1 Departments of Pathobiology, University of Pennsylvania, Philadelphia PA 19104, USA
2 Departments of Biology, University of Pennsylvania, Philadelphia PA 19104, USA
3 Departments of Animal Biology, University of Pennsylvania, Philadelphia PA 19104, USA
4 Department Chemistry and Biochemistry, Signaling Systems Laboratory, University of California at San Diego, La Jolla, CA 92093-0375, USA
Author for correspondence (e-mail: chunter{at}phl.vet.upenn.edu)
Accepted 7 April 2005
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Summary |
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Key words: Toxoplasma gondii, NF-B, Immune regulation, Innate immunity, Host-pathogen interactions, Intracellular signaling
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Introduction |
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Because of the important role played by NF-B signaling in cell survival, innate recognition of microbial products and immunity to infection, this pathway exerts a strong selective pressure on infectious agents. Consequently, numerous parasites, bacteria and viruses have developed strategies to modulate this pathway to promote pathogen survival (Li, 2003
; Santoro et al., 2003
; Tato and Hunter, 2002
). Although the mechanisms utilized by pathogens to exploit NF-
B signaling are diverse, a common theme is the manipulation of I
B degradation. For example, Yersinia pestis inhibits the activation of NF-
B and limits proinflamatory responses by interfering with activation of IKKß and so prevents I
B degradation (Orth et al., 2000
). By contrast, Theileria sp. induce constitutive activation of the IKK signalosome, resulting in NF-
B activity and enhanced proliferation and survival of infected cells (Heussler et al., 2002
).
Toxoplasma gondii is an obligate intracellular parasite, and an important opportunistic pathogen of humans, particularly in patients with defects in T-cell function. This parasite is also a member of the ancient phylum Apicomplexa, which includes multiple pathogens responsible for considerable morbidity and mortality in humans and animals such as Plasmodium (the causative agent of malaria), Theileria and Cryptosposidium. Although previous studies have revealed an important role for NF-B in the development of innate and adaptive immunity to T. gondii (Caamano and Hunter, 2002
), there is evidence that T. gondii can interfere with host cell signaling in the cells it infects and that this represents a parasite strategy to evade the innate immune response (Goebel et al., 2001
; Luder et al., 1998
; Luder et al., 2001
; Shapira et al., 2004
). For example, cells infected with T. gondii are refractory to LPS-induced expression of pro-inflammatory genes such as TNF-
, i-NOS and IL-12, which are required for resistance to this parasite (Butcher et al., 2001
; Denkers, 2003
; Robben et al., 2004
; Shapira et al., 2004
; Shapira et al., 2002
). There are several reports that these genes are regulated by NF-
B (as well as by other pathways) and the failure of infected cells to produce these molecules is consistent with the lack of NF-
B activity in infected cells both in vitro and in vivo (Butcher et al., 2001
; Denkers, 2003
; Robben et al., 2004
; Shapira et al., 2004
; Shapira et al., 2002
). However, despite these studies, the point at which T. gondii interferes with the NF-
B pathway remains unclear (Butcher et al., 2001
; Denkers et al., 2003
; Shapira et al., 2004
; Shapira et al., 2002
).
The experiments described here were aimed at understanding the events that underlie the ability of T. gondii to terminate the NF-B pathway. These studies demonstrate that, despite IKK-dependent degradation of I
B
, infection of mammalian cells with T. gondii does not result in nuclear localization of NF-
B or the upregulation of NF-
B-dependent gene expression. However, this defect is not due to a parasite-mediated block in nuclear import, as general nuclear import and constitutive nuclear-cytoplasmic shuttling of NF-
B remain intact in infected cells. Rather, in T. gondii-infected cells, the termination of NF-
B signaling is associated with reduced phosphorylation of p65/RelA, an event involved in the ability of NF-
B to translocate to the nucleus and bind DNA. Although many pathogens regulate NF-
B by manipulating I
B degradation, the data presented here demonstrate for the first time that the phosphorylation of p65/RelA represents an event downstream of I
B degradation that can be exploited by pathogens to subvert NF-
B signaling.
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Materials and Methods |
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Cell culture and parasites
Human foreskin fibroblasts (HFF) (American Type Culture Collection) were maintained in complete Dulbecco's modified Eagle medium (DMEM, Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (Hyclone Laboratories), 2 mM glutamine, 1000 U/mL penicillin, 10 g/mL streptomycin, 0.25 mg/mL fungizone, 1 mM sodium pyruvate, 1% (vol/vol) nonessential amino acids, and 50 ng/mL ciprofloxacin (Life Technologies). Tachyzoites of the virulent RH strain (expressing RFP where indicated) were maintained in vitro by infection of HFFs and biweekly passage. Tachyzoites from freshly lysed fibroblast cultures were washed once with PBS and resuspended in DMEM for in vitro assays. Parasites were added to cells (10:1 ratio) and at time points indicated (resulting in an 80-90% infection rate), supernatants were removed, cells washed with ice cold PBS, and whole cell extracts prepared.
