The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a common waterborne intestinal disease. In these studies, encystation of Giardia lamblia in vitro was demonstrated by morphologic, immunologic, and biochemical criteria. In the suckling mouse model, the jejunum was shown to be a major site of encystation of the parasite. Small intestinal factors were therefore tested as stimuli of encystation. An antiserum that reacted with cysts, but not with cultured trophozoites was raised in rabbits and used as a sensitive probe for differentiation in vitro. Cultured trophozoites that were exposed to bile salts showed a more than 20-fold increase in the number of oval, refractile cells that reacted strongly with anticyst antibodies, and in the expression of major cyst antigens. Exposure to primary bile salts resulted in higher levels of encystation than exposure to secondary bile salts. These studies will aid in understanding the differentiation of an important protozoan pathogen.

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Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.

SUMMARY Giardia lamblia, a protozoan parasite, infects a wide variety of vertebrates, including humans. Studies indicate that this anaerobic protist possesses a limited ability to synthesize lipid molecules de novo and depends on supplies from its environment for growth and differentiation. It has been suggested that most lipids and fatty acids are taken up by endocytic and non-endocytic pathways and are used by Giardia for energy production and membrane/organelle biosynthesis. The purpose of this article is to provide an update on recent progress in the field of lipid research of this parasite and the validation of lipid metabolic pathways through recent genomic information. Based on current cellular, biochemical and genomic data, a comprehensive pathway has been proposed to facilitate our understanding of lipid and fatty acid metabolism/syntheses in this waterborne pathogen. We envision that the current review will be helpful in identifying targets from the pathways that could be used to design novel therapies to control giardiasis and related diseases.

Although encystation (cyst formation) is important for the survival of Giardia lamblia outside its human host, the molecular events that prompt encystation have not been fully elucidated. Here, we demonstrate that sphingolipids (SLs), which are important for the growth and differentiation of many eukaryotes, play key roles in giardial encystation. Transcriptional analyses showed that only three genes in the SL biosynthesis pathways are expressed and transcribed differentially in nonencysting and encysting Giardia trophozoites. While the putative homologues of giardial serine palmitoyltransferase (gSPT) subunit genes (gspt-1 and -2) are differentially expressed in nonencysting and encysting trophozoites, the giardial ceramide glucosyltransferase 1 gene (gglct-1) is transcribed only in encysting cells. L-Cycloserine, an inhibitor of gSPT, inhibited the endocytosis and endoplasmic reticulum/perinuclear targeting of bodipy-ceramide in trophozoites, and this could be reversed by 3-ketosphinganine. On the other hand, D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthesis, blocked karyokinesis and reduced cyst production in culture. PPMP also altered the expression of cyst wall protein transcripts in encysting cells. Phylogenetic analyses revealed that the gspt genes are paralogs derived from an ancestral spt sequence that underwent gene duplication early in eukaryotic history. This ancestral sequence, in turn, was probably derived from prokaryotic aminoacyl transferases. In contrast, gglct-1 is found in both prokaryotes and eukaryotes without any evidence of gene duplication. These studies indicate that SL synthesis genes are involved in key events in giardial biology and could serve as potential targets for developing new therapies against giardiasis.Giardiasis, a clinical syndrome caused by the intestinal protozoan Giardia lamblia, is a reemerging waterborne infectious illness worldwide. Giardia exists in two morphological forms: (i) actively dividing trophozoites and (ii) relatively inactive cysts. The water-resistant dormant cysts are responsible for transmission of giardiasis via contaminated water and undergo excystation, a stage of the giardial life cycle in which a cyst differentiates into two trophozoites in the stomach. Newly emerged trophozoites then move down the small intestine and colonize below the bile duct (2). Components of the intestinal milieu, including dietary lipids, bile salts, intestinal pH (pH ϳ7.8), and lactic acid, among others, trigger the process of encystation to complete the life cycle of Giardia in the small intestine (10, 16). During encystation, various molecular and cellular changes take place that allow this protozoan to transport cyst wall proteins (CWPs) through regulatory secretory pathways (35). In encysting cells, three encystation-specific CWPs (CWP-1, -2, and -3 encoded by cwp-1, -2, and -3, respectively) are synthesized and concentrated within encystation-specific vesicles (ESVs) before targeting into the cyst wall (17,3...