Date of Award
Doctor of Philosophy (PhD)
Entamoeba histolytica is a protozoan parasite that causes amebic dysentery and amoebic liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable latent cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long-served as a surrogate to study stage conversion. Much remains unclear about stage conversion and the stress response in these parasites and current treatments for amoebiasis are lacking, as they cause severe side effects. Ultimately new therapeutic strategies are needed and the parasite stress response and stage conversion mechanisms may represent targetable vulnerabilities. To gain insight into these cellular processes, we characterized two hypothetical proteins, EIN_059080 (in E. invadens), and EHI_056700 (in E. histolytica). We also characterized two putative eIF2 alpha kinases in E. invadens. In all cases, we used an RNAi-based silencing system to reduce expression of the genes. Reduction of EIN_059080 expression resulted in a decreased rate of encystation and an increased rate of erythrophagocytosis, an important virulence function. Additionally, these mutants were more susceptible to oxidative stress. Similarly, reduction of EHI_056700 resulted in increased susceptibility to oxidative stress and glucose deprivation, but not to nitrosative stress. Interestingly, parasites with decreased expression of EHI_056700 also exhibited decreased erythrophagocytosis and adhesion to host cells. We authenticated the two eIF2α kinases using a heterologous yeast system. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2α and increased sensitivity to oxidative stress. Diminished kinase expression also correlated with an increased rate of encystation, a decreased the rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that these hypothetical proteins and kinases may play a role in various aspects of stage conversion, virulence, and the response to stress.
Walters, Heather Andrews, "Characterization of Kinases and Hypothetical Proteins in the Entamoeba Species" (2022). All Dissertations. 3079.