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Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan, Giardia lamblia*

Open AccessPublished:April 14, 2013DOI:https://doi.org/10.1074/jbc.M112.438416
      The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.
      Background: The production of viable cysts by Giardia is essential for transmitting the infection via contaminated food and water.
      Results: Overexpression and knockdown of glucosylceramide transferase activity affect encystation, cyst viability, and overall lipid balance in Giardia.
      Conclusion: Regulated expression of glucosylceramide transferase is linked to encystation and cyst production.
      Significance: Glucosylceramide synthesis could be targeted for developing novel anti-giardial therapy.

      Introduction

      Giardiasis, caused by Giardia lamblia, is widespread throughout the world. This intestinal parasite exists in two morphologic forms, trophozoites and cysts. Although trophozoites colonize the small intestine and produce infections, cysts transmit the disease via contaminated water and food (
      • Adam R.D.
      Biology of Giardia lamblia.
      ). Stage-specific differentiation of cyst to trophozoite, which takes place in the human stomach, is known as excystation, whereas the transformation from trophozoites to cysts that occurs in the small intestine is called encystation. Giardial cyst walls contain insoluble filamentous materials made of proteins, glycoproteins, and polysaccharides (
      • Das S.
      • Gillin F.D.
      Giardia lamblia: increased UDP-N-acetyl-d-glucosamine and N-acetyl-d-galactosamine transferase activities during encystation.
      ,
      • Gerwig G.J.
      • van Kuik J.A.
      • Leeflang B.R.
      • Kamerling J.P.
      • Vliegenthart J.F.
      • Karr C.D.
      • Jarroll E.L.
      The Giardia intestinalis filamentous cyst wall contains a novel β(1–3)-N-acetyl-d-galactosamine polymer: a structural and conformational study.
      ,
      • Sener K.
      • Shen Z.
      • Newburg D.S.
      • Jarroll E.L.
      Amino sugar phosphate levels in Giardia change during cyst wall formation.
      ,
      • Lauwaet T.
      • Davids B.J.
      • Reiner D.S.
      • Gillin F.D.
      Encystation of Giardia lamblia: a model for other parasites.
      ). At the onset of encystation, encystation-specific vesicles (ESVs)
      The abbreviations used are: ESV, encystation-specific vesicle; CWP, cyst wall protein; GlcCer, glucosylceramide; GlcT1, glucosylceramide transferase 1; gGlcT1, giardial glucosylceramide transferase-1; GCS, glucosylceramide synthase; SL, sphingolipid; GM1, mono-sialotetrahexosylceramide; GM3, mono-sialodihexosylceramide; GD3, di-sialodihexosylceramide; SPT, serine palmitoyltransferase; PPMP, d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; NBD, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]; Bodipy, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; SM, sphingomyelin; FDA, fluorescein diacetate; PI, propidium iodide; FAST Dil, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarboxyanine; ESI, electrospray ionization; GSL, glycosphingolipid; ER, endoplasmic reticulum.
      are synthesized by trophozoites that transport three cyst wall proteins (CWP-1, -2, and -3) to the plasma membrane before laying down the water-resistant cyst wall (
      • Lauwaet T.
      • Davids B.J.
      • Reiner D.S.
      • Gillin F.D.
      Encystation of Giardia lamblia: a model for other parasites.
      ). CWP-1, -2, and -3 are acidic proteins (molecular mass ∼26, ∼39, and ∼27 kDa, respectively) containing similar domains, including N-terminal signal sequences and five tandem, leucine-rich repeats (
      • Mowatt M.R.
      • Luján H.D.
      • Cotten D.B.
      • Bowers B.
      • Yee J.
      • Nash T.E.
      • Stibbs H.H.
      Developmentally regulated expression of a Giardia lamblia cyst wall protein gene.
      ,
      • Luján H.D.
      • Mowatt M.R.
      • Conrad J.T.
      • Bowers B.
      • Nash T.E.
      Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall.
      ). Reports suggest that all three CWPs are essential for forming ESVs and that CWP-2 functions as an aggregation factor by interacting with CWP-1 and CWP-3 via conserved regions (
      • Gottig N.
      • Elías E.V.
      • Quiroga R.
      • Nores M.J.
      • Solari A.J.
      • Touz M.C.
      • Luján H.D.
      Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia.
      ). Besides these three CWPs, high cysteine, nonvariant cyst protein, and cysteine protease-2 were also shown to participate in the process of encystation and cyst production (
      • Lauwaet T.
