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Sateriale, Adam

Cell and Molecular Biology Program

2014

Ph. D.

Entamoeba histolytica is a prominent cause ofdysentery throughout the developing world. This protozoan parasite colonizes the colon of its host, and invades the lamina propria by secreting proteases, and by attacking; killing, and phagocytosing host cells. The phagocytic ability of E. histolytica in vitro has been shown to correlate well with virulence in vivo. In order to better understand amebic phagocytosis, we used microarray analysis to identify gene expression differences between phagocytic and nonphagocytic E. histolytica trophozoites. Using paramagnetic beads coated in host ligand, these two populations were separated, using a magnetic column. 121 genes were identified as having higher expression in the phagocytic population, as compared to the non-phagocytic population.
Using RNAi mediated gene regulation, 15 of the genes found to have higher expression in the phagocytic population of E. histolytica were stably silenced. Of these 15 E. histolytica strains, 5 showed a deficiency in phagocytosis of apoptotic host cells. Silenced strains, however, were proficient in cell monolayer adherence and cytotoxicity.
Post hoc analysis demonstrated that following an initial act of phagocytosis, E. histolytica trophozoites had an increased rate of phagocytosis. In addition, many of the genes identified in our microarray are upregulated in response to this act of phagocytosis. This data has led us to propose a feed-forward mechanism of phagocytosis in E. histolytica; that is, that phagocytosis itself stimulates alterations in gene expression that enhance phagocytic ability. We believe this is a novel mechanism of genetic control of phagocytic ability. Future directions include the use of the newly adapted SNAP-tag, also presented in this thesis, to track proteins encoded by genes discovered to be upregulated during phagocytosis, in order to better understand their involvement in the mechanism.