Identification and characterization of phospholipid metabolic enzymes in Giardia lamblia
As part of our efforts in understanding the mechanism of lipid scavenging and remodeling reactions in Giardia lamblia trophozoites, we have analyzed the location and function of specific phospholipid metabolic enzymes. AU1 epitope tagged constructs were generated for a lysophospholipase enzyme (GLPL) gene and individually transfected into Giardia trophozoites. Confocal analysis revealed that the expression of the AU1 epitope tagged GLPL concentrated in endoplasmic reticulum/perinuclear regions and co-localized with labeled ceramide and sphingomyelin. This is to be expected for a phospholipase A2 enzyme. To test this hypothesis, we generated giardiavirus Hammerhead ribozyme constructs to knock down mRNA for GLPL as well as a second identified enzyme in the lipid-remodeling pathway, lysophosphatydic acid acyl transferase (GLAAT). Giardia trophozoites transfected with either knockout construct demonstrated an initial lower total cell number compared to sham constructs, producing irregularly shaped cells and inhibiting the uptake of fluorescently labeled lipid precursors. After incubation with a radioactively labeled lipid precursor we were able to characterize effects of biochemical activity, including whether GLPL can fulfill some of the normal functions of PLA2 enzymes. Based on our analyses of potential lipid scavenging pathways in Giardia , we predicted that these enzymes would be essential for trophozoite growth, and hence, good targets for new drug development.
Chapoy, Jaime R, "Identification and characterization of phospholipid metabolic enzymes in Giardia lamblia" (2005). ETD Collection for University of Texas, El Paso. AAI3196419.