Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

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-2␣ (TgIF2␣) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2␣ 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) eIF2␣ 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.A well characterized mechanism by which eukaryotic cells respond to environmental stress involves phosphorylation of eukaryotic initiation factor-2 (eIF2) 3 (1-3). The eIF2 combined with GTP delivers Met-tRNA i Met to the translational machinery during initiation of protein synthesis. In mammalian cells, four eIF2 kinases have been described that are each activated by unique stress arrangements. For example, in response to accumulation of malfolded protein in the lumen of the endoplasmic reticulum (so-called ER stress), PEK/Perk (EIF2KA3) phosphorylates the ␣ subunit of eIF2 at serine 51, causing this translation factor to become an inhibitor of its own guanine nucleotide exchange factor, eIF2B. The resulting repression in general translation prevents further synthesis of secretory proteins that would further overload the ER and allows cells sufficient time to trigger the unfolded protein response (UPR) (2). The UPR is a program of mRNA expression involving genes that function in the assembly and transport of secretory proteins (4). In addition to ER stress, three other 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).Very little research has been performed on eIF2 kinase and related stress response pathways in early-diverging eukaryotes, including pathogenic eukaryotes. However, viability, pathogenesis, and transmission of many parasites hinges on their ability to recogn...

Phosphorylation of eukaryotic initiation factor-2α promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

“…Ionophore A23187 elicits ER stress and, consistent with our previous study, this ionophore is a potent inducer of TgIF2α phosphorylation in WT parasites (Fig. 1C) (8). By comparison, phosphorylation is absent in the TgIF2α-S71A mutant parasites (Fig.…”

“…To confirm that the TgIF2α-S71A clone was unable to be phosphorylated, a Western blot of lysates from parasites treated with the ionophore A23187 was carried out using antisera that specifically recognize TgIF2α phosphorylated at Ser-71 or total TgIF2α protein (7,8). Ionophore A23187 elicits ER stress and, consistent with our previous study, this ionophore is a potent inducer of TgIF2α phosphorylation in WT parasites (Fig.…”

“…There are four eIF2 kinases encoded in the Toxoplasma genome, designated TgIF2K-A through -D. TgIF2K-A possesses a transmembrane domain and was found to be localized to the parasite ER (8). Upon treatment with ER stress agents, TgIF2K-A is released from its association with BiP, suggesting shared activation mechanisms with those described in yeast and mammals (14).…”

“…Four eIF2 kinases have been identified in mammals that phosphorylate eIF2α in response to stress (4,6): haem-regulated inhibitor (HRI) (EIF2KA1), which responds to heme deficiency and oxidation stress; double-stranded RNA-dependent protein kinase (PKR) (EIF2KA2), which is involved in antiviral defenses; pancreatic eIF2α kinase/PKR (RNA-dependent protein kinase)-like ER kinase (PEK/PERK) (EIF2KA3), which is activated by endoplasmic reticulum (ER) stress; and general control non-derepessible-2 (GCN2) (EIF2KA4), which responds to nutrient deprivation. We identified and characterized an eIF2α ortholog in Toxoplasma (TgIF2α), as well as four TgIF2α kinases (TgIF2K-A through -D) (7,8). Whereas TgIF2K-C and -D are most closely related to GCN2, TgIF2K-A is localized to the parasite ER and likely mediates the activation of the unfolded protein response analogous to PEK/ PERK (8).…”

“…We identified and characterized an eIF2α ortholog in Toxoplasma (TgIF2α), as well as four TgIF2α kinases (TgIF2K-A through -D) (7,8). Whereas TgIF2K-C and -D are most closely related to GCN2, TgIF2K-A is localized to the parasite ER and likely mediates the activation of the unfolded protein response analogous to PEK/ PERK (8). TgIF2K-B is a unique eIF2 kinase that is not compartmentalized and likely responds to cytoplasmic stresses.…”