High resolution mass spectrometric analysis reveals that Giardia cysts are metabolically active and generates membrane lipids with long chain fatty acyl moieties

Abstract

Giardia lamblia, the causative agent of “giardiasis” affects people throughout the world. Metronidazole (Mtz, Flagyl®), a nitroimidazole group of compounds is the commonly prescribed treatment for giardiasis. However, Mtz is a genotoxic agent and a potential carcinogen. Therefore, it is imperative to identify new and specific targets in Giardia that could be exploited to develop non-Mtz classes of drugs to treat giardiasis. Because of its limited lipid synthetic ability, Giardia was proposed to scavenge phospholipids, sterols and free fatty acids from the hosts or its environment for membrane and organelle biogenesis. In the current study, we utilized a quantitative lipidomic and proteomic approach to further elucidate the lipid metabolic pathways 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 followed by the high-resolution mass spectrometric analysis (Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray (HESI-II) or a Nanospray Flex Ion source, Thermo Fisher). Lipid identification and confirmation was performed on open-source software MS-DIAL and LipidMatch flow. Proteomic analysis was performed on a licensed version of Proteome Discoverer. 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 LPC, P- and O-LPC, LPE, DG, TG, CE, PC, P-PC (Plasmalogen), and ceramides. Lipids, such as SM, PG, 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 several enzymes from lipid elongation pathway i.e., acyl-CoA ligases, long-chain fatty acid elongase, oxidoreductase and enoyl-CoA reductase 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.