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First published online 25 April 2006
doi: 10.1242/jcs.02934

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Toxoplasma gondii triggers G_i-dependent PI 3-kinase signaling required for inhibition of host cell apoptosis

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA

View larger version (36K): [in a new window]   Fig. 1. Toxoplasma induces MØ PKB/Akt activation during in vivo and in vitro infection. (A,B) mice were i.p. infected with 103 YFP-expressing RH tachyzoites. Eight days post-infection, spleen cells were isolated and subjected to staining with Ab to MØ marker F4/80 and phosphorylated-PKB (Ser473). (A) YFP expression in F4/80+ cells from infected animals. Inset shows lack of YFP signal in F4/80+ cells from non-infected mice. (B) Expression of phosphorylated-PKB (Ser473) in infected (YFP+, thick line) and non-infected (YFP-, thin line) F4/80+ cells from infected mice described in A. Dotted line, isotype control. (C) Bone-marrow-derived MØs were infected with RH strain tachyzoites. Then, 15 minutes later, cells were collected for flow cytometric analysis. Cells were permeabilized and subjected to Ab staining for the tachyzoite surface protein p30 (SAG-1). The (a) non-infected and (b) infected populations (percentages are indicated) were examined for PKB expression (thick lines) relative to unstimulated cells (dashed lines). Thin lines in a and b show staining of secondary antibody alone on the total MØ population. Infected and non-infected cells gave the same level of background staining (data not shown). These experiments were repeated twice with the same result.

View larger version (86K): [in a new window]   Fig. 2. Requirement for PI 3-kinase signaling in PKB and MAPK activation differs for T. gondii infection and LPS stimulation. MØs were preincubated 2 hours with PI 3-kinase inhibitor wortmannin (WM, 50 ng/ml) or solvent (DMSO) alone (0), then infected with T. gondii (6:1 ratio of parasites to cells; >90% infected) or stimulated with LPS (100 ng/ml). At the indicated time points, cells were collected for western blot analysis for total and phosphorylated forms of each signaling intermediate. This experiment was performed three times with the same result.

View larger version (88K): [in a new window]   Fig. 3. Signaling mediated by Gi protein-coupled receptors is required for Toxoplasma-induced PKB and ERK1/2, but not p38 MAPK, activation. MØs were set up as described in Fig. 2, after 2 hours preincubation with pertussis toxin (PTx, 50 ng/ml) or solvent (H2O) alone (0) followed by infection with T. gondii (6:1 ratio of parasites to cells) or stimulation with LPS (100 ng/ml). At the indicated time points, cells were collected for western blot analysis measuring total and phosphorylated form of each signaling intermediate. This experiment was repeated three times with essentially identical results.

View larger version (101K): [in a new window]   Fig. 4. MAPK and PKB activation induced by T. gondii and LPS are CCR5 independent. MØs from wild-type (WT) and CCR5 knockout (CCR5 KO) mice were incubated with parasites (6:1 ratio of tachyzoites to cells) or LPS (100 ng/ml), and at the indicated time points, samples were subjected to SDS-PAGE followed by western blotting with antibodies against total and phosphorylated forms of PKB, ERK1/2 and p38 MAPK. The experiment was repeated twice with the same result.

View larger version (101K): [in a new window]   Fig. 5. T. gondii activates ERK1/2 and PKB independently of MyD88, unlike LPS. MØs from wild-type (WT) and MyD88 knockout (KO) mice were incubated with parasites (6:1 ratio of tachyzoites to cells, >90% infected) or LPS (100 ng/ml), and at the indicated time points, samples were collected for western blot analysis measuring total and phosphorylated forms of each signaling intermediate. This experiment was repeated 4 times with the same result.

View larger version (13K): [in a new window]   Fig. 6. Pathway of T. gondii-induced PKB and ERK1/2 activation in MØs. T. gondii infection activated PI 3-kinase through Gi-protein-coupled receptor signaling. In turn, PI 3-kinase stimulates PKB activation and MEK1/2 phosphorylation leading to ERK1/2 activation. Pertussis toxin, which uncouples Gi-protein signaling, and wortmannin and LY294002, which inhibit PI 3-kinase activation, block PKB/Akt and ERK1/2 activation. PTx, pertussis toxin; WM, wortmannin; Ly, LY294002.

View larger version (93K): [in a new window]   Fig. 7. T. gondii infection renders cells resistant to apoptosis. (A-E') MØs were serum-starved for 18 hours, infected with Toxoplasma; 2 hours later staurosporine (1 µM) was added to cell cultures. After 18 hours, cells were subjected to TUNEL-FITC (green) and anti-p30 (SAG-1) Alexa Fluor 594 (red) staining, nuclei were stained with DAPI (blue). (A) Non-infected cell showing DAPI-stained nucleus. (A') Same cell as in A, showing TUNEL- and nuclei-staining (green and blue, respectively, appearing as turquoise). (B-E) Cells from the same population. Examples of infected cells, showing parasite-staining (red) and lack of TUNEL-stained nuclei (therefore remaining blue). Arrows point to tachyzoites in an intracellular location. (F) Quantitation of TUNEL-staining-positive cells. Med, cultured in medium alone; STS, cells cultured with staurosporine; I/STS, infected cells subjected to STS treatment; NI/STS, non-infected cells cultured with STS. *P<0.05; 100 cells were counted in each sample. Bars, 5 µm. This experiment was performed three times with the same result.

View larger version (23K): [in a new window]   Fig. 8. PI 3-kinase inhibition reverses the anti-apoptotic effect of Toxoplasma infection. (A), MØs were serum-starved for 18 hours after which wortmannin (WM), LY294002 (Ly) or solvent alone (DMSO) were added. After 2 hours, cells were infected (6:1 ratio of parasites to cells, >90% infection) or left non-infected in medium (Med). Then, after another 2 hours, staurosporine (STS) was added and 4 hours later cell lysates were prepared for analysis of apoptosis. PARP cleavage, which is indicative of apoptosis, was measured in PARP-specific western blot assays. Apoptotic PARP cleavage generates 89 kDa and 24 kDa fragments (not shown in the blot) from the intact 214 kDa molecule. (B) Cultures were initiated in the presence or absence of WM as described in A. Caspase activity was measured in an ELISA-based assay. Med, cells in medium alone; M+S, cells cultured with staurosporine; Tg, cells infected with T. gondii. Tg+S, cells infected and then triggered with staurosporine. Number above the `Tg+S' group (white bar) indicates percent inhibition relative to the `M+S' group. *P<0.05; ns, not statistically significant. These experiments were performed three times with the same result.

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