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Yersinia Outer Protein P of Yersinia enterocolitica Simultaneously Blocks the Nuclear Factor-B Pathway and Exploits Lipopolysaccharide Signaling to Trigger Apoptosis in Macrophages¹

Klaus Ruckdeschel^2,*, Oliver Mannel^*, Kathleen Richter^*, Christoph A. Jacobi^*, Konrad Trülzsch^*, Bruno Rouot and Jürgen Heesemann^*

^* Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, München, Germany; and Institut National de la Santé et de la Recherche Médicale Unité 431, Université Montpellier II, Montpellier, France

	Abstract

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Exposure of macrophages to bacteria or LPS mediates activation of signaling pathways that induce expression of self defense-related genes. Pathogenic Yersinia species impair activation of transcription factor NF-B and trigger apoptosis in macrophages. In this study, we dissected the mechanism of apoptosis induction by Yersinia. Selectively, Yersinia enterocolitica strains producing the effector protein Yersinia outer protein P (YopP) hampered NF-B activation and subsequently conferred apoptosis to J774A.1 macrophages. Thereby, YopP bound and inhibited the macrophage NF-B-activating kinase IKK. YopP- and Yersinia-, but not Salmonella-induced apoptosis was specifically prevented by transient overexpression of NF-B p65, giving evidence that YopP mediates cell death by disrupting the NF-B signaling pathway. Transfection of J774A.1 macrophages with YopP induced a moderate, but significant degree of apoptosis (40–50% of transfected cells). This effect was strongly enhanced by additional initiation of LPS signaling (80–90%), indicating a synergism between LPS-induced signal transduction and inhibition of NF-B by YopP. This reflects a strategy of a bacterial pathogen that takes advantage of LPS, serving as cofactor, to impair the macrophage.

	Introduction

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Multicellular organisms raise a series of defense responses when encountered with bacterial pathogens. To establish a competitive advantage, pathogenic bacteria have evolved sophisticated strategies for evasion or neutralization of host defense mechanisms. A broad range of bacterial pathogens, including the Gram-negative bacterium Yersinia, uses a so-called type III protein secretion machinery to deliver bacterial effector proteins into the host cell for modulation of cellular functions. The type III protein secretion machinery of Yersinia and a set of six effector Yersinia outer proteins (Yops)³ (YopE, YopH, YopM, YopO/YpkA, YopP/YopJ, YopT) are encoded by a 70-kb virulence plasmid that is common to the three pathogenic Yersinia species (1). Yersinia pestis is the etiological agent of plague; Yersinia enterocolitica and Yersinia pseudotuberculosis cause gastrointestinal syndromes, lymphadenitis, and septicemia. The diverse effector Yops act on different cellular levels to neutralize a multitude of host effector functions (1). Yersinia causes depolymerization of the actin microfilament structure by affecting members of the small GTP-binding proteins Rho, Rac, and Cdc42 (YopE, YopT) (2). YopH tyrosine dephosphorylates host cell proteins, such as p130Cas and focal adhesion kinase FAK (2). By action of these Yops, Yersinia resists phagocytosis and escapes primary killing by phagocytes. Besides these immediate effects, Yersinia suppresses the production of the proinflammatory cytokine TNF- (3, 4, 5, 6) and triggers apoptosis in macrophages (7, 8, 9). Both effects are mediated by YopP in Y. enterocolitica (5, 9), or by its homologue YopJ in Y. pseudotuberculosis (4, 6, 7). A series of studies suggest that Yersinia blocks macrophage TNF- production by down-regulation of mitogen-activated protein kinase (MAPK) activities (3, 4, 5, 6). A recent report from Orth et al. (10) revealed that YopP/YopJ binds and inactivates members of the MAPK kinase superfamily, which function as upstream MAPK activators.

The mechanism of apoptosis induction by Yersinia appears less clear. In a previous study, we revealed a correlation between the abilities of Yersinia to trigger macrophage apoptosis and to suppress activation of transcription factor NF-B (11). The NF-B system is central to innate immunity. It controls the synthesis of cytokines, acute-phase proteins, and adhesion molecules, and mediates cellular survival by prevention of apoptosis (12, 13). Certain extracellular stimuli, such as TNF-, genotoxic agents, and ionizing radiation, simultaneously activate proapoptotic and antiapoptotic pathways in eukaryotic cells (13, 14, 15, 16). NF-B functions to up-regulate synthesis of proteins that counteract the proapoptotic pathways, such as members of the inhibitor of apoptosis protein (IAP), TNFR-associated factor, and Bcl-2 families (17, 18, 19). Accordingly, the activation of NF-B provides protection against apoptotic killing, otherwise induced by these stimuli. In macrophages, after initial NF-B activation, wild-type Y. enterocolitica down-regulates NF-B activities as short as 60–90 min after onset of infection, a lag time necessary for the Yops to reach their targets and to exert their effects (11). As NF-B functions to prevent apoptosis, reduced NF-B activity appears to be responsible for macrophage cell death. However, subversion of NF-B in general is not sufficient to trigger apoptosis in eukaryotic cells. In fact, it requires a secondary stimulus, such as TNF-, which activates an intrinsic cytotoxic pathway leading to apoptosis when NF-B is inhibited (13, 14, 15, 16).

