occurred after only 1 h of cell infection can be explained by the delay necessary for the Yops to reach their targets and to exert their effects on the host cell (5, 16, 19). The initial stimulation of the three types of MAPKs followed by selective inhibition with the virulent Y. enterocolitica strain was also observed with macrophages derived from human monocytes (data not shown).

A link between MAPK activation and TNFalpha production induced by LPS has been widely documented (41-42, 58-60). Deactivation of the MAPKs p38, JNK, and ERK1/2 induced by virulent Y. enterocolitica, therefore, might be related to its inhibitory effect on macrophage TNFalpha secretion. Indeed, we found that all investigated Y. enterocolitica strains capable of inhibiting MAPK activities also prevented TNFalpha production, and reciprocally, all strains inhibiting TNFalpha release also deactivated MAPKs. This finding strongly supports the hypothesis that inhibition of TNFalpha release by Y. enterocolitica originates from shortening p38, ERK1/2, and JNK activation by reducing their levels of tyrosine phosphorylation. Furthermore, our evaluation of TNFalpha mRNA levels by reverse transcriptase-PCR indicated that LPS stimulation and infection with the nonvirulent Y. enterocolitica strain dramatically enhanced the amount of TNFalpha mRNA, while no accumulation of this messenger occurred after infection with the virulent strain. This indicates that the inhibitory effect of the virulent Y. enterocolitica strain on TNFalpha release is probably not due to alteration of TNFalpha maturation or secretion but rather to a lack of TNFalpha mRNA accumulation. The absence of mRNA may be due to inhibition of TNFalpha gene transcription or due to mRNA instability. A role of p38 in post-transcriptional control of TNFalpha gene expression has been clearly shown by the group of Lee (41, 42) using the anti-inflammatory drug SB203580. It is thus possible that the accelerated dephosphorylation of p38 is partially responsible for the inhibition of TNFalpha synthesis.

The fact that not only p38, but also ERK1/2 and JNK, are deactivated by the virulent Y. enterocolitica strain raises the question of their potential role in TNFalpha suppression. Indeed, it was recently shown that blockage of the ERK pathway by the MEK inhibitor PD98059 prevents TNFalpha mRNA synthesis induced by Fcgamma R stimulation (58). No specific drugs for the JNK pathway are available yet. However, TNFalpha gene expression is stimulated by AP-1 (61, 62), a transcription factor composed of c-Jun and c-Fos, which are activated through phosphorylation by ERK and JNK (63). Deactivation of p38, ERK, and JNK induced by the virulent Y. enterocolitica strain may thus together contribute to inhibition of TNFalpha synthesis. The similar time courses of deactivation of all the MAPK, ERK1/2, JNK, and p38 suggests that yet unidentified bacterial virulence factors might act at a step that is common to the three pathways, i.e. upstream of MAPKs. When investigating this possibility, we found that Raf-1 activity was lowered after infection with the virulent Y. enterocolitica strain compared with the nonvirulent strain. This finding indicates that at least part of the TNFalpha -inhibitory action takes place upstream of the MAPKs. However, it remains to be determined whether Y. enterocolitica inhibits MAPK signaling cascades via reduction of upstream kinase activities only or whether it also causes dephosphorylation of MAP kinases themselves. It cannot be ruled out that bacterial factors trigger or accelerate the expression of an endogenous macrophage phosphatase, such as the specific MAPK phosphatase-1 (64, 65) or HVH1 or HVH2, two human homologs of the vaccinia virus dual specific phosphatase VH1 (66, 67).

In an attempt to identify the potential virulence factors involved in MAPK deactivation and TNFalpha inhibition, we compared the characteristics of several Y. enterocolitica mutants. Analysis of mutants with impaired tyrosine phosphatase YopH expression or secretion clearly excluded participation of YopH in the inhibitory effect on MAPK tyrosine phosphorylation and TNFalpha production. Furthermore, experiments on a mutant with defective secretion of all Yops (LcrD- mutant) demonstrated that indeed one or several released Y. enterocolitica proteins other than YopH mediate inhibition of MAPK activities and TNFalpha production. Analysis of Y. enterocolitica strains capable of producing individual virulence factors revealed, in agreement with our previous data on granulocytes (16), that YopH and YopE confer resistance to phagocytosis and suppression of the J774A.1 cell oxidative burst to Y. enterocolitica; a strain, capable of producing YopH, YopE, and the adhesin YadA, as well as YopD, YopB, YopN, and the V antigen, suppressed macrophage phagocytosis and oxidative burst, in contrast to a strain secreting only the latter proteins, which are necessary for expression, secretion, and translocation of active Yops (2, 4, 7-8, 10). The fact that both strains were unable to inhibit MAPK activities and TNFalpha production clearly demonstrates that the anti-TNFalpha effect is not a consequence of the ability of Y. enterocolitica to inhibit phagocytosis and to suppress the oxidative burst. Furthermore, this finding implies that the Yops released by this strain (YopD, YopB, YopN, YopH, YopE, V antigen) are not, or at least not solely, responsible for the inhibition of TNFalpha production, although such an effect was previously attributed to YopB (28) and the V antigen (26, 27).

In summary, we demonstrated for the first time that virulent Y. enterocolitica mediates disruption of eukaryotic signal transduction. Moreover, our study highlights a correlation between the inhibition of macrophage TNFalpha production by Y. enterocolitica and deactivation of MAPK pathways. The virulence factors responsible for these inhibitory effects are released Y. enterocolitica proteins other than YopH or YopE. The cellular target from which the different MAPK pathways are affected seems to be located at the MAPK kinase kinase level, i.e. Raf-1, or upstream. These characteristics point to the Ras superfamily of small G proteins, among which Cdc42, Rac, and Rho appear to be activated in cascade, with subsequent activation of multiple pathways including MAPK modules (68). Studies presently under way address this question and should provide new insight into the pathogenesis of yersiniosis.


FOOTNOTES

*   This work was supported in part by the Human Capital and Mobility program of the European Union (Grant CHRX-CT94-0451, to K. R. and R. Z.), a fellowship from the Deutsche Forschungsgemeinschaft (to J. M.), and the Association pour la Recherche contre le cancer (ARC No. 6497).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed. Tel.: 33 467 14 42 44; Fax: 33 467 14 33 38; E-mail: rouot{at}crit.univ-montp2.fr.
1   The abbreviations used are: Yop, Yersinia outer protein; TNFalpha , tumor necrosis factor; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase (MAPK is used here in a general sense and includes ERK, JNK, and p38 kinases); ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase (also termed SAPK); SAPK, stress-activated protein kinase; p38, murine homologue of the Saccharomyces cerevisiae high osmolarity glycerol protein kinase HOG1; LPS, lipopolysaccharide of Gram-negative bacteria; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PVDF, polyvinyldifluoride; beta 2m, beta 2-microglobuline; PMSF, phenylmethylsulfonyl fluoride; MEK, MAPK/ERK kinase; ATF2, activating transcription factor 2.

ACKNOWLEDGEMENTS

We thank M. Karin, B. Dérijard, and A. Nordheim for providing GST-protein vectors and J. Favero, J. Dornand, S. Köhler, and J.-P. Toutant for constructive discussions. We also thank M. David for purification of the histidyl-tagged MEK and ERK2, J. Armand for technical assistance, and M. Passama for the drawings.


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