Date of Award
Master of Science (MS)
Dr. Lesly Temesvari, Committee Chair
Dr. Lisa Bain
Dr. Lukasz Kozubowski
Entamoeba histolytica is a food- and water-borne intestinal parasite responsible for amoebic dysentery and amoebic liver abscess. The life cycle of E. histolytica alternates between the host-restricted trophozoite form and the highly infective latent cyst stage that is able to persist in the environment. Throughout its life cycle, which may include invasion of tissues in the human host, the parasite is subjected to a variety of stressful conditions. In other systems, stress can trigger the activation of kinases that phosphorylate a serine residue on eukaryotic translation initiation factor-2Î± (eIF2Î±). This modification inhibits the activity of eIF2 resulting in a general decline in protein synthesis, and, paradoxically, an up-regulation of the expression of certain genes that permit the cell to counter the stress. Genomic data reveal that E. histolytica possesses eIF2Î± with a conserved phosphorylatable serine at position 59. Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed E. histolytica trophozoites to six different stress conditions and assessed viability, as well as the level of total and phospho-EheIF2Î± via Western blot of cell lysates. Long term serum starvation induced an increase in the level of phospho-EheIF2Î±, but no other stress condition caused a significant change. Long term serum starvation also showed a decrease in polyribosome abundance as observed through sucrose gradient ultracentrifugation; this is consistent with the observation that this condition also induces phosphorylation of EheIF2Î±. This suggests that the eIF2Î±-dependent stress response system is operational in E. histolytica and that the system may be activated only by certain stresses. To further examine the role of phosphorylation of EheIF2Î± during stress, three transgenic cell lines were created. EheIF2Î±-S59 over-expresses wild type eIF2Î± protein. EheIF2Î±-S59A expresses eIF2Î± with the serine-59 residue mutated to an alanine, creating a non-phosphorylatable subunit. EheIF2Î±-S59D expresses eIF2Î± with the serine-59 residue mutated to an aspartic acid to mimic a phosphorylated residue. EheIF2Î±-S59 exhibited a high level of phosphorylation of the exogenous protein, leading to a decreased growth and polyribosome abundance when compared to the control cell line. EheIF2Î±-S59A had the highest growth rate and retained a high abundance of polyribosome. EheIF2Î±-S59D exhibited the slowest growth rate and had a decrease in polyribosome when compared to control; however, EheIF2Î±-S59D did exhibit the highest survival rate in over half the stress conditions tested. This may indicate the protective nature of phosphorylation of EheIF2Î± during times of stress.
Hendrick, Holland M., "Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Regulates Stress in the Human Protozoan Parasite Entamoeba Histolytica" (2016). All Theses. 2340.