Translational Control of Encystation in the Entamoebae

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 DESCRIPTION (provided by applicant): Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess, and is classified as a Category B bioterrorism agent. Latent food- and water-borne cysts are the infectious form of this parasite. E. histolytica does not readily encyst in axenic culture. Thus, E. invadens, a reptilian parasite that encysts in vitro has been used as a model. The inability to reproducibly induce E. histolytica encystation in vitro represents a significant gap in the research tool-set and has hampered studies of E. histolytica latency. In other parasites latency is regulated, in part, by translational control. Stress can activate kinases that phosphorylate the a subunit of eukaryotic initiation factor-2 (eIF2a). eIF2a is part of a complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2a is inhibitory and leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to induce latency. Genome analyses indicate that the Entamoebae possess the components of this stress response system. Furthermore, preliminary data show an increase in the level of phospho-eIF2a during encystation in E. invadens cells. Thus, the central hypothesis is that Entamoeba encystation is regulated, in part, by phosphorylation of eIF2a, which in turn, down-regulates translation. The role of phospho-eIF2a in encystation will be defined using both species (E. invadens and E. histolytica) in two specific aims. In the first aim, encystation will be tracked in E. invadens cel lines expressing non-phosphorylatable and phosphomimetic mutant versions of eIF2a. This will reveal if phospho-eIF2a is necessary and sufficient for stage conversion. Two presumptive E. invadens eIF2a kinases will also be characterized. In the second aim, the levels of phospho-eIF2a will be manipulated genetically in E. histolytica in an attempt to induce encystation- and excystation-like events in the human pathogen. If successful, it would represent the first protocol for reproducibly recapitulating the life cycle of this parasite in vitro. Given that encystation ofE. invadens can be induced in culture, it is likely that a role for phospho-eIF2a in Entamoeba latency can be defined. Inclusion of E. histolytica represents the high-risk/high- reward aspect of the project. Establishing an in vitro system for encystation and excystation of E. histolytica would provide an innovative tool to the community of researchers that would inspire numerous novel studies of E. histolytica latency.