The intracellular parasite Theileria parva protects infected T cells from apoptosis

Abstract

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.

Figure 1

The inhibitors of IκB phosphorylation TPCK and BAY 11–7082 block NF-κB activation and induce apoptosis in T. parva-transformed T cells. (A) TPCK and BAY 11–7082 inhibit IκBα phosphorylation Whole-cell protein extracts were prepared from cells treated with TPCK (25 μM) or BAY 11–7082 (50 μM) for different lengths of time, and immunoblot analysis was carried out by using anti-IκBα antibodies. (B) T. parva-transformed cells were transfected with the −121/+232 HIV-CAT construct and cultured in the presence or absence of TPCK and CAT activity determined after 16 h. Error bars represent the SD for three independent experiments. (C) Logarithmically growing T. parva-infected T cells were treated with TPCK (25 μM) or BAY 11–7082 (50 μM) for 5 h, and annexin-V-FITC binding was analyzed by FACScan.

Figure 2

The inhibition of NF-κB DNA binding activity correlates with the induction of apoptosis. Cells were treated with TPCK (25 μM) for different times as indicated. NF-κB complexes present in nuclear extracts were demonstrated by electrophoretic mobility-shift assay (Upper) and quantitated by PhosphorImager analysis (Lower, gray bars). Cells also were analyzed by FACScan, and the percentage of annexin-V-FITC binding cells were determined at each time point (Lower).

Figure 3

Dominant negative mutant forms of IκBα and p65 inhibit NF-κB transcriptional activity in T. parva-transformed T cells. CAT activity was monitored in cells that were cotransfected with −121/+232 HIV-CAT (10 μg) and either the empty expression vectors pcDNA3 and pCMV, or the expression vectors pcDNA3-I^S32A or pCMV-p65ΔC (40 μg each). Data are presented as percentage of CAT activity measured for cells transfected with the corresponding control plasmid constructs. Error bars represent the SD for three independent experiments.

Figure 4

Analysis of GFP expression in T. parva-transformed T cells undergoing apoptosis. Cells were transfected with the plasmid pCMV-GFPsg25 (50 μg) and treated for 5 h with TPCK as indicated. GFP expression was monitored in a counting chamber by using a fluorescence microscope. Error bars represent the SD for three independent experiments.

Figure 5

Expression of IκBαS32A or p65(Δ)C induces apoptosis in T. parva-transformed T cells. (A) Immunofluorescence micrographs (final magnification ×100) of cells cotransfected with pCMV-GFPsg25 and either the empty expression vectors pCDNA3 and pCMV, or vectors expressing IκBαS32A or p65(Δ)C. (B) Quantitation of GFP-expressing cells. T. parva-transformed T cells were transfected as described above, and the number of cells expressing GFP was monitored in a counting chamber by using a fluorescence microscope. Error bars represent the SD for three separate experiments.