Abstract
A key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2alpha (TgIF2alpha) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2alpha remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Furthermore, we have characterized novel eIF2 kinases; one in the endoplasmic reticulum and a likely regulator of the unfolded protein response (TgIF2K-A) and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2alpha phosphorylation is employed by cells to maintain a latent state, and 3) endoplasmic reticulum and cytoplasmic stress responses evolved in eukaryotic cells before the early diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.
Highlights
A well characterized mechanism by which eukaryotic cells respond to environmental stress involves phosphorylation of eukaryotic initiation factor-23 [1,2,3]
In addition to ER stress, three other eukaryotic initiation factor-2 (eIF2) kinases have been described that recognize different forms of cytoplasmic stress in mammalian cells. These include: GCN2 (EIF2KA4), which responds to nutrient deprivation and is well conserved among eukaryotes [3, 5], HRI (EIF2KA1), which is reported to be activated by heme deficiency, oxidative stress induced by arsenite treatment, and heat shock [6, 7], and PKR (EIF2KA2), which is involved in the antiviral defenses [8, 9]
Serine residue that is phosphorylated in 2B). These results support the idea that T. gondii activates response to alkaline pH or heat shock, stress conditions that the eIF2 kinase pathway in response to both ER and cytoplasinduce parasite differentiation to bradyzoites in vitro [11, 14]. mic stress arrangements
Summary
Parasite Lines and Culture Methods—T. gondii tachyzoites (RH and ME49 strains) were cultivated in confluent monolayers of human foreskin fibroblast cells. In experiments in which parasites were radiolabeled, equal numbers of extracellular tachyzoites were resuspended in labeling media, Dulbecco’s modified Eagle’s medium without L-methionine, L-cysteine, L-glutamine, or sodium pyruvate (Invitrogen #21013-024) supplemented with 5% fetal bovine serum, 1 mM L-glutamine, 0.5 mM sodium pyruvate. Parasites were collected by centrifugation, washed twice in PBS containing 50 g/ml cycloheximide, and resuspended in Breaking Solution (20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 10 mM MgCl2, 0.1% Triton, 50 g/ml cycloheximide, 0.04 units/l RNase Out, and 250 g/ml heparin) in the presence of protease inhibitors listed above. The parasites were lysed in 20 mM Tris (pH 7.6), 150 mM NaCl, and 0.5% Triton supplemented with protease inhibitors, and the suspension was briefly sonicated 3 times on ice. The lysates were precleared with protein-A-agarose (Invitrogen) followed by immunoprecipitation with affinitypurified TgIF2K-A or TgIF2K-B antibodies and protein-A-agarose for 16 h at 4 °C. Treatment of T. gondii with arsenite led to a ϳ60% reduction in protein synthesis compared with the mock-treated cells (Fig. 1C)
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