In this study, we focused on the mechanism of apoptosis induction by Y. enterocolitica. We wondered whether YopP/YopJ alone is able to trigger apoptosis in macrophages, or whether it requires a secondary signal. Macrophages are supposed to be the major target cells of YopP/YopJ, because Yersinia selectively triggers apoptosis in macrophages (8). Our first aim was to analyze the impact of YopP/YopJ on the NF-B signaling pathway in macrophages. NF-B is activated by the I-B (inhibitory protein that dissociates from NF-B) kinase (IKK) complex in response to a large spectrum of stimulation conditions, including bacterial infection and LPS treatment. The IKK complex is composed of at least three proteins, IKK, IKK, and IKK, and phosphorylates I-Bs, inhibitory proteins that sequester NF-B in the cytoplasm (12). Phosphorylation is followed by I-B ubiquitination and degradation, enabling liberation of NF-B, which translocates to the nucleus and activates transcription (12). Orth et al. (10) demonstrated binding of YopP/YopJ to GST-fused IKK in embryonic kidney 293 cells. However, interaction of YopP/YopJ with the IKK complex in its major target cell, the macrophage, has not yet been investigated. We chose the J774A.1 macrophage cell line as an established infection model, which closely reflects the situation in primary macrophages (11). We report that YopP from Y. enterocolitica interacts with IKK in vivo to trigger apoptosis in macrophages. LPS-initiated signal transduction strongly enhances the apoptosis-inducing capacity of YopP, indicating that LPS actively promotes apoptosis due to YopP. This points out sophisticated devised tactics of a bacterial pathogen, which affects a key effector pathway of innate immunity. By injection of YopP, Yersinia reverses the LPS-induced defense response in macrophages, which leads to macrophage apoptosis.

	Materials and Methods

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Bacterial strains, cell culture, and stimulation conditions

Y. enterocolitica strains, listed in Table I, were grown as described previously (11). Salmonella typhimurium strains were cultured until they entered the early stationary growth phase, a stage by which Salmonella acquires the capability to induce apoptosis (20). The S. typhimurium strains were kindly provided by M. Hensel (Institute for Clinical Microbiology, Immunology, and Hygiene, Erlangen, Germany). We analyzed two S. typhimurium wild-type strains (ATCC14028 and SL1344) and their respective mutants (ATCC14028-MvP215 and SL1344-EE659), which are impaired in secretion of virulence proteins of Salmonella pathogenicity island 1 (SPI-1) due to transposon mutation of prgK (21, 22). The murine macrophage cell line J774A.1 was grown as described (11), treated with LPS from Escherichia coli O55:B5 (Sigma, St. Louis, MO), polymyxin B sulfate (Life Technologies, Karlsruhe, Germany), or infected as indicated. Infections were performed at a ratio of 50 bacteria per cell. For incubation times longer than 90 min, bacteria were killed by addition of gentamicin (100 µg/ml) after 90 min of infection.

The yopP-negative mutant was constructed by performing a transposon mutagenesis on a 10-kb EcoRI DNA fragment of Y. enterocolitica virulence plasmid pYVO8. This fragment harbors the yopO/yopP operon and has been subcloned into vector pLAFR2 (23). Transposon mutagenesis was accomplished using the TnMax25 mini-transposon system (24). Mutagenized plasmids were mobilized in strain WA-C-pLCR and screened on their inability to trigger macrophage apoptosis in comparison with the nonmutated control. WA-C-pLCR harbors a plasmid encoding the Y. enterocolitica secretion and translocation machinery (23) and additionally the gene for the adhesin YadA. Several clones unable to induce apoptosis were isolated, and locations of the transposons in the yopP gene were analyzed by DNA sequencing. An internal 7.5-kb KpnI DNA fragment encompassing the yopP gene inclusive of the transposon in the N-terminal coding region of yopP was subcloned into suicide vector pKAS46. The resulting mutator plasmid was transferred into strain WA-314, and the endogenous yopP gene was replaced by the yopP::TnMax25 construct by double recombination (resulting in strain WA-314yopP). Location of the transposon insertion was confirmed by PCR, and Yop production and secretion were checked as described previously (23). For complementation of WA-314yopP, a DNA fragment encompassing the entire yopP open reading frame was amplified by PCR and subcloned in plasmid pCJYE138-G3 (25), replacing the gfp gene of pCJYE138-G3 in frame by yopP (pCJYE138-yopP). In a second approach, the amplified yopP DNA fragment was expanded at the C-terminal coding region by the sequence, which encodes the 9E10 c-myc tag epitope MEQKLISEEDL, by PCR (pCJYE138-yopPmyc). All PCR products were checked for sequence accuracy by sequencing. In the resulting plasmids, yopP is located downstream of the yopE promoter and fused to regions encoding the first 138 aa of YopE. Induction of gene expression mediates production of a YopE138-YopP hybrid protein that is intrabacterially stabilized by the YopE-specific chaperone SycE. Translocated YopE138 hybrid proteins do not exert a YopE effect (25). The yopP-negative mutant WA-314yopP was complemented with plasmid pCJYE138-yopP (resulting in strain WA-314yopP/P⁺). To generate a Yersinia strain that secrets and translocates YopP as the unique effector Yop, plasmid pCJYE138-yopPmyc was introduced in strain WA-C-pLCR (resulting in strain WA-C-pLCRyopP).

