Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species

Abstract

Entamoeba histolytica is an enteric pathogen responsible for amoebic dysentery and liver abscess. It alternates between the host-restricted trophozoite form and the infective environmentally-stable cyst stage. Throughout its lifecycle E. histolytica experiences stress, in part, from host immune pressure. Conversion to cysts is presumed to be a stress-response. In other systems, stress induces phosphorylation of a serine residue on eukaryotic translation initiation factor-2α (eIF2α). This inhibits eIF2α activity resulting in a general decline in protein synthesis. Genomic data reveal that E. histolytica possesses eIF2α (EheIF2α) with a conserved phosphorylatable serine at position 59 (Ser59). Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed cells to different stress conditions and measured the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildtype or EheIF2α-S59D exhibited increased or decreased translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced translation. Cells expressing EheIF2α-S59D were more resistant to long-term serum starvation underscoring the significance of EheIF2α phosphorylation in managing stress. Finally, phospho-eIF2α accumulated during encystation in E. invadens, a model encystation system. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in Entamoeba species.

Flow cytometric characterization of encystation in Entamoeba invadens

“…E. histolytica does not readily encyst in axenic culture; thus, less is known about encystation than invasive disease. Therefore, E. invadens , a reptilian parasite that encysts in vitro , has been used as a model [3–7]. E. invadens exhibits efficient stage conversion in culture after simultaneous reduction of osmolarity, glucose, and serum in the medium [6].…”

“…The latter is complemented by the published transcriptome of encysting E. invadens cells [3]. Another method of tracking encystation efficiency is incubation of cells in detergent, such as sarkosyl, followed by manual counting using a hemacytometer [7]. Since detergent lyses trophozoites and immature cysts (pre-cysts), the surviving detergent-resistant cells (DRC) are thought to be terminally-differentiated mature cysts.…”

“…E. invadens trophozoites were incubated in nutrient-rich medium (control) or encystation medium as described [6,7]. Cells were collected over time, stained with the fluorescent chitin stain, Congo Red [18], fixed, and analyzed by flow cytometry by collecting ten-thousand individual events.…”

Translational Control in the Latency of Apicomplexan Parasites

“…It is notable that eIF2 phosphorylation and mRNP granules accompany differentiation in the kinetoplastids such as Trypanosoma cruzi and Leishmania infantum , and eIF2 phosphorylation has also been implicated in Entamoeba spp. encystation [89,107–111]. Key priorities for the future include the identification of downstream ISR effectors in these parasites as well as determining which kinases and naturally occurring environmental signals mediate each specific developmental transition (see Outstanding Questions).…”

The combination of Radix Astragali and Radix Angelicae Sinensis attenuates the IFN-γ-induced immune destruction of hematopoiesis in bone marrow cells

“…The activation of eIF2 can downregulate the protein synthesis of BM cells under condition of several stresses [29]. The levels of eIF2α and phospho-eIF2α increased significantly compared with those of the normal group (P < 0.01 or P < 0.05, respectively).…”