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First published online December 9, 2005
doi: 10.1242/10.1242/jcs.02709

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Host and parasite-derived IKK activities direct distinct temporal phases of NF-B activation and target gene expression following Toxoplasma gondii infection

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536, USA

View larger version (74K): [in a new window]   Fig. 1. Kinetics of phospho-IB localization at the T. gondii PVM as an indicator of TgIKK activity. (A-F) IKK–/–ß–/– MEFs were cultured on glass coverslips and infected with freshly passaged T. gondii tachyzoites at an m.o.i. of 5:1 for the times indicated. Double immunofluorescence was performed with mouse monoclonal anti-phospho-IB Ser32 antibody (P-IB; green) and rabbit polyclonal anti-T. gondii GRA3 antibody (red). Different levels of phospho-IB coverage at the PVM allowed the classification of vacuoles into three groups: group I showed 0-25% of the PVM covered with phospho-IB (yellow arrows), group II displayed 50-75% coverage (orange arrows), and group III showed 75-100% of their surface covered with phospho-IB (blue arrows). Bars, 6 µm. (G-J) Quantification of the different populations of vacuoles indicated a time-dependent increase in phospho-IB localization at the PVM. WT, IKK–/–, IKKß–/– and IKK–/–ß–/– MEFs were cultured on glass coverslips and infected with T. gondii at an m.o.i. of 5:1 for the times indicated. A minimum of 300 vacuoles were counted under 100x magnification in a blinded fashion for each cell line and time point. Data represent means ± s.d. of three separate experiments individually quantified by both authors.

View larger version (47K): [in a new window]   Fig. 2. Kinetic analysis of IB phosphorylation in total cell populations reveals a requirement for host IKK. WT, IKK–/–, IKKß–/– and IKK–/–ß–/– MEFs were infected at an m.o.i. of 5:1 for the times indicated. Immunoblots were performed with antibodies against phospho-IB (Ser32) and IB as described in the Materials and Methods. Elevated levels of phospho-IB (P-IB) were apparent by 1 hour p.i. in WT (A) and IKK–/– (B) cells. Phosphorylation of IB was maximal at 9 hours p.i. (A,B), which is concurrent with the increase in TgIKK activity at the PVM observed in Fig. 1. Contrary to this, only a slight elevation in IB phosphorylation was detected in IKKß–/– cells (C) and a lack of phosphorylation was observed in IKK–/–ß–/– cells (D). The levels of IB in all cell lines examined were comparable between uninfected (0 hour time point) and T. gondii-infected cells.

View larger version (28K): [in a new window]   Fig. 3. A disruption in the integrity of the host IKK complex causes defective NF-B translocation in response to infection. Nuclear extracts were prepared from WT, IKK–/–, IKKß–/– and double-knockout IKK–/–ß–/– MEFs after different periods of infection with T. gondii. Cells were infected at an m.o.i. of 5:1. Translocation of p50 (A) and p65 (C) was examined by immunoblotting, and densitometric analysis of corresponding protein bands (B,D) was performed as described in the Materials and Methods. The kinetics of p50 (A,B) and p65 (C,D) translocation in WT cells paralleled the robust response seen with the phosphorylation of IB shown in Fig. 2. Contrary to this, only moderate increases in p50 and p65 translocation were observed in all IKK mutant cell lines at early stages of infection. IDV, integrated densitometric value.

View larger version (82K): [in a new window]   Fig. 4. IFA analysis of NF-B localization in WT, IKK–/–, IKKß–/– and IKK–/–ß–/– MEFs infected with T. gondii for 24 hours. Double immunofluorescence labeling was performed with antibodies against p50 (A) and p65 (B), and anti-T. gondii SAG1. Translocation of p50 and p65 is observed in infected WT, IKK–/– and IKKß–/– cells but not in uninfected cells. A proportion of infected IKKß–/– cells fails to show p65 translocation at a similar extent as WT and IKK–/– cells (yellow arrow). Infected IKK–/–ß–/– cells do not display nuclear localization of p50 or p65. Bars, 20 µm.