      • Davids B.J.
      • Reiner D.S.
      • Gillin F.D.
      Encystation of Giardia lamblia: a model for other parasites.
      ,
      • DuBois K.N.
      • Abodeely M.
      • Sakanari J.
      • Craik C.S.
      • Lee M.
      • McKerrow J.H.
      • Sajid M.
      Identification of the major cysteine protease of Giardia and its role in encystation.
      ).
      Emerging reports indicate that the enzymes and products of sphingolipid (SL) biosynthesis pathways act as regulators of various cellular processes in both lower and higher eukaryotes. For instance, ceramide synthase activity is associated with increased tumor growth and progression in head and neck cancers (
      • Separovic D.
      • Breen P.
      • Joseph N.
      • Bielawski J.
      • Pierce J.S.
      • VAN Buren E.
      • Gudz T.I.
      Ceramide synthase 6 knockdown suppresses apoptosis after photodynamic therapy in human head and neck squamous carcinoma cells.
      ). Sphingosine 1-phosphate has been shown to generate initial signals for apoptotic cell extrusion (
      • Gu Y.
      • Forostyan T.
      • Sabbadini R.
      • Rosenblatt J.
      Epithelial cell extrusion requires the sphingosine 1-phosphate receptor 2 pathway.
      ). Ceramidase expression is important for normal retinal function, and the targeted expression of neutral ceramidase in mutant fruit flies rescues photoreceptor degeneration (
      • Acharya U.
      • Patel S.
      • Koundakjian E.
      • Nagashima K.
      • Han X.
      • Acharya J.K.
      Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration.
      ). The mutation in the long chain sphingoid base subunit-1 (LCB1) of serine palmitoyltransferase causes autosomal, dominant, peripheral, and sensory neuropathy (
      • Gable K.
      • Gupta S.D.
      • Han G.
      • Niranjanakumari S.
      • Harmon J.M.
      • Dunn T.M.
      A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity.
      ). In plants, the synthesis of four different classes of SLs and glycosphingolipids (GSLs) was observed, including glycosylinositolphosphoceramides, glycosylceramides, ceramides, and free LCBs. Several enzymes of plant SL metabolic pathways have also been cloned and characterized (
      • Pata M.O.
      • Hannun Y.A.
      • Ng C.K.
      Plant sphingolipids: decoding the enigma of the Sphinx.
      ). SLs are also critical for regulating various biological processes in unicellular organisms like fungi and protozoa. In budding yeast, SLs contain C26 acyl moieties and are connected with the target of rapamycin complex 2 (TORC2) and phosphoinositide signaling (
      • Dickson R.C.
      Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast.
      ). In Leishmania and Trypanosoma parasites, the synthesis of large amounts of nonglycosylated inositol phosphorylceramides, and ethanolamine phosphorylceramides, along with other SLs, have been reported. These SLs in trypanosomatids contribute greatly to the growth and survival of parasites within the host and have important functions during host-parasite interactions (
      • Zhang K.
      • Bangs J.D.
      • Beverley S.M.
      Sphingolipids in parasitic protozoa.
      ).
      As far as giardial SLs are concerned, only five genes of the entire SL metabolic pathway have been annotated in the Giardia Genome Database, and they are all transcribed differentially between trophozoites and cysts (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ). These genes are as follows: (i) giardial serine-palmitoyltransferase-1 and -2 subunit genes (gspt-1 and gspt-2), (ii) glucosylceramide synthase or glucosylceramide transferase 1 (gglct-1), and (iii) two acid sphingomyelinase genes (gasmase 3b and gsmase B). It was reported that d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide (GlcCer) synthase (GCS or GlcT1), interferes with encystation and cyst production by Giardia, suggesting that GlcCer synthesis is important for encystation (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ,
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ). Despite this information, the exact mechanisms by which this enzyme and its product regulate the ESV biogenesis and cyst production are still not clear.
      This study focuses on understanding the unique and precise function of gGlcT1 and its role in encystation and cyst formation. Here, we report that modulation of gGlcT1 activity by overexpression and knockdown affects the ESV biogenesis and cyst viability of Giardia. We also provide evidence that gGlcT1 overexpression destabilizes lipid balance in this parasite by increasing the influx of lipids, cholesterol, and fatty acids from the culture medium.