To construct a mammalian yopP expression vector, a DNA fragment encompassing the yopP open reading frame fused to a C-terminal c-myc tag epitope was amplified and inserted into the multicloning side of pcDNA3.1(-) (Invitrogen, San Diego, CA), using XbaI and XhoI restriction sides. This procedure placed the yopPmyc gene under control of the CMV promoter (pYopP).

Coimmunoprecipitations and Western immunoblotting

A total of 10⁸ cells was infected with bacteria for 60 min, washed, scraped, and lysed in lysis buffer containing 10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1% Nonidet P-40, 1 mM DTT, and phosphatase and protease inhibitors (Boehringer Mannheim, Indianapolis, IN). These conditions selectively lyse the cells, but not the bacteria. Cell lysates were diluted 1/1 with buffer TN (20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT, and phosphatase and protease inhibitors) and precleared with an irrelevant polyclonal rabbit Ab and protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). For immunoprecipitation, cell lysates were incubated with a polyclonal rabbit anti-YopE Ab (25) for 4 h. Immune complexes were collected with protein A-agarose, washed three times with buffer TN, and resuspended in sample buffer. Proteins were separated by 7.5% SDS-PAGE, electrotransferred to polyvinylidene difluoride (PVDF) membrane (DuPont-NEN, Boston, MA), and probed with either rabbit anti-YopE, or polyclonal rabbit anti-IKK Ab (which exhibits partial cross-reactivity with IKK; Santa Cruz Biotechnology). Immunoreactive bands were visualized by incubation with goat anti-rabbit Abs conjugated to HRP (Amersham Pharmacia, Piscataway, NJ) using enhanced chemiluminescence reagents (Amersham Pharmacia).

EMSA and IKK assay

To analyze nuclear translocation of NF-B, nuclear proteins were extracted and EMSAs were performed as described (11), with minor modifications: The nuclear proteins (7 µg) were preincubated with 1.5 µg poly(dI-dC) (Amersham Pharmacia) on ice before addition of 2–5 ng of the radiolabeled NF-B oligonucleotide probe (Santa Cruz Biotechnology). DNA-protein complexes were separated by 5% PAGE and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitations with anti-IKK Ab and kinase assays were conducted with 12.5 x 10⁶ cells per sample, according to the method described by C. Fischer et al. (26). The kinase reactions were performed with 1 µg GST/I-B for 30 min at 30°C in the presence of 20 mM HEPES, pH 8, 10 mM MgCl₂, 50 mM NaCl, 2 mM DTT, phosphatase and protease inhibitors, and 20 µM ATP (4 µCi of [-³²P]ATP per sample). The plasmid encoding GST/I-B was kindly provided by U. Siebenlist (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Proteins were separated by 7.5% SDS-PAGE and electrotransferred to PVDF membrane. The upper part of the membrane was immunoblotted with anti-IKK Ab to determine the amount of precipitated IKK; the lower part, including GST/I-B, was analyzed by PhosphorImager.

Transfection of J744A.1 cells and analysis of quantity and morphology of transfected cells

A total of 5 x 10⁵ cells/well was transfected with 4 µl ExGen 500 according to the manufacturer’s instructions (Fermentas, Hanover, MD). Transfections were conducted as cotransfection experiments, using 0.33 µg pSV--galactosidase expression vector (Promega, Madison, WI) and 0.66 µg of the plasmid of interest. Expression plasmids used in this study include p50 and p65 (kindly provided by H. W. L. Ziegler-Heitbrock, Institute for Immunology, Munich, Germany), IKK and IKK (Ref. 27 ; kindly provided by H. Nakano, Juntendo University School of Medicine, Tokyo, Japan), phosphorylation-defective GST/I-B (kindly provided by P. J. Nelson, Medical Policlinic, Munich, Germany), and YopP (described above). Empty expression vectors containing no inserts were used as negative controls. Eighteen hours after transfection, cells were infected with Yersinia strains or treated with LPS, as indicated. To identify transfected macrophages, cells were stained with 5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal) after certain points of time. For assessment of cell death of transfected cells, blue transfected cells were counted, and morphology of transfected cells was determined using light microscopy (14, 15, 17, 18). Every single transfected cell was analyzed for an apoptotic appearance. A minimum of eight microscopic fields was investigated for each sample. The degree of cell death of transfected cells after bacterial infection was calculated as percentage of dead blue cells of infected vs noninfected cells (=100%). To quantify cell death induced by transfections with the different expression vectors independently of bacterial infection, the number of apoptotic blue cells was assayed in relation to the total number of transfected cells. Results are expressed as mean percentages ± SD from at least three independent experiments.

