(Received for publication, November 19, 1996, and in revised form, March 3, 1997)
From 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
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- (TNF
) 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 TNF
release, (ii) the suppressor effect
on TNF
production, which originates from the lack of TNF
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 TNF
production by inhibiting ERK1/2, p38, and JNK kinase activities.
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
TNF (26-29). Released TNF
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 TNF
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
TNF
, 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 TNF 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
TNF production but is not required for the inhibition of macrophage
phagocytosis and oxidative burst.
|
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 StimulationThe 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 AntibodiesAntibodies 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.
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 -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.
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 -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
-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
-glycerophosphate, 0.1 mM Na3VO4, 20 mM
p-nitrophenyl phosphate, 20 µM ATP (4 µCi of
[
-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 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
-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
[
-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).
Cells dispatched in plastic culture
plates (1 × 106 cells/sample for TNF quantitation
and 1 × 107 cells/sample for analysis of TNF
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 TNF
quantitation, the cell culture supernatants were removed after a final
120-min incubation. The TNF
cytokine level in the culture
supernatant was evaluated by a cytotoxic assay performed with the
TNF
-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 TNF
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 TNF
(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
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
2m was used as a control. The PCR
products were run on a 1.5% agarose gel supplemented with ethidium
bromide.
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 Assays5 × 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.
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).
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.
Inhibition of TNF
Since the virulent Y. enterocolitica
strain abolished MAPK activation, we wondered whether MAPK deactivation
was related to the inhibition of macrophage TNF secretion. Fig.
3 (lanes 2-4) confirms that LPS and
nonvirulent yersiniae induced a strong TNF
-response in J774A.1
cells, while the virulent Y. enterocolitica strain, which
prevented p38 and ERK1/2 tyrosine phosphorylation, completely blocked
TNF
secretion (Fig. 3A) as well as TNF
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 TNF
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 TNF
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 TNF
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 TNF
-production of J774A.1 cells (Fig. 3, lanes
7 and 8).
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 TNF
-production.
Inhibition of TNF
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
TNF 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 TNF
production. Interestingly, the YopH protein, which possesses tyrosine phosphatase activity (12), seems not to be involved in p38 and ERK1/2
dephosphorylation and TNF
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 TNF
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.
|
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.
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 TNF 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 TNF
secretion. Indeed, we found that all investigated Y. enterocolitica strains capable of inhibiting MAPK activities also
prevented TNF
production, and reciprocally, all strains inhibiting
TNF
release also deactivated MAPKs. This finding strongly supports
the hypothesis that inhibition of TNF
release by Y. enterocolitica originates from shortening p38, ERK1/2, and JNK
activation by reducing their levels of tyrosine phosphorylation.
Furthermore, our evaluation of TNF
mRNA levels by reverse
transcriptase-PCR indicated that LPS stimulation and infection with the
nonvirulent Y. enterocolitica strain dramatically enhanced
the amount of TNF
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 TNF
release is probably not due to alteration of TNF
maturation or secretion but rather to a lack of TNF
mRNA
accumulation. The absence of mRNA may be due to inhibition of
TNF
gene transcription or due to mRNA instability. A role of p38
in post-transcriptional control of TNF
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
TNF
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 TNF suppression. Indeed, it was recently shown that blockage of the ERK pathway by the MEK inhibitor PD98059 prevents TNF
mRNA synthesis induced by Fc
R stimulation (58). No specific drugs for the JNK pathway are available yet. However, TNF
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 TNF
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 TNF
-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 TNF 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 TNF
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 TNF
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 TNF
production clearly demonstrates that the anti-TNF
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 TNF
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 TNF 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.
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.