Molecular and Cellular Characterizations of Glucosylceramide Transferase (Synthase) in Giardia lamblia

Giardiasis, caused by an intestinal protozoan, Giardia lamblia, is a major public health problem worldwide. Giardia infection is spread through the fecal-oral route via contaminated water and food. It is estimated that over 280 million people are infected with Giardia across the globe. Giardia is a non-invasive parasite and consists of a two-stage life cycle: trophozoites and cysts. Ingested cysts undergo excystation in the proximal small intestine, releasing two trophozoites (excyzoites). Upon reaching the distal small intestine and colonizing there, trophozoites transform into cysts by encystation. It has been reported earlier that the process of encystation induced the synthesis of giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of the sphingolipid pathway that participates in various biological processes including cell division, cytokinesis, ESV biogenesis, cyst production, and maintaining the cellular lipid homeostasis. The modulation of its activity using molecular tools (overexpression, knockdown, and rescue) interfered with cyst production, cyst viability, and infectivity in laboratory animals. gGlcT1 is unique because it is sufficiently diverged from other eukaryotic GlcT enzymes and consists of two active catalytic domains (CGlcT and CGalT) that catalyze the synthesis of glucosylceramide (GlcCer) and galactosylceramide (GalCer), respectively in a non-competitive manner. The goal of my dissertation is to further analyze these two catalytic domains and elucidate their roles in growth, encystation, and cyst production by Giardia. To address this goal, I used homologous recombination and overexpression techniques and created deletion mutant Giardia cell lines. I found that both catalytic domains are active and participate in cyst production and maintaining cyst wall structures. I also observed that Giardia expresses a long-chain fatty acid elongase (gFAELO) enzyme, which is involved in the remodeling of membrane lipids by exchanging fatty acyl moieties (the Land’s cycle). Since gFAELO is mostly expressed in cysts and gGlcT1 overexpression affects lipid uptake, I investigated whether the interplay between gGlcT1 and gFAELO is important for cyst production, morphology, and viability as these events dictate the parasite’s ability to transmit the disease process. Therefore, the goal of my project was to elucidate the molecular mechanisms behind gGlcT1 and gFAELO as they pertain to the life cycle of Giardia. I hypothesize that The Specific Aims of the project were to: (1) use molecular techniques to determine which domain/s of gGlcT1 were important for its function; (2) and to investigate the how the domains of gGlcT1 and its interaction with gFAELO would modulate cyst morphology. I hypothesize that elucidating the function of glucosylceramide transferase will reveal the mechanisms of giardial differentiation and could be exploited to design novel drugs against this pathogen. Therefore, The Specific Aims of the project are to: (1) use molecular techniques to determine which domain/s of gGlcT1 were important for its function; (2) and to investigate the how the domains of gGlcT1 and its interaction with gFAELO would modulate cyst morphology. It is anticipated that the findings of this study will elucidate new knowledge about gGlcT1 and gFAELO interplay and the mechanisms of cyst production by Giardia