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First published online 9 September 2003
doi: 10.1242/jcs.00683

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Activation of NF-B by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IB to the parasitophorous vacuole membrane

¹ Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536, USA
² Infectious Diseases Section, Yale University School of Medicine, New Haven, CT 06520, USA

View larger version (48K): [in a new window]   Fig. 1. T. gondii infection induces nuclear translocation of NF-B. MEFs were incubated in medium alone (uninfected), treated with 20 ng ml–1 of TNF for 20 minutes or infected with T. gondii at an m.o.i. of 5:1 for 20 hours. Confocal immunofluorescence microscopy was performed with primary rabbit polyclonal antibodies against NF-B p50 (A) and p65 (B), and mouse monoclonal anti-T.-gondii SAG1. Proteins were localized using species-specific Oregon Green (NF-B) or Texas Red (SAG1) conjugated secondary antibodies. Uninfected cells displayed mainly cytoplasmic staining of p50 and p65 (A,B, top, respectively). Stimulation with TNF caused translocation of both subunits to the nucleus. Infection with T. gondii resulted in nuclear localization of p50 and p65 (A,B, middle, respectively). Uninfected cells in the same field failed to exhibit NF-B translocation. Treatment of the mixed population of uninfected and infected MEFs resulted in translocation of p50 and p65 in all cells (A,B, bottom, respectively). Scale bar, 20 µm. (C) NF-B translocation induced by T. gondii was confirmed in nuclear fractions of infected MEFs by immunoblotting. Cells were infected at increasing m.o.i. for 20 hours. Treatment of uninfected cells with TNF (20 ng ml–1, 20 minutes) was used as a positive control. Nuclear fractions were prepared as described in the Methods section and immunoblots were performed with polyclonal rabbit antibodies to p50 and p65. A dose-dependent increase in host cell nuclear p50 and p65 localization was observed in response to T. gondii infection.

View larger version (18K): [in a new window]   Fig. 2. The T.-gondii-mediated translocation of NF-B occurs early in infection. MEFs were infected at a m.o.i. of 5:1 for 1 hours, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours. Double immunofluorescence was performed with primary rabbit polyclonal antibodies against NF-B p50 (A) and p65 (B) and mouse monoclonal anti-T.-gondii SAG1. Translocation of p50 and p65 occurred as early as 1 hour p.i. (A,B, top, respectively). Scale bar, 20 µm. Single-cell analysis of NF-B translocation during the 6 hour incubation period showed significant differences in the proportions of infected cells displaying p50 and p65 nuclear staining compared with uninfected cells (A,B, bottom, respectively). Cells were counted in a minimum of 20 microscopic fields under 100x and numerical data was collected from three separate experiments. *, P<0.05.

View larger version (90K): [in a new window]   Fig. 3. T. gondii infection results in an increase in NF-B DNA binding activity. (A) Binding reactions for EMSAs were performed with a radiolabeled oligonucleotide probe containing the NF-B consensus sequence and nuclear protein extracts from uninfected, infected or TNF-treated wild-type (WT) MEFs. Antibodies to NF-B members p50, p65, RelB, c-Rel and p52 were used for supershift assays. An increase in NF-B binding activity was observed in infected MEFs and TNF-treated cells compared with uninfected controls. Supershifts of NF-B complexes confirmed the presence of p50/p65 heterodimers in infected and TNF-treated cells. Supershifts were also observed with antibodies to RelB and p52 in infected cells. (B) Analysis of NF-B translocation by EMSA in p65–/– MEFs infected with T. gondii. Nuclear extracts from uninfected, infected and TNF-treated p65–/– cells revealed similar levels of NF-B binding activity. Supershift assays of NF-B complexes showed the presence of p50/p50 homodimers in the three experimental conditions, but p52 was only observed in T.-gondii-infected cells. As expected, no supershift was detected with anti-p65 antibodies. Abbreviation: n.s., non-specific complex.