      RESULTS

      gGlcT1 Activity Is Elevated during Encystation

      Earlier reports from this laboratory and other laboratories have indicated that GlcCer, a precursor of wide varieties of GSLs, play an important role during encystation by Giardia (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ,
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ). It was observed that the transcript of the gGlcT1 gene (gglct1) is up-regulated in encysting cells and that PPMP, an inhibitor of GlcCer synthesis (
      • Kovács P.
      • Pintér M.
      • Csaba G.
      Effect of glucosphingolipid synthesis inhibitor (PPMP and PDMP) treatment on Tetrahymena pyriformis: data on the evolution of the signaling system.
      ,
      • Shukla A.
      • Radin N.S.
      Metabolism of d-[3H]threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis, and the synergistic action of an inhibitor of microsomal monooxygenase.
      ), inhibits the cyst production in culture (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ). In this study, we asked if Giardia synthesizes the active gGlcT1 enzyme and uses it to drive the process of encystation. Therefore, gGlcT1 activity was measured in the cell-free extracts (1500 × g supernatant) of nonencysting and encysting trophozoites as well as in water-resistant cysts using UDP-[14C]glucose and ceramide (
      • Uchida Y.
      • Murata S.
      • Schmuth M.
      • Behne M.J.
      • Lee J.D.
      • Ichikawa S.
      • Elias P.M.
      • Hirabayashi Y.
      • Holleran W.M.
      Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress.
      ,
      • Couto A.S.
      • Caffaro C.
      • Uhrig M.L.
      • Kimura E.
      • Peres V.J.
      • Merino E.F.
      • Katzin A.M.
      • Nishioka M.
      • Nonami H.
      • Erra-Balsells R.
      Glycosphingolipids in Plasmodium falciparum. Presence of an active glucosylceramide synthase.
      ). Results show that the basal gGlcT1 activity is low in nonencysting trophozoites and up-regulated in 12- and 24-h encysting cells and increases severalfold in cysts (Fig. 1A). We found that the gGlcT1 activities in trophozoites and encysting cells were inhibited by PPMP, which that bears a resemblance to both the ceramide and GlcCer (
      • Kovács P.
      • Pintér M.
      • Csaba G.
      Effect of glucosphingolipid synthesis inhibitor (PPMP and PDMP) treatment on Tetrahymena pyriformis: data on the evolution of the signaling system.
      ,
      • Shukla A.
      • Radin N.S.
      Metabolism of d-[3H]threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis, and the synergistic action of an inhibitor of microsomal monooxygenase.
      ). However, unlike trophozoites and encysting cells, the cyst enzyme was completely resistant to PPMP (Fig. 1A). Next, we generated GlcT1 knockdown Giardia where the gglct1 gene was silenced by morpholino oligonucleotide. Knockdown by morpholino analogs is a faster and more efficient way to silence genes in Giardia (
      • Carpenter M.L.
      • Cande W.Z.
      Using morpholinos for gene knockdown in Giardia intestinalis.
      ). In recent years, various laboratories have successfully used antisense morpholino oligonucleotides to knock down giardial kinesin and flagellar genes. The effects of antisense morpholinos are transient and last for 72 h, which is sufficient to carry out one round of the encystation cycle (
      • House S.A.
      • Richter D.J.
      • Pham J.K.
      • Dawson S.C.
      Giardia flagellar motility is not directly required to maintain attachment to surfaces.
      ). As shown in Fig. 1B, knockdown by anti-gGlcT1 morpholino reduced the gGlcT1 activity in cysts by ∼60%. This result suggests that although resistant to PPMP, cyst gGlcT1 could be inhibited by anti-gGlcT1 morpholino, which blocks the translation initiation of gGlcT1 by targeting the 5′ region of the gglct1 gene. However, PPMP competes with ceramide for the catalytic site of gGlcT1 and is thereby expected to inhibit the enzymatic reaction (
      • Kovács P.
      • Pintér M.
      • Csaba G.
      Effect of glucosphingolipid synthesis inhibitor (PPMP and PDMP) treatment on Tetrahymena pyriformis: data on the evolution of the signaling system.
      ).
      Figure thumbnail gr1
      FIGURE 1GlcT1 activity in encysting Giardia. A, gGlcT1 activity in nonencysting, encysting (12- and 24-h), and water-resistant cysts was measured in the cell-free extracts using [14C]UDP-glucose (∼100,000 cpm/assay) and ceramide as substrates. The reaction was carried out for 5 h at 37 °C in the presence and absence of PPMP (10 μm). 14C-Labeled GlcCer was extracted following the protocol described under “Experimental Procedures.” Data represent means ± S.D. of three separate experiments, and the experiments were carried out in duplicate (**, p < 0.01). B, activity of gGlcT1 in control, PPMP-treated, and gGlcT1-knockdown cysts. Control and anti-gGlcT1 morpholino-transfected trophozoites were subjected to encystation. In vitro-derived cysts were isolated by centrifugation and ruptured by sonication and homogenization, and cell-free extracts (1500 × g) were used to measure the enzyme activity. To assess the effect of the inhibitor, PPMP (10 μm) was added to the assay mixture as described under “Experimental Procedures.” The data represent means ± S.D. of three separate experiments (**, p < 0.01). C, synthesis of NBD-GlcCer by encysting (12 and 24 h) trophozoites. Live cells were labeled with NBD-ceramide following the method described by Gupta et al. (
      • Gupta V.
      • Patwardhan G.A.
      • Zhang Q.J.
      • Cabot M.C.
      • Jazwinski S.M.
      • Liu Y.Y.
      Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.
      ). Sphingolipids were extracted and analyzed by TLC as described under “Experimental Procedures.” The experiment was carried out three times, and the representative result from a single experiment is shown here.
      Synthesis of GlcCer, the reaction product of gGlcT1, was also monitored by assessing NBD-GlcCer synthesis by encysting cells. For this, both nonencysting and encysting Giardia trophozoites were labeled with NBD-C6-ceramide, and the product was analyzed by TLC. Fig. 1C shows that the synthesis of NBD-GlcCer is extremely low (almost nonvisible in the photograph) in trophozoites and increases during encystation. However, we found that the formation of NBD-GlcCer in 12-h encysting cells is slightly higher than in 24-h encysting cells, which could be due to the fact that cells at the later stage of encystation (i.e. 18 h onwards) start forming cyst walls and become increasingly impermeable to NBD-ceramide and other molecules (data not shown). Therefore, in this study, no attempt was made to label the cyst with NBD-GlcCer. The increased synthesis of NBD-GlcCer during 12- and 24-h encysting Giardia supports our enzymatic results (Fig. 1A) that encystation stimuli induce gGlcT1 activity, leading to the synthesis of GlcCer.