Assessment of apoptosis and fluorescence microscopy

To quantify apoptosis of J774A.1 cells in response to bacterial infection, apoptotic cells were specifically labeled with fluorescein-conjugated annexin V (Boehringer Mannheim), as described (8). The rate of apoptosis was determined by counting a minimum of 100 cells per sample in a fluorescence microscope. Results are expressed as mean percentages of apoptotic cells ± SD from three independent experiments.

For immunofluorescent labeling of transfected cells, the cells were stimulated as indicated, washed twice with PBS, fixed with 3.7% paraformaldehyde, permeabilized with 0.02% Nonidet P-40, and blocked with goat serum. To specifically label single proteins, the following primary Abs were used: goat and rabbit polyclonal anti-p65 (Santa Cruz Biotechnology), rabbit polyclonal anti--galactosidase, and mouse monoclonal anti-c-myc (Clontech). Primary Abs were stained with appropriate fluorescein- or rhodamine-conjugated secondary Abs (Sigma). The TUNEL reaction, for labeling free 3' OH ends of DNA fragments with fluorescein, was performed according to the manufacturer’s instructions (Boehringer Mannheim).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
YopP impairs activation of NF-B and subsequently triggers apoptosis in J774A.1 macrophages

To identify the apoptosis-inducing virulence factor in our infection model (11), we performed a transposon mutagenesis on a 10-kb DNA fragment of the Y. enterocolitica virulence plasmid that conferred the capability to trigger apoptosis when introduced into strain WA-C-pLCR. WA-C-pLCR solely harbors the Yersinia type III protein secretion machinery, but does not produce any effector Yop. Disruption of the yopP gene in the DNA fragment by the transposon compelled the strain unable to cause apoptosis. Using this construct, we generated a yopP-negative mutant of the Y. enterocolitica wild-type strain WA-314, designated WA-314yopP. In accordance with our previous work (11), the wild-type strain WA-314 efficiently impaired nuclear translocation of transcription factor NF-B within 90 min of infection, and subsequently triggered macrophage apoptosis (Fig. 1, lane 4). In contrast, WA-314yopP (lane 5) elicited a NF-B response that was even stronger than that triggered by the virulence plasmid-cured Yersinia strain WA-C (lane 3). Both strains were unable to kill the macrophages by apoptosis. The NF-B responses induced by WA-314yopP and WA-C showed increasing tendencies within 90 min of infection (11 , and data not shown), whereas the NF-B response due to LPS treatment, exhibiting its maximum after 30 min of stimulation (11), was already noticeably reduced after 90 min (lane 2). The capabilities to suppress activation of NF-B and to induce apoptosis could be restored by introduction of the yopP gene in trans into WA-314yopP. The resulting strain, WA-314yopP/P⁺, synthesizes YopP as fusion protein with the first 138 aa of YopE controlled by the yopE promoter (25). Similar to wild-type Yersinia, this strain blocked NF-B activation and efficiently induced apoptosis (lane 6). We furthermore compared strain WA-C-pLCR, bearing the type III protein secretion apparatus, with strain WA-C-pLCRyopP, additionally harboring a yopP-encoding plasmid. This strain secretes the YopE138-YopP hybrid protein as single effector Yop. In contrast to the original strain WA-C-pLCR (lane 7), WA-C-pLCRyopP strongly suppressed NF-B activation and triggered apoptosis (lane 8). The NF-B response mediated by WA-C-pLCR was reduced compared with NF-B stimulations induced by WA-314yopP or WA-C. This probably is the result of lytic affection of a portion of the infected cells, which is conferred by pore-forming proteins of the Yersinia translocation apparatus under conditions, when no effector Yops are translocated (28). Together, the analysis of the different Yersinia strains indicates that YopP of Y. enterocolitica impairs NF-B activation and subsequently mediates apoptosis in J774A.1 macrophages.