NF-B reporter cells
NF-B reporter cells were generated as follows: NIH 3T3 cells were transfected using FuGENE transfection reagent (Roche, Indianapolis, IN), with pNF-
B-hrGFP (Stratagene, La Jolla, CA) plasmid containing a GFP reporter driven by a basic promoter element (TATA box) joined by 5 tandem repeats of NF-
B binding elements. Hygromycin selection was used to generate a population of cells that were then used to generate clones by limiting dilution. Clones used for experiments were selected based on high GFP expression following TNF-
treatment and low GFP expression under resting conditions.
Microscopy
For indirect immunofluorescence, coverslips were fixed for 10 minutes in 4% paraformaldehyde, permeabilized for 10 minutes in 0.25% Triton-X 100, blocked for 1 hour in PBS (pH 7.4) + 3% BSA fraction V (Fisher), incubated for 1 hour with primary antibody (in blocking solution), washed, and incubated for 1 hour in secondary antibody. Secondary antibodies used were Alexa goat anti-mouse 488 (Molecular Probes; 1:500) and Alexa goat anti-rabbit 594 (Molecular Probes; 1:500). Finally, parasite and host nuclear DNA were stained with 49,6-diamidino-2-phenylindole (DAPI) for 5 minutes (in PBS), samples were washed in PBS, and mounted on glass slides using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) for examination using either a Zeiss Axiovert 35 equipped with a heated stage or a Leica DM IRBE equipped with a motorized filter wheel. Both inverted microscopes were equipped with motorized stage, 100W Hg-vapor lamp and Orca-ER digital camera (Hamamatsu, USA). Images were captured using Openlab 3.1 software (Improvision, Lexington, MA).
Analysis of the NF-B pathway
All immunoprecipitation (IP) and immuno-blot (IB) analysis was performed as described previously (Zhong et al., 1997). Cells were lysed in buffer containing 200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM DTT, and protease inhibitors (Roche). For in vitro kinase assays, GST-I
B was generated and assays were performed as previously described (Zhong et al., 1997
). Briefly, cells were stimulated with TNF-
(10 ng/ml), or infected for the times indicated, and whole cell lysates were prepared. Lysate proteins (approx. 500 µg) were immunoprecipitated with anti-IKK-
antibody (Santa Cruz Biotechnology), and the immunoprecipitates were assayed for kinase activity using 3 µg recombinant GST-I
B
(1-54) as a substrate as described (Ruland et al., 2001
). Electrophoretic mobility shift assays (EMSAs) were performed as described previously (Zhong et al., 1997
). Briefly, samples contained 10 µg of whole cell extracts and were incubated (15 min; 26°C) with 32P-labeled double-stranded oligonucleotides corresponding to the palindromic
B site (5'-GGGAATTCCC-3'), electrophoresed on a 5.5% polyacrylamide gel and then visualized by autoradiography. To asses phosphorylation of p65, HFF cells were labeled in vitro with 32Pi as previously described (Zhong et al., 1997
). Briefly, cells were labeled with 0.5 mCu 32Pi for 2 hours before stimulation for 15 minutes. The cells were lysed, p65 was immunoprecipitated and separated on SDS-PAGE, and the dried gel was exposed for autoradiography over night or 3 hours for total phosphorylation (unprecipitated whole cell extracts).
Microinjections
Alexa-488-BSA and Alexa-594-BSA were from Molecular Probes (Eugene, OR). NLS-containing peptides were generated by Bioworld (Dublin, Ohio), and were crosslinked to Alexa-BSA as previously described (Adam et al., 1990). A pressure injector system (World Precision Instruments, Sarasota, FL) was used to load Alexa-594-conjugated peptide into cells bathed in an external solution containing (in mM) 145 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 10 glucose, pH 7.3. Cells to be injected were visualized using DIC optics in a chamber mounted on the stage of an inverted fluorescence microscope (Leica DMIRBE). Microinjection pipettes were fabricated with a tip diameter of 0.5 µM from boroscillicate capillary glass using a horizontal pipette puller (Sutter Instruments, Novato, CA). Fluorescence images of peptide localization were acquired using an intensified CCD camera (XR Mega 10, Stanford photonics) attached to the microscope side port and images acquired using QED (Pittsburgh, PA) imaging software.
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Results |
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Infection with T. gondii does not induce NF-B dimers capable of binding DNA and abrogates phosphorylation of p65/RelA
A normal consequence of IB degradation is the liberation of NF-
B dimers in the cytosol that can bind to NF-
B consensus cites on DNA. To determine whether T. gondii-induced degradation of I
B yields active NF-
B dimers capable of binding DNA, electrophoretic mobility shift assays (EMSA) were performed on whole cell extracts from TNF-
treated or infected HFF cells. Whereas TNF-
stimulation led to increased levels of NF-
B binding activity (Fig. 5A, left), infection with T. gondii did not result in the expected appearance of NF-
B complexes capable of binding DNA (Fig. 5A, right; to view the entire gel, including unbound probe, see supplementary material Fig. S4). The lack of NF-
B activity in infected cells was not due to a reduction in cellular p65/RelA, as total levels of p65/RelA remained the same through the course of infection (Fig. 5B).
