Sphingolipid and Mechanism of Cyst Formation by Giardia

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DESCRIPTION (provided by applicant): Giardia lamblia, a waterborne parasite, is responsible for intestinal infections in both developed and developing countries. Giardiasis, which is also a zoonotic disease, is transmitted via infective cysts through contaminated water. Exposures of cysts to gastric acid during passage through the human stomach trigger excystation, while factors in the small intestine, where trophozoites colonize, induce encystation or cyst formation. The hallmark of encystation is the biogenesis of encystation-specific vesicles (ESVs), which transport cyst-wall materials that later merge with the plasma membrane and lay down the cyst wall. However, it is not clear how ESV biogenesis is regulated and how viable or infective cysts are produced. Results from our laboratory have indicated that Giardia expresses fewer sphingolipid biosynthesis genes, which are differentially regulated during encystation. The overexpression of glucosylceramide transferase-1 (gGlcT1), one of the important enzymes of SL biosynthesis, produces enlarged and aggregated ESVs and alters cellular lipid homeostasis. The knockdown of gGlcT1, on the other hand, interferes with ESV formation that leads to the generation of cryptic cysts with reduced viability. Based on these observations, we hypothesize that the regulated expression of gGlcT1 is essential for ESV biogenesis and the production of infective cysts that transmit the disease. In Specific Aim 1, the mechanism by which gGlcT1 regulates lipid homeostasis and ESV biogenesis will be investigated using gGlcT1 overexpressed and knockdown Giardia. We will determine whether gGlcT1 coordinates with other enzymes of sphingolipid biosynthetic pathway to regulate ESV formation. The lipid components involved in assembly of functional ESVs will be identified by mass spectrometry and molecular/cellular methodologies. In Specific Aim 2, the role of gGlcT1 in regulating cyst morphology, viability, and infectivity will be determined. The biological activity and infectivity of cryptic cysts produced by gGlcT1 knockdown will be tested in in vitro excystation and in vivo animal model, respectively. The cyst- wall components that are responsible for altering cyst morphology and viability will be determined. The proposed study will identify gGlcT1 as a novel regulator of cyst formation by Giardia and could be exploited for the development of new therapies to control giardiasis, which affects millions of children worldwide each year. PUBLIC HEALTH RELEVANCE: Giardia, a Class B Biodefense Agent, can infect millions of people annually, parasitizing the intestinal tract and causing diarrhea, malabsorption, and related abnormalities. Although giardiasis can be treated with the metronidazole group of compounds, these drugs can be problematic, resulting in unpleasant side effects and ultimately leading to drug-resistant parasites. The proposed study will identify sphingolipids as key molecules for the production of viable and infective cysts (that transmit giardiasis), and could be targeted for developing new therapies to control the disease process.