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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02428

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Initiation and termination of NF-B signaling by the intracellular protozoan parasite Toxoplasma gondii

Sagi Shapira¹, Omar S. Harb², Juan Margarit^1,*, Mariana Matrajt^2,*, Jerry Han³, Alexander Hoffmann⁴, Bruce Freedman³, Michael J. May³, David S. Roos² and Christopher A. Hunter^1,

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

View larger version (49K): [in a new window]   Fig. 1. Infection with T. gondii fails to induce NF-B-dependent gene expression and does not result in nuclear accumulation of NF-B. (A) Clonal 3T3 cells expressing GFP driven by a promoter containing NF-B binding sites were used to assess NF-B responsiveness during T. gondii infection. Cells were either left untreated (left panel), stimulated with TNF- (10 ng/ml) for 18 hours (middle panel), or infected with p30-RFP-expressing Rh parasites for 18 hours (right panel); parasites were generated as previously described (Striepen et al., 2001). Expression of GFP and RFP was assessed by flow cytometry using BD FACS Calibur and analyzed using Cell Quest (Becton Dickinson, Mountain View, CA). (B) Indirect immunofluorescence analysis of T. gondii-infected HFF cells demonstrates lack of nuclear accumulation of NF-B. Anti-p65 (red) staining reveals cytoplasmic localization in uninfected cells and nuclear accumulation in cells stimulated with TNF- for 1 hour. `DAPI' indicates staining of host cell and parasite nuclei (blue). Yellow triangles indicate parasite-containing vacuoles, and infection times are indicated in the top left corner of each panel. The absence of nuclear accumulation of NF-B in infected cells correlates with the lack of GFP expression in panel A.

View larger version (41K): [in a new window]   Fig. 2. Infection with T. gondii induces IKK-dependent degradation of IB. (A,B) Whole cell extracts from HFF cells (A) or IKK/ß dKO 3T3 cells (B) treated with TNF- (left panels), or infected with T. gondii (right panels) were prepared at the times indicated. To assess IKK activity, in vitro kinase assays were performed as previously described (Zhong et al., 1997) (A, top panels) and gels were Coomassie Blue stained to confirm equal loading (A, middle panels). Immunoblots with anti-IB antibody (A, bottom panels) were performed to assess total levels in treated cells.

View larger version (74K): [in a new window]   Fig. 3. The ability of T. gondii to prevent the nuclear accumulation of NF-B does not depend on IB family members. Indirect immunofluorescence analysis of T. gondii-infected 3T3 cells doubly deficient in IBß and IB demonstrates lack of nuclear accumulation of NF-B. Anti-p65 (red) staining reveals cytoplasmic localization in uninfected cells and nuclear accumulation in cells stimulated with TNF- for 1 hour. `DAPI' indicates staining of host cell and parasite nuclei (blue). Yellow triangles indicate parasite-containing vacuoles, and infection times are indicated in the top left corner of each panel. Similarly, nuclear accumulation was not observed following infection of IB/IBß or IB/IB dKO cells (data not shown). Yellow triangles indicate parasite-containing vacuoles, and infection times are indicated in the top left corner of each panel.

View larger version (71K): [in a new window]   Fig. 4. T. gondii does not inhibit general nuclear import mechanisms or interrupt the constitutive nucleo-cytoplasmic shuttling of NF-B. To address whether T. gondii regulates general nuclear import, infected HFFs were microinjected with SV40-NLS crosslinked to BSA (A) and assessed using real-time imaging. Microinjection of uninfected cells with this construct resulted in rapid accumulation in the nucleus, reaching nucleo-cytoplasmic equilibrium within 10 minutes following microinjection (data not shown) (A). Microinjection of T. gondii-infected cells with BSA-NLS complex. PV, parasitopharous vacuole; host cell nucleus indicated by arrow. (B) Treatment of uninfected cells (B, top panels) or infected cells (B, bottom panels; parasite containing vacuoles indicated by yellow triangles) with LMB for 4 hours resulted in the nuclear accumulation of p65/RelA.

View larger version (29K): [in a new window]   Fig. 5. Infection with T. gondii blocks the phosphorylation of p65/RelA and DNA binding. (A,B and C) Whole cell extracts from HFF cells treated with TNF- (left panels), or infected with T. gondii (right panels) were prepared at times indicated. EMSA was performed to determine whether the degradation of IB yields active NF-B dimers (A). (B) Immunoblots with anti-IB antibody (A, bottom panels) were performed to assess total levels in treated cells. (C) HFFs were labeled with 32Pi orthophosphate as previously described (Zhong et al., 1997) and were treated with TNF- or infected with T. gondii for 15 minutes. Whole cell extracts were either immunoprecipitated overnight with anti-p65 antibody to determine p65/RelA-specific phosphorylation (upper panel) or separated on SDS-PAGE to determine total cellular phosphorylation (lower panel).

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