      Modulation of gGlcT1 Activity Affects ESV Biogenesis

      Giardia has a relatively simple life cycle, i.e. replicative trophozoites and relatively dormant cysts. During encystation in the small intestine, trophozoites synthesize ESVs that are necessary to transport CWPs to plasma membranes (
      • Bittencourt-Silvestre J.
      • Lemgruber L.
      • de Souza W.
      Encystation process of Giardia lamblia: morphological and regulatory aspects.
      ). ESVs are synthesized in the endoplasmic reticulum (ER), located in the perinuclear membranes/cytoplasm in early encysting cells, and then distributed extensively in the periphery of the cell (in close proximity to peripheral vacuoles) in the late encystation phase (
      • McCaffery J.M.
      • Gillin F.D.
      Giardia lamblia: ultrastructural basis of protein transport during growth and encystation.
      ). Because of the presence of Golgi-like cisternae in ESVs, these sorting vesicles are also considered as a primitive Golgi complex of Giardia (
      • Stefanic S.
      • Morf L.
      • Kulangara C.
      • Regös A.
      • Sonda S.
      • Schraner E.
      • Spycher C.
      • Wild P.
      • Hehl A.B.
      Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.
      ). Over the years, many investigators have studied encystation and identified various proteins and genes that are associated with the process (see Ref.
      • Lauwaet T.
      • Davids B.J.
      • Reiner D.S.
      • Gillin F.D.
      Encystation of Giardia lamblia: a model for other parasites.
      for review). However, nothing is known about how the ESV biosynthesis is regulated and participates in viable and infective cyst formation. Because gGlcT1 activity is up-regulated during encystation (Fig. 1A), we had thought that gGlcT1 was linked to ESV biogenesis. To test this, control and gGlcT1-knockdown trophozoites were subjected to encystation for 18 h, and ESV synthesis was monitored using an anti-cyst antibody that recognizes early and late ESVs as well as the cyst walls (
      • Hehl A.B.
      • Marti M.
      Secretory protein trafficking in Giardia intestinalis.
      ). It was observed that anti-gGlcT1 morpholino oligonucleotide, which inhibited gGlcT1 activities in trophozoites and cysts, was found to block ESV biogenesis completely (Fig. 2A). However, ESV synthesis by encysting cells that were transfected with scrambled morpholino oligonucleotide (labeled as control (scrambled) in Fig. 2A) was not affected at all. To further elucidate the role of gGlcT1 in ESV biogenesis, we overexpressed the gglct1 gene in trophozoites and subjected them to encystation. Fig. 2B shows that gGlcT1 overexpression produces aggregated and enlarged ESVs as compared with control cells that were transfected with empty plasmid. This indicates that the modulation of gGlcT1 activity either by knockdown or overexpression affects ESV biosynthesis in Giardia. The specificity of anti-cyst antibody used in this study is shown in Fig. 2C, which reveals that this antibody recognizes cyst wall proteins (CWP1, CWP2, and CWP3) that are synthesized during encystation.
      Figure thumbnail gr2
      FIGURE 2Knockdown and overexpression of gGlcT1 affects ESV biogenesis. A, Giardia trophozoites were transfected with anti-gGlcT1 morpholino oligonucleotide (50 μm) (
      • Carpenter M.L.
      • Cande W.Z.
      Using morpholinos for gene knockdown in Giardia intestinalis.
      ) and subjected to encystation for 24 h before ESVs were analyzed by confocal microscopy. Control trophozoites were transfected with a scrambled morpholino oligonucleotide sequence (50 μm) supplied by the manufacturer. Discrete ESVs are visible in control cells but are not present in anti-gGlcT1 morpholino-transfected (i.e. gGlcT1 knockdown) cells. Arrows show ESVs, and arrowheads indicate nuclei. Bar, 10 μm. B, gGlcT1-overexpressing (+gGlcT1) trophozoites were subjected to encystation for 24 h, and ESVs were analyzed as described above. gGlcT1 overexpression causes an aggregation/enlargement of the ESVs when compared with control cells that were transfected with empty plasmids. Arrows show ESVs, and arrowheads indicate nuclei. Bar, 10 μm. Inset shows the magnified images of ESVs. C, immunoblot analysis reveals that anti-cyst antibody recognizes cyst-specific proteins (i.e. CWP1, CWP2, and CWP3) expressed during encystation. Troph denotes trophozoites; Pre-En indicates pre-encysting trophozoites; 6 h, denotes 6-h encysting trophozoites; 12 h indicates 12-hr encysting trophozoites; 24 h gGlcT1-knockdown 24-h encysting trophozoites; 48 h indicates 48-h encysting trophozoites.
      Fig. 3A demonstrates that gGlcT1 activity in overexpressed trophozoites increased ∼3-fold as compared with the activity present in control trophozoites (i.e. specific activities increased from 0.5 pmol/min/mg protein in control to 1.4 pmol/min/mg protein in gGlcT1-overexpressing cells). Knockdown by anti-gGlcT1 morpholino oligonucleotide inhibits gGlcT1 activity in trophozoites by ∼50% (from 0.5 to 0.24 pmol/min/mg protein). Furthermore, the rescue experiment (i.e. transfecting gGlcT1-overexpressing trophozoites with anti-gGlcT1 morpholino oligonucleotide) lowered the gGlcT1 activity by ∼40%. Fig. 3B demonstrates that overexpression of gGlcT1 in nonencysting trophozoites causes an increased synthesis of NBD-GlcCer, further suggesting that gGlcT1 activity is directly linked to GlcCer synthesis in Giardia.
      Figure thumbnail gr3