View larger version (48K): [in this window] [in a new window]   FIGURE 1. YopP suppresses NF-B and mediates J774A.1 cell apoptosis. J774A.1 cells were left untreated (lane 1) or stimulated with LPS (lane 2), virulence plasmid-cured WA-C (lane 3), wild-type WA-314 (lane 4), yopP-negative WA-314yopP (lane 5), complemented WA-314yopP/P+ (lane 6), WA-C-pLCR bearing the type III secretion apparatus (lane 7), or WA-C-pLCRyopP secreting only YopP (lane 8). The NF-B activities were determined 90 min after infection by EMSA (upper panel). Only sections of the autoradiogram containing the NF-B-DNA complexes are shown. The upper panels (EMSA) show data from one experiment representative for five performed. Apoptosis was assayed 6 h after onset of infection by staining cells with annexin V and counting apoptotic cells in a fluorescence microscope (lower panel). Results are expressed as mean percentages ± SD from three independent experiments.

YopP interacts with macrophage IKK in vivo and suppresses IKK activities

We wondered whether YopP down-regulates NF-B activity in macrophages by interference with the IKK complex. J774A.1 cells were infected with the yopP-negative mutant WA-314yopP or with two Yersinia strains secreting the YopE138-YopP hybrid protein (WA-314yopP/P⁺ and WA-C-pLCRyopP). Immunoprecipitation of YopP was conducted by using a polyclonal anti-YopE Ab, which recognizes the N-terminal 138 aa of the YopE138-YopP fusion protein (25). Precipitation with this Ab effectively accumulated translocated YopP in case of infection with WA-314yopP/P⁺ and WA-CpLCRyopP (Fig. 2A, lanes 2 and 3), but not WA-314yopP (lane 1). Immunostaining of the separated precipitates with Abs directed against IKK and IKK revealed that IKK, but not IKK, coprecipitated with YopP (Fig. 2A). These results demonstrate that translocated YopP interacts with endogenous IKK in vivo during infection of macrophages.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. YopP coimmunoprecipitates IKK and suppresses IKK activities. A, Coimmunoprecipitation of IKK with YopP. Cells were infected with yopP-negative WA-314yopP (lane 1), complemented WA-314yopP/P+ (lane 2), or WA-C-pLCRyopP (lane 3), the latter two secreting YopP as YopE138-YopP fusion protein. After 60 min, cells were lysed and YopP was immunoprecipitated with polyclonal anti-YopE Ab. Immunocomplexes were subjected to SDS-PAGE and transferred to PVDF membrane. One part of the membrane was immunoblotted with anti-YopE Abs, recognizing the YopP fusion protein (lower panel), the other part with anti-IKK Abs (upper panel). YopP in lane 3 exhibits slower electrophoretic mobility than in lane 2 because of the c-myc tag. B, IKK activity assay. Cell extracts from untreated cells (lane 1), or cells treated with LPS (lane 2), wild-type WA-314 (lane 3), yopP-negative WA-314yopP (lane 4), or complemented WA-314yopP/P+ (lane 5) for 30 min were incubated with anti-IKK Abs to precipitate IKK. IKK activities were assayed by measuring the abilities of the immunocomplexes to radioactively phosphorylate rGST/I-B. Kinase reaction samples were subjected to SDS-PAGE and transferred to PVDF membrane. The upper part of the membrane was immunoblotted with anti-IKK Abs (upper panel). The double band appearing in lanes 2–4 may reflect phosphorylated and nonphosphorylated forms of IKK. The lower part of the membrane, including GST/I-B, was analyzed by autoradiography and quantified by PhosphorImager (lower panel). The results shown are from one representative experiment of three performed. (WB, Western blot; KA, kinase assay.)

NF-B p65 and IKK exert a protective effect against Yersinia- but not Salmonella-induced apoptosis

To substantiate a possible relation between NF-B inhibition and apoptosis induction, we checked the impact of overexpression of NF-B subunits p50 and p65 on YopP-mediated apoptosis. NF-B consists as a heterodimer from two subunits, p50 and p65, but solely the p65 subunit harbors a transactivation domain (12). J774A.1 macrophages were transiently transfected with eukaryotic expression vectors encoding the p50 or p65 subunit, or the empty CMV vector control. To identify the transfected cells, the plasmids were cotransfected with a -galactosidase-encoding reporter vector (14, 15, 17, 18). After Yersinia infection, cells were fixed and stained with X-gal to detect -galactosidase-expressing blue cells. The number of transfected cells and their morphology were scored by microscopy. Apoptotic death was characterized by typical condensed, misshapen appearance of transfected cells and by reduction of the number of viable blue cells in the samples (14, 15, 17, 18). Fig. 3A displays a typical microscopic picture of transfected and infected J774A.1 macrophages. In the noninfected control, single transfected cells exhibit blue color and a normal cellular shape. Cells transfected with empty expression vector and treated with wild-type Yersinia WA-314 in majority assumed a characteristic apoptotic appearance, being condensed and shapeless, similar to the nontransfected cells in the surroundings. The apoptotic response was associated with a decrease in viable -galactosidase-positive cells. In contrast, when cells were transfected with NF-B p65, transfected blue cells kept their normal cellular shape and did not undergo apoptosis, while the surrounding nontransfected cells died. This suggests that p65 rescues the cells from Yersinia-induced apoptosis. The yopP-negative mutant WA-314yopP was neither able to trigger cell death in transfected nor in nontransfected cells. To quantify the degree of apoptosis induced by the different Yersinia strains in transfected cells, the number of apoptotic, -galactosidase-expressing cells was determined in relation to the noninfected control (14, 15, 17, 18). As shown in Fig. 3B, transfection of the p65 expression vector resulted in marked inhibition of apoptosis induced by wild-type Yersinia, while transfection with the empty expression vector or transcriptionally inactive p50 did not provide considerable protection. These findings suggest that p65 can function as a suppressor of apoptosis induced by YopP-producing Y. enterocolitica. A similar result was obtained for IKK, because transfection with IKK, but not with IKK, was able to confer protection against YopP-mediated apoptosis (Fig. 3B).

