Eluication of Lipid Metabolic Pathways in Differentiating Giardia lamblia Using High Resolution Mass Spectrometry
Abstract
Giardia lamblia is an intestinal protozoan found worldwide, including the U.S. This parasite exists in two morphologic stages—a replicative trophozoite and a relatively dormant yet viable cyst. While exposures of cysts to gastric acid during passage through the human stomach induces excystation, factors in the small intestine, where trophozoites colonize trigger encystation or cyst formation. Transformation into cyst stage is essential for Giardia to survive in the environment for months before infecting new hosts. Because of its small genome size (11.7 Mb), metabolic pathways in Giardia are highly reduced. As far as lipid metabolism is concerned, only limited number of lipid genes are present in this parasite. Therefore, to compensate, Giardia scavenges lipids from its environment, remodels and use them to assembles membranes and vesicles. Earlier, using ESI-LTQ-MS, we have demonstrated that phosphatidylglycerols (PGs), phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) are major glycerol-based phospholipids in Giardia and abundant in non-encysting and encysting trophozoites, as well in cysts. The GC/MS analysis identified palmitate (16:0), palmitoleate (16:1), oleate (18:1), and linoleate (18:2) as major fatty acids in Giardia. In the current study, we employed high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) for quantitative lipidome and proteome analysis to further elucidate the lipid metabolic pathways that is operative in Giardia. Vegetative and encysting trophozoites as well as osmotically resistant cysts were cultured in the laboratory, collected, and subjected to lipid extraction or tryptic digestion. Lipidomic analysis revealed the dynamic changes of overall lipid and fatty acid (mostly C16, C18 moieties) profiles throughout the life stages of Giardia. Lipid classes that were found in cysts included lyso-phosphatidylcholine (LPC, P- and O-), lyso-phosphatidylethanolamine (LPE), diacylglycerol (DG), triacylglycerol (TG), cholesterol ester (CE), PC, P-PC (Plasmalogen), and ceramides. Sphingomyelin (SM), PG, bis (monoacylglycerol) phosphate (BMP) were present mostly in encysting trophozoites rather than cysts. Ether linked PC species revealed to be most abundant in trophozoites. Proteomic analysis identified increased levels of enzymes from lipid elongation pathway—i.e., acyl-CoA ligases, long-chain fatty acid elongase (LCFAELO), oxidoreductase and enoyl-CoA reductase in cysts. To further explore how fatty acid elongase influences the lipid metabolism, giardial fatty acid elongase (gFAELO) was overexpressed, followed by lipidomic analysis. It was observed that the overexpression of gFAELO induced the generation of long chain fatty acids and upregulated Cer, PE, LPC, LPE, and LPA in cysts. These studies suggest that although limited, Giardia maintains an active and robust lipid metabolism in all stages including the water-resistant non-motile cysts. It is possible that active lipid metabolism in cysts is required for the successful excystation to produce infective membranous trophozoites.
Subject Area
Biochemistry|Biology|Parasitology
Recommended Citation
Ellis, Cameron Conner, "Eluication of Lipid Metabolic Pathways in Differentiating Giardia lamblia Using High Resolution Mass Spectrometry" (2022). ETD Collection for University of Texas, El Paso. AAI29258567.
https://scholarworks.utep.edu/dissertations/AAI29258567