      FIGURE 3Modulation of gGlcT1 activity by overexpression and knockdown. A, stable Giardia cell lines overexpressing GlcT1 enzyme (designated as +GlcT1) were generated by transfecting trophozoites with pNT5-gglct1 (tagged with a small peptide, called AU1) plasmid as described under “Experimental Procedures.” Overexpression increased the synthesis of gGlcT1 activity by ∼3-fold, which could be reduced by transfecting the overexpressed cells with anti-gGlcT1 morpholino oligonucleotide (i.e. gGlcT1-rescued cells). The enzyme activities in gGlcT1-overexpressing, -knockdown, and -rescued cells were measured and the results are presented in mean values ± S.D. of three separate experiments (**, p < 0.01). B, synthesis of NBD-GlcCer by control and gGlcT1 overexpressed (+gGlcT1) trophozoites. Fractions containing NBD-GlcCer were extracted as described under “Experimental Procedures,” dried under N2, and re-dissolved in chloroform. 10 μl of sample was spotted in each lane and visualized under a UV lamp. It was noted that gGlcT1-overexpressing cells take up more ceramides than the control cells. Although the experiments were carried out twice separately with different cell preparations, results (TLC) shown here are from a single study. C, ESI-MS/MS analysis of GSLs. GSLs from control (containing empty plasmid) with, gGlcT1-knockdown (KD), overexpressed (+GlcT1), and rescued trophozoites were extracted and analyzed as described under “Experimental Procedures.” Mono-hexosylceramide (HexCer), di-hexosylceramide (di-HexCer); tri-hexosylceramide (tri-HexCer); the results (pmol/106 cells) presented here are the mean of fold changes (compared with control trophozoites) ± S.D. of three technical replicates (*, p < 0.05; ***, p < 0.001). D, trophozoites expressing AU1-tagged gGlcT1 were monitored by reacting with anti-AU1 antibody followed by labeling with FITC-conjugated anti-mouse antibody and examination under an immunofluorescence confocal microscope. The figure shows that overexpressed gGlcT1-AU1 (shown as +gGlcT1 in the figure) forms structures that are granular, aggregated, and localized throughout the cytoplasm and, to some extent, in the perinuclear regions. However, the overexpressed gGlcT1 granules are also localized in the ventral groove and lateral shield areas (
      • House S.A.
      • Richter D.J.
      • Pham J.K.
      • Dawson S.C.
      Giardia flagellar motility is not directly required to maintain attachment to surfaces.
      ) of the trophozoites. White arrowheads indicate DAPI-stained nuclei (control) and long yellow arrowheads denote ventral groove (vg), lateral shield (ls), and plasma membrane (pm) in overexpressed trophozoites. Bar, 10 μm.
      Next, we asked whether gGlcT1 modulation by overexpression and knockdown also affects the GSL profiles in Giardia. For this, lipids were extracted from overexpressed, knockdown, and rescued trophozoites and subjected to ESI-MS/MS analysis as described under “Experimental Procedures.” Using total ion map and precursor ion scans, we identified both neutral and acidic GSLs that were present in control and gGlcT1-modulated trophozoites. Among the neutral lipids, mono-, di-, and tri-hexosylceramides were identified and found to be altered by gGlcT1 modulation. Although gGlcT1 overexpression increased the amount of mono-hexosylceramide by ∼28-fold, the effect was somewhat less (∼2-fold) on di-hexosylceramides. Interestingly, no effects of gGlcT1 overexpression were observed in the case of tri-hexosylceramides (Fig. 3C). gGlcT1 knockdown, however, elevated the level of mono-hexosylceramides (∼5-fold), and the rescue experiment had no effect, i.e. same as the knockdown (Fig. 3C). Knockdown also decreased the levels of di- and tri-hexosylceramide slightly, and rescue treatment appeared to reverse these effects (Fig. 3C). The analysis of acidic fractions revealed that GM1, GM3, and GD3 lipids are present in control, overexpressed, knockdown, and rescued trophozoites; however, the changes of their levels were not consistent with gGlcT1 modulation (data not shown).
      Because ESV synthesis is linked to gGlcT1 activity (Fig. 2), and the fact that PPMP blocks encystation (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ,
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ), we asked if the modulation of gGlcT1 activity (shown in Fig. 3A) also regulates ESV biogenesis. Fig. 4A, panels b and c, demonstrates that although gGlcT1 overexpression induces the synthesis of aggregated and enlarged ESVs, knockdown of gGlcT1 activity by anti-gGlcT1 morpholino oligonucleotide completely blocks ESV synthesis. More interestingly, the rescue experiment reverses the effects of overexpression and knockdown and generates ESVs (Fig. 4A, panel d) that are comparable with control cells (Fig. 4A, panel a). Again, Fig. 4A, panel e, represents ESVs generated by encysting cells that were treated with scrambled morpholino oligonucleotide, which shows the similar pattern of ESVs found in nontransfected control encysting cells shown in Fig. 4A, panel a. Analysis of individual ESVs is shown in Fig. 4B, demonstrating that gGlcT1 overexpression increases the perimeters and areas of ESVs by ∼6- and ∼10-fold, respectively. The perimeter and area analyses of individual ESVs were carried out with the help of Zeiss Zen 2009 software (Carl Zeiss) as shown in Fig. 4B.
      Figure thumbnail gr4
      FIGURE 4Modulation of gGlcT1 activity affects ESV biogenesis in Giardia. A, ESV production was monitored in the following:(panel a, control; panel b, GlcT1-overexpressing (+gGlcT1); panel c, antisense, morpholino oligonucleotide-treated gGlcT1-knockdown cells; panel d, rescued cells, and panel e, cells transfected with scrambled morpholino oligonucleotides. Arrows indicate ESVs, and arrowheads denote nuclei. Bar, 10 μm. B and C, changes of perimeters and areas of ESVs are shown in the bar graphs. The analyses were carried out by measuring the perimeters and the areas of individual ESVs using Zeiss Zen 2009 confocal software. *, p < 0.05; **, p < 0.01.