View larger version (29K): [in a new window]   Fig. 4. Expression of host cell genes involved in the regulation of apoptosis in response to T. gondii infection. (A) Wild-type (WT) and p65–/– MEFs were infected with T. gondii for 20 hours and total RNA was isolated as described in the Materials and Methods section. Radiolabeled cDNA probes were hybridized to nylon membranes of the Mouse Apoptosis GEArray Q Series, which consist of a panel of 96 genes associated with apoptosis and four housekeeping genes (Superarray). Signals corresponding to the cDNAs hybridized to each gene were detected by phosphorimaging. Integrated densitometric values were calculated from each spot and normalized to actin values. On the y axis, ratios between infected and mock-infected values for each gene were calculated to depict changes in gene expression after infection. Bars represent the means of three experiments corresponding to the 100 genes in the array, which were grouped into families on the x axis. The identity of the complete set of genes and the levels of expression as a result of infection are shown in Table 1. A comparative analysis of wild-type and p65–/– MEFs revealed distinct patterns of gene expression in response to T. gondii infection. (B) Expression of apoptosis-related genes belonging to the Bcl-2 and IAP families. Differences between WT (black bars) and p65–/– (white bars) infected MEFs were most evident in the expression of the Bcl-2 anti-apoptotic gene Bfl-1 (top) and the IAP members NAIP1 and IAP2 (bottom). Levels of expression of Bcl-2 proapoptotic genes were similar in both cell lines (middle). Bars denote ratios between infected and mock-infected gene expression levels and represent the means ± standard deviations of three separate experiments.

View larger version (58K): [in a new window]   Fig. 5. RT-PCR analysis of gene expression in response to T. gondii infection. Total RNA (1 µg) from mock-infected and infected MEFs was used in a RT-PCR reaction in the presence of [-32P]dCTP as described in Materials and Methods. Signals corresponding to the different PCR products were detected by phosphorimaging. Results depict a representative experiment of three performed.

View larger version (27K): [in a new window]   Fig. 6. Effects of T. gondii infection on the phosphorylation of IB. MEFs were infected at increasing m.o.i. for 20 hours. Treatment of uninfected cells with TNF (20 ng ml–1, 20 minutes) was used as a positive control. Immunoblots were performed with mouse monoclonal anti-P-IB, polyclonal rabbit anti-IB, and polyclonal rabbit anti-actin as described in the Materials and Methods section. (A) Elevated levels of P-IB were observed in infected MEFs but, unlike the TNF control, levels of IB were not decreased. (B) Treatment with 50 µM MG132 proteosome inhibitor for 2 hours prior to TNF stimulation caused an accumulation of P-IB and prevented degradation of the protein. In contrast to this, no apparent increase in P-IB accumulation was observed in T.-gondii-infected cells treated with 50 µM MG132 for 2 hours prior to immunoblot analysis.

View larger version (59K): [in a new window]   Fig. 7. Localization of P-IB to the T. gondii PVM. MEFs grown on glass coverslips were infected at a m.o.i. of 5:1 for 20 hours. Double immunofluorescence was performed with mouse monoclonal anti-P-IB (green) and mouse monoclonal anti-T.-gondii SAG1 (red). A distinctive pattern of P-IB was observed around the T. gondii PVM (A,C, white arrows). Uninfected cells (D, black arrows) displayed little, if any, cytoplasmic P-IB staining. Blocking experiments with a peptide corresponding to a short amino acid sequence containing phosphorylated Ser 32 of IB eliminated labeling by the anti-P-IB antibody but had no effect on the parasite marker SAG1 (E-G). Treatment with TNF resulted in high levels of P-IB in both infected and uninfected cells (I,K). Staining for P-IB was observed in the host cell cytoplasm, nucleus and around the PVM. Incubation with the blocking peptide resulted in the loss of P-IB staining both at the PVM and host cell (M-O). Scale bar, 20 µm.

View larger version (101K): [in a new window]   Fig. 8. Confirmation of P-IB localization to the T. gondii PVM by immunoelectron microscopy. Infected fibroblasts were labeled with rabbit anti-P-IB antibodies and 5 nm protein-A/gold particles. The reactivity of gold-labeled anti-P-IB antibodies was detected mainly around the PVM (circles), with no significant staining of the parasite being observed. Abbreviations: ER, host endoplasmic reticulum; M, host mitochondria; P, parasite; PVM, parasitophorous vacuolar membrane. The localization pattern of P-IB in the PVM of infected cells suggests a highly specific mechanism is involved in the activation of NF-B by T. gondii. Scale bars, 0.5 µm.