Yonsei Med J.  2006 Dec;47(6):862-869. 10.3349/ymj.2006.47.6.862.

Toxoplasma gondi Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-kappaB Activation

• Kim JY
• Ahn MH
• Jun HS
• Jung JW
• Ryu JS
• Min DY

• KMID: 1777177
• DOI: http://doi.org/10.3349/ymj.2006.47.6.862

Abstract

Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracellular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/ 10% FBS at 37(i)E, in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-kappaB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respectively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-kappaB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis- associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-kappaB activation in mouse spleen cells.

Keyword

Toxoplasma; apoptosis; Bcl-2; family; caspase; NF-kappaB; TEM

MeSH Terms

bcl-2-Associated X Protein/metabolism
Toxoplasma/*physiology
RNA, Messenger/metabolism
Poly(ADP-ribose) Polymerases/antagonists & inhibitors
NF-kappa B/*metabolism
Mice
Gene Expression Regulation
Flow Cytometry
DNA Fragmentation
Cells, Cultured
Caspase 3/*antagonists & inhibitors
Apoptosis/*physiology
Animals

Figure

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  Fig. 1 Fragmentation patterns of DNA from mouse spleen cells treated with actinomycin-D and/or T. gondii for 18 h. Fragmentation of DNA was down regulated in cells treated with AD/T. gondii (lane 6). Spleen cells infected with T. gondii exhibited only weak DNA fragmentation (lane 5). Lane 1. spleen cells treated with actinomycin-D (5 µg/mL); 2. spleen cells treated with actinomycin-D (10 µg/mL); 3. normal spleen cells for 18h incubation; 4. fresh normal spleen cells; 5. T. gondii infected spleen cells (cell:parasite = 1:10); 6. spleen cells infected with T. gondii and treated with actinomycin-D (5 µg/mL). In repeated experiments, similar results were shown.

• 

  Fig. 2 Flow cytometric analysis of apoptosis in mouse spleen cells treated with actinomycin-D and/or T. gondii for 18 h. (%) indicates level of apoptosis. (A) actinomycin-D (5 µg/mL), 80.3%; (B) actinomycin-D (10 µg/mL), 81.2%; (C) normal spleen cells for 18h incubation, 39.6%; (D) freshly isolated spleen cells, 23.8%; (E) T. gondii infected, 18.2%; (F) T. gondii and actinomycin-D treated spleen cells, 30.1%. Flow cytometry was repeated more than three times and similar results were observed.

• 

  Fig. 3 RT-PCR analysis of Bcl-2 family genes in mouse spleen cells. Expression of pro-apoptotic Bax mRNA was down regulated and levels of anti-apoptotic Bcl-2 and Bcl-XL mRNA remained constant in spleen cells treated with AD/T. gondii. Spleen cells were co-cultured with either actinomycin-D and/or T. gondii for 30 min, 60 min, or 120 min followed by extraction of total cellular RNA.. (M): marker; (-): negative; (AD): actinomycin-D (5 µg/mL); (AD + T.g): actinomycin-D + T. gondii infection.

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  Fig. 4 Western blot analysis of Bcl-2 family proteins, caspase 3, caspase 8 and PARP in mouse spleen cells in the presence or absence of T. gondii and actinomycin-D after incubation for 0 min, 60 min or 120 min. Production of Bax protein was decreased in spleen cells treated with AD/T. gondii, while levels of Bak protein was unchanged. Down regulation of cleaved caspase 3 at 120 min post infection and reduced production of PARP in T. gondii infected cells was detected by Western blotting. (AD): actinomycin-D (5 µg/mL); (AD + T.g): actinomycin-D + T. gondii infection.

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  Fig. 5 Increased NF-κB and decreased IκBα of mouse spleen cells by T. gondii infection was observed. (A) NF-κB activity was apparent at 120 min in spleen cells treated with AD and infected with T. gondii on EMSA. (B) Decreased cytosolic IκBα, pNF-κB and increased nuclear NF-κB proteins were detected in spleen cells treated with AD/T. gondii by Western blot. (+): TNF-α (20 ng/mL); (AD): actinomycin-D; (AD + T. gondii): actinomycin-D + T. gondii; (CP): cytosolic proteins; (NP): nuclear proteins.

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  Fig. 6 Ultrastructural morphology of mouse spleen cells treated with actinomycin-D and T. gondii after cultivation in vitro for 6h. Condensation of nuclear chromatin and fragmentation of the nucleus were observed in spleen cells treated with AD (A & B); however, these events were not observed in cells treated with AD/T. gondii tachyzoites (C & D) or T. gondii only(E). A, B: Spleen cells treated AD (5 µg/mL); C, D: Spleen cells treated with AD and T. gondii; E: Spleen cells treated with T. gondii; F: Normal spleen cells. A, B, E and F: 6000 ×, C: 7000 ×, D: 4000 ×, Bar = 2 µm. The arrows and arrow heads indicate apoptotic spleen cells (A & B) and T. gondii tachyzoites, respectively (C, D & E).

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