Sphingolipids as Potential Targets for Anti-Giardial Therapy

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Giardia lamblia is a non-invasive parasite that can cause waterborne infection in humans. This flagellated protozoan exists in two forms, i.e., the trophozoite and the cyst. The morphological transformation from cyst to trophozoite (excystation) takes place in the stomach, while factors in the small intestine trigger the transformation from trophozoite to cyst (encystation). The major goal of this proposal is to elucidate the novel role of sphingolipids in regulating the encystation-excystation cycle of Giardia. It is well known that sphingolipids play an important role in signaling, differentiation and apoptosis in all eukaryotic cells. However, Giardia has a limited ability to synthesize sphingolipids and depends on an exogenous supply of sphingolipids for energy production and membrane biosynthesis. Our studies indicate that Giardia uses actin/clathrin-dependent pathways to import and target ceramide, the major precursor of sphingolipids, to the ER/perinuclear membranes. Molecular analysis revealed that only two sphingolipid metabolic genes are differentially expressed in trophozoites and encysting Giardia. SPT-2 (serine-palmitoyl transferase-2) mRNA is expressed predominantly in trophozoites, while GlcT-1 (ceramide-glucosyl transferase) transcript is expressed predominantly in encysting cells. As a result of SPT-2 gene upregulation in trophozoites, we hypothesize that 3-keto-sphinganine, which is synthesized by SPT and required for actin polymerization and endocytosis, increases the endocytic traffic in trophozoites. For cyst wall biosynthesis, however, we hypothesize that exogenous ceramide is taken up by Giardia and used as a scaffold to assemble complex saccharide-containing ceramide/sphingolipids with the help of the GlcT-1 enzyme (encoded by the GlcT-1 gene). To test these hypotheses we propose the following aims, i.e., Aim-1: Determine whether SPT-2 and GlcT-1 genes and enzymes that are differentially regulated in excysting and encysting Giardia; Aim-2: Determine whether post-transcriptional silencing of SPT-2 and GlcT-1 genes will interrupt the excystation-encystation cycle; and Aim-3: Determine whether SPT-2 and GlcT-1 mRNA or gene products are essential for ceramide endocytosis and metabolism in trophozoites, and synthesis of encystation-specific vesicles and cyst wall in the encysting cell. These studies will yield valuable information regarding the role of sphingolipids in regulating the giardial life cycle, and will lay the foundation for future efforts to develop new therapies against this waterborne pathogen.