Sphingolipid synthesis and metabolism as a target for anti Giardial therapy
Giardia lamblia, a parasitic protozoan and a major cause of waterborne enteric disease worldwide, exists in two morphologic forms: (1) the infective trophozoite, and (2) the transmissible water-resistant cyst. Exposure of cysts to gastric acid during passage through the human stomach triggers excystation, while factors in the small intestine, where trophozoites colonize, induce encystation or cyst formation. Successful operation of this excystation-encystation cycle is important for this pathogen to survive, multiply, and differentiate in the human small intestine. Sphingolipids and their metabolites have been shown to modulate a wide variety of cellular events, including apoptosis, cell signaling, and synthesis of membrane rafts. More recently, 3-keto-sphinganine, synthesized by serine-palmitoyl transferase (SPT) as the first product of sphingolipid biosynthesis, was shown to be involved in endocytosis in yeast. Interestingly, however, Giardia has a very limited ability to synthesize sphingolipids de novo and depends on exogenous sources for energy production and other metabolic activities. I hypothesize that sphingolipids scavenged by this parasite from its micro-environment or synthesized de novo (although this is limited) play key roles in regulating the life cycle of Giardia. To test my hypothesis, I used radioactive and fluorescent sphingolipid probes and various metabolic/pathway inhibitors to study the mechanism of sphingolipid internalization and targeting. Studies indicated that Giardia trophozoites have the ability to internalize and target intracellular ceramide, the major sphingolipid precursor, through clathrin- and cytoskeleton-based endocytic pathways. I speculated that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide and sphigomyelin molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation. In the second phase of my investigation, I used molecular and bioinformatic approaches to analyze sphingolipid metabolic genes in Giardia. Analyses revealed that within all sphingolipid biosynthesis pathways only five gene transcripts---serine-palmitoyltransferase 1 & 2 (spt-1 and 2), ceramide-glucosyltransferase (glcT-1), and sphingomyelinase B & 3b (smase b and 3b)---are differentially expressed in trophozoites and encysting cells. The spt-1 and spt-2 genes are expressed predominately in trophozoites, while glcT-1 and smase mRNAs are increased in encysting cells. Phylogenic and protein-family (Pfam) databases have indicated that genes for both SPT subunits (i.e., spt-1 and 2) in Giardia are closely related to prokaryotic amino acid transferases. Giardial glcT-1, on the other hand, belongs to the glucosyltransferase family 2 that is distributed widely in prokaryotes and eukaryotes. To test whether these genes and gene products are important for the growth and encystation of this waterborne pathogen, I used L-cycloserine (SPT inhibitor) and two newly available GlcT-1 inhibitors--- D-threo-1-phenyl-2-decanoyl-3-morpholinopropanol (PDMP) and D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP)---on trophozoite and encysting cells. The results suggested that the inhibition of SPT by L-cycloserine induced morphological alterations and inhibited the endocytosis and intracellular targeting of fluorescently labeled ceramide dramatically, suggesting that the product of SPT enzyme (i.e., 3-keto-sphinganine) is in fact regulated the endocytosis in Giardia, as in Saccharomyces cerevisiae. On the other hand, PDMP and PPMP, two inhibitors of GlcT-1, reduced the production of vitro-derived cysts and damaged the cyst wall structures, as evidenced by DIC/confocal microscopy. These studies suggest that sphingolipids are important for giardial growth and encystation and that they could serve as potential targets for developing new therapies against Giardia and related mucosal parasites.
Hernandez, Yunuen, "Sphingolipid synthesis and metabolism as a target for anti Giardial therapy" (2007). ETD Collection for University of Texas, El Paso. AAI3262908.