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 eIF2 kinases that phosphorylate the -subunit of eukaryotic initiation factor-2 (eIF2). eIF2 is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2 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-eIF2 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 i sto define the functions of phospho-eIF2 and to authenticate eIF2 kinases as they relate to the E. histolytica stress response. This will involve tracking phospho-eIF2 during a variety of stressful conditions. Furthermore, cells expressing dominant negative eIF2 will be subjected to stress and their viability will be measured.
The second aim i s 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 eIF2 in E. histolytica. Thus, there is high probability of success in defining a role for phospho-eIF2 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.
Entamoeba histolytica is the causative agent of amoebic dysentery and is classified as a Category B bioterrorism agent. The proposed studies will examine how the parasite responds to stress, and in particular, how protein translation in the parasite is altered during stress. An appropriate stress-response is necessary for survival of the parasite in the host; therefore, targeting this process is a logical approach for therapy.
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