View larger version (78K): [in this window] [in a new window]   FIGURE 3. NF-B protects against Yersinia-induced apoptosis. A, X-gal staining of transfected cells. J774A.1 cells were cotransfected with cDNA expressing -galactosidase and either empty expression vector (vector) or p65 expression vector (p65), thereafter infected with wild-type (WA-314) or yopP-negative Yersinia (WA-314yopP). After 8 h, cells were stained with X-gal. The arrows indicate transfected cells with apoptotic morphology. The results shown are from one representative experiment of five performed. B, Protection of J774A.1 cells from YopP-induced apoptosis by p65 and IKK. Cells were transfected with -galactosidase reporter vector and either empty expression vector or expression vectors for p50, p65, IKK, or IKK, infected with WA-314 or WA-314yopP, and stained with X-gal, as indicated above. To assess cell death of transfected cells, the number of apoptotic blue cells was determined. Data show mean percentages of dead cells of infected vs noninfected cells ± SD from three independent experiments.

LPS signaling enhances the capacity of YopP to trigger apoptosis in J774A.1 macrophages

To determine whether YopP is sufficient to induce apoptosis in the absence of bacteria, or whether a cofactor is required, we inserted yopPmyc into a eukaryotic expression vector under control of the CMV promoter. The resulting plasmid pYopP was transfected in J774A.1 macrophages, and morphology of transfected cells was assessed at single cell level. In cotransfection experiments with the -galactosidase-encoding reporter vector, YopP-transfected cells exhibited a heterogeneous morphology (Fig. 4A, middle). Some cells had normal appearance similar to cells transfected with the empty vector control. Other cells were remarkably shrunken and condensed, features that resemble characteristics of apoptosis (14, 15, 17, 18). Interestingly, the misshapen apoptotic phenotype of YopP-transfected macrophages was remarkably enhanced by subsequent treatment with LPS, resulting in almost complete destruction of YopP-transfected cells (Fig. 4A, right). To precisely characterize the process occurring in these macrophages, we specifically labeled apoptotic cells by the TUNEL reaction, which allows detection of apoptotic DNA breaks at the single cell level. TUNEL-positive cells show a typical condensed nuclear morphology by fluorescence microscopy (green fluorescence; Fig. 4B). Simultaneous application of anti--galactosidase Abs enabled distinct identification of transfected cells (red fluorescence; Fig. 4B). Individual YopP-transfected cells were also stained using an anti-c-myc Ab, but immunofluorescent labeling was less effective as compared with -galactosidase staining in cotransfected cells (data not shown). As anticipated, the apoptotic phenotype of YopP-transfected and LPS-challenged macrophages correlated with positive TUNEL staining in 80–90% of transfected cells. On the contrary, cells transfected with the empty control vector conserved their healthy appearance and became TUNEL positive in only less than 20%. This indicates that the morphological changes occurring in YopP-transfected and LPS-stimulated cells indeed reflect execution of apoptosis.

View larger version (100K): [in this window] [in a new window]   FIGURE 4. LPS signaling enhances YopP-induced apoptosis. A, X-gal staining of YopP-transfected cells. J774A.1 cells were cotransfected with -galactosidase reporter vector and either empty expression vector (vector) or YopP expression vector (YopP). Transfected cells were left untreated or exposed to LPS (10 µg/ml) for 8 h, thereafter stained with X-gal. The arrows indicate transfected cells with apoptotic morphology. B, Detection of YopP-induced and LPS-enhanced apoptosis by TUNEL staining. J774A.1 cells were cotransfected with -galactosidase reporter vector and either empty expression vector or YopP expression vector. Transfected cells were exposed to LPS (10 µg/ml) for 6 h. Thereafter, cells were processed for apoptosis-specific TUNEL staining (green fluorescence) and immunofluorescence staining using anti--galactosidase and rhodamine-conjugated secondary Abs to detect transfected cells (red fluorescence). The arrow indicates a YopP-transfected, TUNEL-positive cell. C, LPS signaling reinforces apoptosis due to YopP. J774A.1 cells were cotransfected with -galactosidase reporter vector and either empty expression vector or YopP expression vector. Transfected cells were left untreated or exposed to LPS (1 or 10 µg/ml) for 8 h in presence or absence of polymyxin B (5 µg/ml), as indicated. In some cases, the supernatant from YopP-transfected and LPS-treated cells (lane 6) was removed, divided in two aliquots, and reused to stimulate YopP-transfected cells (lanes 6a and 6b). To one of these aliquots polymyxin B (5 µg/ml) was added (lane 6b). Cells were stained with X-gal, and single transfected cells were analyzed for an apoptotic morphology. Results are expressed as mean percentages ± SD from three independent experiments. A and B, The results shown are from one representative experiment of three performed.