View larger version (50K): [in a new window]   Fig. 5. T. gondii infection results in different levels of NF-B DNA binding activity depending on the IKK background of the host cell. (A) Binding reactions for EMSA were performed with a radiolabeled oligonucleotide probe containing the NF-B consensus sequence and nuclear protein extracts from uninfected (U) or infected (I) cells. Diverse NF-B binding activities of three complexes (C1, C2 and C3) are observed among WT, IKK–/–, IKKß–/– and IKK–/–ß–/– cells with infection. Increases in binding activity of complexes C2 and C3 are observed in infected WT and, to a lesser extent, in IKK–/– cells compared with uninfected controls (dashed box). Levels of NF-B binding activity of C2 and C3 complexes are minimal in infected IKKß–/– cells and absent in infected IKK–/–ß–/– cells. (B) Supershift analysis of infected WT, IKK–/– and IKKß–/– cells determined the presence of p50/p65 and p50/p50 dimers in the C2 and C3 complexes, respectively (arrowheads). The disappearance or marked reduction of C1 with the p50 but not the p65 antibody suggests the presence of p50 subunits in this complex, which might also consist of additional unidentified proteins.

View larger version (40K): [in a new window]   Fig. 6. Modulation of host cell genes involved in the regulation of the NF-B pathway by T. gondii. Densitometric values were normalized to actin, and levels of gene expression are shown as infected/uninfected ratios (see color key showing relative fold change). Data represent the means of three experiments performed separately. The complete data sets with means ± s.d. are shown in Table S1 (supplementary material). Target genes of NF-B are shown in bold. Pos. indicates position in the array.

View larger version (26K): [in a new window]   Fig. 7. Temporal analysis of gene expression by T. gondii infection. WT, IKK–/–, IKKß–/– and IKK–/–ß–/– MEFs were infected with T. gondii at an m.o.i. of 5:1 and total RNA was isolated at the time points indicated. Expression of IL-6 (A), GRO1 (B) and IAP-2 (C) was determined by real-time RT-PCR analysis as described in the Materials and Methods. Infection of WT cells resulted in fluctuating patterns of IL-6 (A) and GRO1 (B) activation; these fell into two major phases determined by an early host IKK response (1-3 hours) and an intermediate-to-late TgIKK response (9-24 hours). Contrary to this, a marked decrease of IL-6 and GRO1 expression was observed in IKK-knockout cells. A disruption of the IKK complex did not abrogate the expression of IAP2 to the same extent as observed with IL-6 and GRO1 (C), thus the temporal analysis of IAP2 lacked the characteristic biphasic response observed with early response genes. *, P<0.05 when comparing the levels of expression at the 9 hours time point of WT cells to all other time points in IKK–/–, IKKß–/– and IKK–/–ß–/– cells.

View larger version (29K): [in a new window]   Fig. 8. A model representing the modulation of NF-B-dependent gene expression by T. gondii. (A) Early in infection, the parasite triggers activation of the host IKK complex, resulting in a primary wave of NF-B translocation and induction of gene expression (early phase). An increase in TgIKK activity at the PVM phosphorylates a subset of IB molecules, inducing a secondary wave of NF-B activation (intermediate phase). The extent of this response is abrogated owing to a disruption in the host IKK signalosome. The magnitude of the NF-B transcriptional response might also depend on an amplification loop through autocrine effectors (i.e. TNF-, IL-1), resulting in sustained levels of gene expression at late stages of infection (late phase). (B) Signals deriving from the host IKK signalosome and TgIKK affect the strength and duration of the NF-B-dependent transcriptional response. Levels of gene expression shown in the blue line are for representation purposes only and do not reflect actual quantitative data. The regulation of specific NF-B target genes displays oscillatory patterns early in infection (blue line, early phase). An increase in TgIKK activity at the PVM (green dashed line) causes an activation of the response during mid-infection, resulting in sustained levels of NF-B-dependent gene expression (blue line, intermediate-to-late phases). An impaired IKK complex in the host cell abrogates this response significantly owing to a failure to attain an `activation threshold' (black dashed line) required by TgIKK for the induction of sustained levels of NF-B activation (orange line).

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