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Together with the observation that general nuclear import is intact in infected cells, the results described above indicate that T. gondii targets the ability of NF-B to accumulate in the nucleus and bind DNA. Although our understanding of the events that regulate the strength and duration of NF-
B transcriptional activity is in its infancy, recent evidence indicates that, in response to various stimuli, the phosphorylation of p65/RelA is critical in regulating DNA binding and subsequent transactivation (Chen and Greene, 2004
; Zhong et al., 2002
; Zhong et al., 1997
). Consistent with these observations, stimulation of HFF cells with TNF-
resulted in an increase in total levels of phosphorylated proteins and specific phosphorylation of p65/RelA (Fig. 5C). However, although infection with T. gondii also stimulated an increase in the total levels of phosphorylation in these cells, specific phosphorylation of p65/RelA was not detected (Fig. 5C). These findings represent the first identification of a specific step in the NF-
B signaling pathway that is deficient in cells infected with T. gondii.
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Discussion |
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Although much is known about the immune mechanisms that regulate resistance to T. gondii, it remains unclear how the initial interaction between the parasite and the host cell affects the development of protective immune responses. It is clear, however, that the development of these responses is dependent on various transcription factors including NF-B. Thus, the utilization of various NF-
B-knockout mice has identified critical roles for these transcription factors in resistance to T. gondii (Caamano et al., 1999
; Caamano et al., 2000
; Franzosa et al., 1998
). However, the observation that T. gondii fails to activate NF-
B has been controversial because of reports that infection of cells with T. gondii results in activation of this pathway at late time points, that this is required to prevent apoptosis of infected cells (Molestina et al., 2003
) and that less virulent strains of T. gondii can induce low levels of NF-
B nuclear translocation (Robben et al., 2004
). Although these results may represent differences between virulent (type I) and less virulent (type II) strains of T. gondii, we have not found any evidence for these observations (data herein and not shown). Furthermore, while Sibley and colleagues observed that the RH (type I) strain of T. gondii did not activate NF-
B (similar to the results reported here), they did observe that the less virulent (type II) Pruigniaud strain induced low levels of NF-
B that correlated with increased production of IL-12 (Robben et al., 2004
). However, it is important to note that, in the assays used in this report, infection with tachyzoites of Pruigniaud did not lead to nuclear accumulation or phosphorylation of p65 (data not shown). Nevertheless, although the reasons for these differences remain unclear, the data presented here are consistent with previous studies showing that infection with this parasite does not lead to the activation of NF-
B (Butcher and Denkers, 2002
; Butcher et al., 2001
; Luder et al., 2001
; Robben et al., 2004
; Shapira et al., 2002
). Also, one interpretation of these results is that the inhibition of NF-
B signaling by T. gondii may in fact protect from the pro-apoptotic signals mediated by this transcription factor. Moreover, since infected cells, in vitro or in vivo, have a reduced capacity to produce pro-inflammatory cytokines such as IL-12, it seems likely that the failure to activate NF-
B represents a strategy that promotes the early expansion and growth of this parasite.
While these findings identify p65/RelA phosphorylation as a novel target that can be manipulated by pathogens to limit NF-B-dependent signaling, they also highlight the role of this post-translational event in the regulation of DNA binding and suggest that this process may influence nuclear import and retention of NF-
B. Several phosphorylation sites as well as multiple kinases are required for p65/RelA activation, association with CBP/p300, and activation of gene transcription. For example, phosphorylation of p65/RelA at S536 is mediated by IKKß and/or IKK
and occurs in the cytoplasm, whereas phosphorylation of p65/Rel at S276 is regulated by PKA and cAMP (Mattioli et al., 2004
; Yang et al., 2003
; Zhong et al., 1997
). Furthermore, the phosphorylation status of p65/RelA is determined by both kinases and phosphotases. For example, recent evidence suggests that protein phosphatase 2A (PP2A) is physically associated with the p65/RelA-I
B complex and can dephosphorylate p65/RelA under appropriate conditions (Yang et al., 2001
). Signal-induced cytosolic p65/RelA phosphorylation provides a mechanism that ensures that only activated NF-
B can induce transcription, thereby maintaining NF-
B as an inducible transcription factor. Given the role of NF-
B signaling in the development of inflammatory and autoimmune disease, as well as cancer, much effort has been directed at the identification of drugs that can modulate this pathway. Elucidating the mechanism by which T. gondii interferes with the phosphorylation of p65/RelA will extend our understanding of the host-pathogen relationship, and should also yield novel insights into the molecular mechanisms of NF-
B signaling in mammalian cells and provide potential targets for drug design.
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Footnotes |
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* Present address: University of Vermont, Department of Microbiology and Molecular Genetics, Burlington, VT 05405, Canada
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