      Anti-gGlcT1 Morpholino Oligonucleotide Does Not Interfere with the Growth and Replication of Trophozoites

      PPMP is a common inhibitor of GlcT1 enzyme that has been extensively used to evaluate GlcCer functions in various organisms (
      • Atilla-Gokcumen G.E.
      • Bedigian A.V.
      • Sasse S.
      • Eggert U.S.
      Inhibition of glycosphingolipid biosynthesis induces cytokinesis failure.
      ). It has been reported that PPMP blocks replication and cytokinesis of giardial trophozoites and is thereby thought to inhibit the encystation and cyst production in culture (
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ,
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ). We have shown earlier that PPMP interferes with the formation of cysts when it is added in the culture medium during encystation (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ). Because our current results show that anti-gGlcT1 morpholino oligonucleotide inhibits gGlcT1 activity and ESV biogenesis (FIGURE 3, FIGURE 4), and because the activity of this enzyme is low in trophozoites and up-regulated in encysting cells, we thought that the effect of PPMP-inhibiting replication and cytokinesis on nonencysting trophozoites might not occur via GlcCer synthesis, as thought previously (
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ,
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ). In fact, there are reports in which PDMP (an analog of PPMP) has been shown to block cell cycle progression in mammalian cells by inhibiting cyclin-dependent kinases, which could be independent of GlcT1 inhibition and ceramide accumulation (
      • Rani C.S.
      • Abe A.
      • Chang Y.
      • Rosenzweig N.
      • Saltiel A.R.
      • Radin N.S.
      • Shayman J.A.
      Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases.
      ). To address this possibility in Giardia, trophozoites were transfected with morpholino oligonucleotide (anti-gglct1), and the growth was measured as shown in Fig. 5A. Side by side, the growth of the trophozoites in the presence of PPMP (10 μm) was also conducted, and the results showed that although PPMP affects the growth of trophozoites, anti-gGlcT1 morpholino exhibits no effects (Fig. 5A). Fig. 5B, panel b, demonstrates that PPMP, as shown earlier (
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ,
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ), blocks the replication of trophozoites and generates undivided doublet cells. In contrast, anti-gGlcT1 morpholino did not affect the replication of trophozoites (Fig. 5B, panel c). When morpholino-transfected, gGlcT1-knockdown trophozoites were treated with PPMP, the formation of undivided doublets was observed again (Fig. 5B, panel d). The analysis revealed that although ∼25% of trophozoites form undivided doublets after PPMP treatment, less than ∼5% of doublets was observed in control and anti-gGlcT1 morpholino-treated trophozoites (Fig. 5C). The combined effect of anti-gGlcT1 morpholino and PPMP generated ∼20% doublets, as shown in Fig. 5C. These results indicate that PPMP may not act via gGlcT1 in Giardia and that the blocking of cytokinesis in trophozoites by this inhibitor could be through other mechanisms.
      Figure thumbnail gr5
      FIGURE 5Effects of PPMP and anti-gGlcT1 morpholino on growth and replication of trophozoites. A, assessing the growth of trophozoites. Approximately 2 × 105 cells were inoculated into 4-ml tubes containing TYI-S-33 medium, pH 7.1, supplemented with adult bovine serum and bile. Trophozoites were allowed to grow for 35 h (the doubling time of trophozoites is ∼7 h) and were counted under a microscope. Each experiment was carried out in triplicate, and the experiment was repeated three times. B, differential interference contrast confocal images show the effects of anti-gGlcT1 morpholino oligonucleotide and PPMP on cell division and cytokinesis. Nuclei were stained with DAPI. Control, panel a; 10 μm PPMP, panel b; knockdown with morpholino, panel c; morpholino + PPMP cells, panel d. C, quantitative assessment (e.g. microscopic counts) of doublet trophozoites that are produced by control, PPMP-treated, anti-gGlcT1 morpholino-induced knockdown trophozoites (knockdown) and knockdown (KD) trophozoites + PPMP. At least 100 singlet, doublet, and triplet (the number of undivided triplet trophozoites were small and therefore are not shown in the figure) trophozoites from 10 different fields were viewed and counted under the Zeiss LSM 700 confocal microscope using Zen 2009 software. The percentage values of doublet cells were calculated based on the total number of singlet, doublet, and triplet cells. *, p < 0.05; **, p < 0.01.

