Stress Induced Control of Protein Translation in Entamoeba Histolytica

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DESCRIPTION (provided by applicant): Entamoeba histolytica is the causative agent of amoebic dysentery and is classified as a Category B bioterrorism agent. E. histolytica infections are contracted by ingestion of latent cysts from fecally contaminated food or water. Amoeboid trophozoites emerge in the small intestine and subsequently move to the large bowel. The parasite may also cross the gut epithelial layer and establish extra-intestinal infections, the mos common of which is amoebic liver abscess. During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments (e.g., small intestine, large intestine, circulation, liver) and by the host immune response. To survive, the parasite must circumvent these exogenous pressures. Thus, it may be useful to target E. histolytica's response to stress for therapy. In other parasites, stress can activate eIF2α kinases that phosphorylate the α-subunit of eukaryotic initiation factor-2 (eIF2α). eIF2α is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2α inhibits this activity which, in turn, leads to a decline in protein synthesis. This allows clls to conserve resources and reconfigure gene expression to effectively manage stress. Genome analyses indicate that E. histolytica possesses all of the components of this stress response system; however, the premise that phospho-eIF2α controls stress and translation in this pathogen has not been tested. This represents a profound gap in knowledge. Thus, the goal of this application is to characterize this stress response system in E. histolytica. The first aim isto define the functions of phospho-eIF2α and to authenticate eIF2α kinases as they relate to the E. histolytica stress response. This will involve tracking phospho-eIF2α during a variety of stressful conditions. Furthermore, cells expressing dominant negative eIF2α will be subjected to stress and their viability will be measured. The second aim is to determine if translational control plays a role in the E. histolytica stress response. To answer this question, cells will be subjected to stress and the abundance of polyribosomes will be quantified. Preliminary data demonstrate that at least one condition of stress, serum-starvation, can induce phosphorylation of eIF2α in E. histolytica. Thus, there is high probability of success in defining a role for phospho-eIF2α in control of translation in E. histolytica. If inhibition of translation accompanies stress, it would represent the first example of translational control in this pathogen.