The NF-B pathway protects against apoptosis induced by YopP and LPS treatment

We analyzed nuclear translocation of NF-B by fluorescence microscopy using anti-NF-B p65 Abs. Fig. 5A displays the impact of YopP transfection on migration of endogenous p65 upon LPS treatment. The cells were cotransfected with the -galactosidase reporter vector and processed by immunofluorescent labeling with anti--galactosidase and anti-p65 Abs. In Fig. 5A, transfected cells are characterized by a bright fluorescence due to superimposition of red (-galactosidase) and green (p65) fluorescence. The location of endogenous p65 was assessed in transfected cells. In nonstimulated cells, the major amount of NF-B p65 was localized in the cytoplasm (Fig. 5A, left). Upon treatment with LPS, endogenous p65 moved to the nucleus and exhibited a nuclear staining pattern in cells transfected with the empty vector control (Fig. 5A, middle). In contrast, endogenous p65 was impeded in its ability to translocate to the nucleus in YopP-transfected cells, and p65 was predominantly detected in the cell cytoplasm (Fig. 5A, right). This is in agreement with the results obtained by EMSA in Yersinia-infected macrophages (Fig. 1), showing impairment of nuclear translocation of NF-B by YopP. In a second set of experiments, the cells were cotransfected with the NF-B p65 expression plasmid, and immunofluorescence staining was solely performed on NF-B p65 (Fig. 5B). Overexpressed p65 was strongly labeled in the transfected cells, whereas endogenous p65 stained only very weak. Thus, only the transfected, but no nontransfected cells are visible in Fig. 5B. Staining of p65 revealed that LPS treatment induced nuclear translocation of overexpressed p65 in both cells, either cotransfected with the empty vector control or the YopP expression vector (Fig. 5B, middle and right). In addition, in correlation with the infection experiments (Fig. 3), overexpressed p65 rescued the cells from undergoing apoptosis after YopP transfection (Fig. 5C, left panel). The protective effect of p65 held true under both conditions, with and without additional treatment with LPS. The protection was unique to p65, because neither p50 nor IKK provided survival (Fig. 5C, left panel). IKK only slightly attenuated apoptosis, implying that transfected YopP inhibits transfected IKK to a certain extent. The prevention of cell death by overexpressed p65 gives evidence that subversion of the NF-B signaling pathway is the major mechanism by which YopP triggers apoptosis in macrophages. Furthermore, this result confirms crucial importance of the NF-B pathway in mediating survival of macrophages when challenged with LPS. Comparable results as with transfected YopP were obtained with a phosphorylation-defective I-B mutant that inhibits release and nuclear translocation of NF-B (14, 15). As soon as NF-B activation was blocked by YopP (Fig. 5C, left panel) or by the I-B mutant (Fig. 5C, right panel), LPS stimulation triggered severe apoptosis in J774A.1 macrophages. This indicates that LPS exposure mediates macrophage cell death when activation of NF-B is impaired.

