Date of Award
Master of Science (MS)
Temesvari, Lesly A
Paul , Kimberly S
Hersh , Bradley M
Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Currently, the World Health Organization estimates that amoebic dysentery results in 40,000-100,000 deaths annually worldwide, which places amoebiasis second only to malaria as a leading cause of death due to parasitic infection. The pathogenesis of invasive amoebiasis depends on the parasite's ability to carry out endocytosis in the host's bowel lumen enabling it to colonize the digestive tract. The pathogen is contracted by the ingestion of its multinucleate cysts. Upon excystation in the small intestine, motile trophozoites are released into the bowel lumen, where bacteria, erythrocytes, and cellular debris serve as their food source. Phagocytosis in E. histolytica has been directly linked to its ability to destroy host tissues. Thus, understanding the molecular mechanisms of cell movement and nutrient uptake will provide insight into factors that contribute to the pathogen's virulence and reveal potential molecular targets for interventions.
The goal of this Master's thesis research project is to examine the role of a second messenger lipid, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), during endocytosis in E. histolytica trophozoites. Phosphoinositides (PIs) represent a dynamic pool of membrane-associated lipids, whose composition is regulated by several isoforms of phosphatidylinositol 3-kinase (PI 3-kinase). A number of recent studies have implicated PIs in regulating virulence-associated cellular functions in E. histolytica. For instance, a putative PI3-kinase was identified among the signaling molecules that were physically associated with E. histolytica phagosomes, and pharmacological inhibition of PI3-kinase blocked the uptake of erythrocytes by this amoeba. Moreover, a singly phosphorylated product of PI3-kinase activity, PI3P, was shown to accumulate in the erythrophagosomal cups in E. histolytica. These observations provided the incentive to investigate the distribution of PIP3 during endocytosis in E. histolytica. We implemented two approaches utilizing GST- and GFP- tagged biosensors to examine the spatial and temporal aspects of PIP3 distribution during endocytosis in E. histolytica.
We have shown that PIP3 accumulated in pseudopodia and erythrophagosomal cups but not in fluid phase pinosomes in E. histolytica trophozoites. We also observed high steady-state levels of PIP3 in the plasma membrane of E. histolytica trophozoites, which were not abolished by serum withdrawal. To our knowledge, this is the first report describing PIP3 distribution during phagocytosis in E. histolytica that also elucidates a unique aspect of biology of this parasite, namely the stability of PIP3 lipid in plasma membrane. This distinguishes the physiology of this parasite from that of mammalian host cells. This study provides insight into the molecular mechanisms of E. histolytica pathogenicity, since nutrient uptake represents an important virulence function in this parasite.
Byekova, Yevgeniya, "Characterization of phosphatidylinositol (3,4,5)-trisphosphate subcellular localization during endocytosis in Entamoeba histolytica" (2008). All Theses. 404.