      Regulated Expression of gGlcT1 Is Critical for Maintaining Cyst Viability

      The production of viable cysts is essential for Giardia to establish infection in the small intestine of humans. Earlier reports suggested that cyst morphology is directly correlated with cyst viability, and only viable cysts of Giardia produce infection in mice (
      • Schupp D.G.
      • Erlandsen S.L.
      A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.
      ,
      • Schupp D.G.
      • Erlandsen S.L.
      Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy.
      ). Gillin et al. (
      • Gillin F.D.
      • Boucher S.E.
      • Rossi S.S.
      • Reiner D.S.
      Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro.
      ) reported that in vitro-derived G. lamblia cysts are of two kinds, i.e. type I and type II. The type I cysts are oval shaped with uniform and refractive cyst walls, being mostly (∼90%) viable. In contrast, type II cysts lack the morphological characteristics of type I, and a majority (∼70%) of them are nonviable. In a heterogeneous cyst population (i.e. a population composed of both type I and type II cysts), ∼10–30% of cysts exhibit type I morphology (
      • Gillin F.D.
      • Boucher S.E.
      • Rossi S.S.
      • Reiner D.S.
      Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro.
      ,
      • Boucher S.E.
      • Gillin F.D.
      Excystation of in vitro-derived Giardia lamblia cysts.
      ). Because we found that the gGlcT1 activity increased during encystation (Fig. 1) and that the modulation of its function (by overexpression and knockdown) regulated ESV biogenesis (FIGURE 2, FIGURE 3, FIGURE 4), we asked whether gGlcT1 activity is also important for maintaining the cyst morphology and viability. Therefore, trophozoites from various conditions, i.e. control and gGlcT1-overexpressing, gGlcT1 knockdown, and gGlcT1-rescued, were subjected to encystation for 72 h, and the cysts were isolated by centrifugation as described under “Experimental Procedures.” Fig. 6A, panel a, shows control cysts with type I morphology that react with the anti-cyst antibody, which labels the oval-shaped cyst wall. However, this changes significantly in gGlcT1-overexpressing and gGlcT1-knockdown cells (Fig. 6, A, panels b and c). Cysts produced by gGlcT1-overexpressing cells appear to be type II because they are incomplete, clustered, have thin cyst walls, and show minimum reactivity to anti-cyst antibody. Knockdown of gGlcT1 also produces aggregated cryptic cysts; the majority of these cyst-like structures exhibit no reaction to anti-cyst antibody. The rescue treatment, shown in Fig. 6A, panel d, however, produces cysts with well formed cyst walls that react with the cyst antibody. Because the morphology could be an indicative of viability (
      • Schupp D.G.
      • Erlandsen S.L.
      A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.
      ,
      • Boucher S.E.
      • Gillin F.D.
      Excystation of in vitro-derived Giardia lamblia cysts.
      ), our next goal was to test if the viability of cysts is affected by gGlcT1 overexpression and knockdown. The viable and nonviable cysts were identified by staining with fluorogenic dyes, i.e. cell-permeable esterase substrate FDA and the cell impermeant nucleic acid stain PI, which were earlier used by other laboratories to determine the viability of giardial cysts (
      • Gillin F.D.
      • Boucher S.E.
      • Rossi S.S.
      • Reiner D.S.
      Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro.
      ,
      • Schupp D.G.
      • Erlandsen S.L.
      A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.
      ,
      • Schupp D.G.
      • Erlandsen S.L.
      Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy.
      ). Fig. 6B indicates that although in control samples ∼12% of cysts were viable, only ∼8 and ∼3% of cysts were viable in gGlcT1-overexpressing and gGlcT1-knockdown cells, respectively. Most importantly, the rescue experiment, in which the effect of gGlcT1 was neutralized by the anti-gGlcT1 morpholino analog, recovers the cyst and increases the viability up to ∼10%. This is an important observation and strongly suggests that gGlcT1 activity in Giardia not only regulates ESV biogenesis but also maintains the cyst viability. Although it appears that these numbers of viable cysts are somewhat low, our calculation is based on the total number of water-resistant cysts that contain all types of cysts, including type I and type II. Thus, viability estimations, shown here, are within the expected range and in accordance with the report published by Gillin et al. (
      • Gillin F.D.
      • Boucher S.E.
      • Rossi S.S.
      • Reiner D.S.
      Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro.
      ) and Boucher et al. (
      • Boucher S.E.
      • Gillin F.D.
      Excystation of in vitro-derived Giardia lamblia cysts.
      ).
      Figure thumbnail gr6
      FIGURE 6gGlcT1 activity regulates the morphology and viability of in vitro-derived cysts. A, trophozoites were subjected to encystation as described under “Experimental Procedures” for 72 h. Cysts were harvested by centrifugation, kept in cold water (6–10 °C) for 48 h, treated with anti-cyst antibody, and examined under differential interference contrast and confocal microscopy. Panel a, control. Panel b, gGlcT1-overexpressing (+gGlcT1); panel c, gGlcT1-knockdown; and panel d, rescued. Arrows indicate the cyst wall. Cyst nuclei were stained with DAPI. Bar, 10 μm. B, testing the same cyst samples (shown in A) for viability using FDA and PI stains. The cells were also analyzed using differential interference contrast and confocal microscopy with green indicating live and red denoting dead cysts. Panel e, control; panel f, +gGlcT1; panel g, gGlcT1 knockdown, and panel h, rescued. Bar, 10 μm. C, quantification of the viability assay. Approximately 100–150 cysts were examined from 5 to 10 different fields to identify and count the viable cysts. The results were presented in mean values ± S.D. of three separate experiments conducted in different days with different sample preparations. *, p < 0.05.