View larger version (76K): [in this window] [in a new window]   FIGURE 5. NF-B provides protection against YopP- and LPS-mediated apoptosis. A, Transfected YopP inhibits nuclear translocation of endogenous NF-B p65 in LPS-treated J774A.1 cells. J774A.1 macrophages were cotransfected with -galactosidase reporter vector and either empty expression vector or YopP expression vector. Transfected cells were left untreated or exposed to LPS (10 µg/ml, 60 min), then processed for double immunofluorescence staining using 1) rabbit anti--galactosidase and rhodamine-conjugated secondary Abs, to detect transfected cells, and 2) goat anti-p65 and fluorescein-conjugated secondary Abs, to detect location of endogenous p65. The arrows indicate transfected cells, exhibiting a bright fluorescence that results from superimposition of green (staining of endogenous p65) and red fluorescence (staining of -galactosidase). The nontransfected cells solely stained with anti-p65 Abs and thus exhibit a reduced fluorescence as compared with the transfected cells. B, Overexpressed NF-B p65 moves to the nucleus in YopP-transfected and LPS-treated cells. J774A.1 cells were cotransfected with p65 and either empty expression vector or YopP expression vector. Transfected cells were left untreated or exposed to LPS (10 µg/ml, 60 min), then processed for immunofluorescence staining using anti-p65 Abs to detect location of p65. The arrows indicate nuclear location of overexpressed p65 in cotransfected cells. C, NF-B p65 protects against YopP-induced and LPS-enhanced apoptosis. J774A.1 cells were cotransfected with -galactosidase reporter vector, YopP expression vector, and vectors for p50, p65, IKK, IKK, or empty expression vector (left panel), or cotransfected with -galactosidase reporter vector and empty vector control or phosphorylation-defective I-B (right panel). Transfected cells were left untreated or exposed to LPS (10 µg/ml) for 8 h, as indicated. Thereafter, cells were stained with X-gal, and single transfected cells were analyzed for an apoptotic morphology. Results are expressed as mean percentages ± SD from three independent experiments. A and B, The results shown are from one representative experiment of three performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Use of the yopP-encoding eukaryotic expression vector allowed us to dissect the mechanism by which YopP triggers macrophage apoptosis. As apoptosis of eukaryotic cells is an active process of cell suicide initiated by an activator signal, inhibition of a survival-mediating pathway, i.e., NF-B, alone may not explain occurrence of apoptosis (13, 14, 15, 16). It rather may additionally require an inducer of a death-promoting signal that activates the apoptotic pathway. In fact, inhibition of NF-B by Yersinia in epithelial HeLa cells is not sufficient to trigger apoptosis, unless they are subsequently treated with TNF- (11). Apparently, TNF- provides the appropriate death signal mediating apoptosis in HeLa cells when NF-B is inhibited. Transfection of J774A.1 cells with solely YopP induced a moderate, but significant degree of apoptosis (40–50% of transfected cells). This level was strongly enhanced by additional treatment with LPS, covering 80–90% of YopP-transfected cells. The synergistic effect was directly mediated by LPS-induced cell signaling, because LPS detoxification with polymyxin B and cell culture transfer experiments could not produce comparable results. In the same context, administration of TNF--neutralizing Abs together with LPS did not confer a protective effect. This rules out crucial involvement of TNF-, potentially released from LPS-treated macrophages in an autocrine manner, as apoptotic trigger. Together, this implies that LPS serves as a cofactor in mediating macrophage apoptosis by YopP. Apparently, YopP suppresses LPS-induced NF-B activation, and simultaneously exploits proapoptotic LPS signaling to efficiently kill the macrophage. In fact, Yersinia activates a multitude of LPS-responsive signaling pathways in macrophages (3, 11). Like TNF- in HeLa cells, LPS obviously activates death-inducing and -preventing pathways in macrophages the same time. When the antiapoptotic NF-B pathway is blocked by YopP, the cytotoxic pathways dominate, and the macrophage undergoes apoptosis. This points out initiation of a death-inducing signal by LPS at a level upstream from NF-B. Studies presently underway address implications of proximal LPS-signaling intermediates in the regulation of apoptotic signal networks due to LPS treatment.

In summary, our study provides new insights into the mechanism by which Y. enterocolitica triggers apoptosis in macrophages. By injection of YopP, Yersinia affects the signaling networks of a highly conserved host defense system. The NF-B signaling pathway is remarkably shared in vertebrates and invertebrates, and represents a key effector pathway of innate immunity. YopP binds to IKK and subverts IKK activities, which disrupts the NF-B pathway in macrophages. Together with the initiation of LPS signaling, originally thought to mount a protective response against the invading organism, the action of YopP compels the macrophage to undergo apoptosis. By that way, YopP exploits the intrinsic mechanisms of host self defense to establish a competitive advantage to a bacterial pathogen.

	Acknowledgments

 
We thank H. W. L. Ziegler-Heitbrock for p50 and p65 cDNAs; H. Nakano for IKK and IKK cDNAs; P. J. Nelson for phosphorylation-defective I-B cDNA; U. Siebenlist for GST/I-B cDNA; M. Hensel for Salmonella strains; and K. Brand, C. Fischer, G. Grassl, S. Fessele, and R. Haas for informative and technical help. We thank G. Pfaffinger for expert technical assistance, and C. Barz, M. Aepfelbacher, W. D. Hardt, R. Zumbihl, and C. Pelludat for constructive discussions.

	Footnotes

 
¹ This work was supported by grants from the Bundesministerium für Forschung und Technologie and the Deutsche Forschungsgemeinschaft (Grant DFG Ru788). Moreover, this work was supported by the German-French exchange program PROCOPE and by the Association pour la Recherche contre le Cancer (Grant ARC 5566).

² Address correspondence and reprint requests to Dr. Klaus Ruckdeschel, Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstr. 9a, 80336 Munich, Germany.

³ Abbreviations used in this paper: Yop, Yersinia outer protein; I-B, inhibitory protein that dissociates from NF-B; IKK, I-B kinase; MAPK, mitogen-activated protein kinase; PVDF, polyvinylidene difluoride; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside.

Received for publication July 11, 2000. Accepted for publication November 15, 2000.

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