      gGlcT1 Overexpression Alters Lipid Balance in Trophozoites

      As proposed earlier, Giardia trophozoites have limited abilities to synthesize membrane lipids, cholesterol, and fatty acids de novo and thus depend on supplies from the small intestines, where the trophozoites colonize (
      • Jarroll E.L.
      • Muller P.J.
      • Meyer E.A.
      • Morse S.A.
      Lipid and carbohydrate metabolism of Giardia lamblia.
      ,
      • Das S.
      • Stevens T.
      • Castillo C.
      • Villasenõr A.
      • Arredondo H.
      • Reddy K.
      Lipid metabolism in mucous-dwelling amitochondriate protozoa.
      ). Studies suggest that most of the lipids and fatty acids are taken up by this parasite from outside sources and are utilized for energy metabolism and biogenesis of organelles and vesicles (
      • Lujan H.D.
      • Mowatt M.R.
      • Nash T.E.
      Lipid requirements and lipid uptake by Giardia lamblia trophozoites in culture.
      ,
      • Hernandez Y.
      • Castillo C.
      • Roychowdhury S.
      • Hehl A.
      • Aley S.B.
      • Das S.
      Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.
      ,
      • Yichoy M.
      • Duarte T.T.
      • De Chatterjee A.
      • Mendez T.L.
      • Aguilera K.Y.
      • Roy D.
      • Roychowdhury S.
      • Aley S.B.
      • Das S.
      Lipid metabolism in Giardia: a post-genomic perspective.
      ). Because gGlcT1 overexpression induces the synthesis of enlarged vesicles and produces cysts with reduced viability (Figs. 2, 4, and 6), we investigated whether gGlcT1 expression is associated with increased lipid uptake by Giardia trophozoites. We asked whether gGlcT1 expression is linked to an increased influx of membrane lipids and whether the intracellular levels of cholesterol and fatty acid are altered by gGlcT1, which in turn would affect the membrane fluidity and lipid uptake.

      gGlcT1 Overexpression Affects the Internalization and Intracellular Targeting of Fluorescent Lipids

      Using fluorescently labeled (Bodipy or NBD) lipid and fatty acid analogs, we have shown earlier that trophozoites have the machinery to recruit lipids from their environment and target them into specific cellular locations, including the plasma membrane and endomembranes, as well as the cytoplasm (
      • Stevens T.L.
      • Gibson G.R.
      • Adam R.
      • Maier J.
      • Allison-Ennis M.
      • Das S.
      Uptake and cellular localization of exogenous lipids by Giardia lamblia, a primitive eukaryote.
      ). For example, ceramide and PG were found to be localized at ER/perinuclear membranes, and PC was incorporated into plasma and flagellar membranes of trophozoites. We found exogenous SM were targeted into nuclear and plasma membranes and PE in the inner layer of the plasma membrane (
      • Gibson G.R.
      • Ramirez D.
      • Maier J.
      • Castillo C.
      • Das S.
      Giardia lamblia: incorporation of free and conjugated fatty acids into glycerol-based phospholipids.
      ,
      • Das S.
      • Castillo C.
      • Stevens T.
      Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.
      ). Furthermore, it was shown by us earlier that ceramide is taken up by Giardia via a clathrin-dependent pathway and is likely to be regulated by giardial serine palmitoyltransferase, a rate-determining enzyme of SL biosynthesis (
      • Hernandez Y.
      • Shpak M.
      • Duarte T.T.
      • Mendez T.L.
      • Maldonado R.A.
      • Roychowdhury S.
      • Rodrigues M.L.
      • Das S.
      Novel role of sphingolipid synthesis genes in regulating giardial encystation.
      ,
      • Hernandez Y.
      • Castillo C.
      • Roychowdhury S.
      • Hehl A.
      • Aley S.B.
      • Das S.
      Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.
      ). The internalization of various fluorescent lipid probes, including Bodipy-ceramide, NBD-SM, Bodipy-PG, Bodipy-PC, NBD-PE, and FAST Dil (a common lipid-labeling dye), was studied in control and gGlcT1-overexpressing trophozoites. The results demonstrated that gGlcT1 overexpression increased the uptake and labeling intensities of ceramide, SM, PG, PE, and FAST Dil (Fig. 7A). Interestingly, PE, which is located in the inner plasma membranes, migrates inside the cells and is concentrated in the ER/perinuclear membranes (Fig. 7, A, panels i and j), whereas PC, another major lipid of the plasma membrane, is not affected by gGlcT1 (Fig. 7A, panels e and f). The labeling by FAST Dil (which labels the lipids in plasma and intracellular membranes) is also increased in gGlcT1-overexpressing cells (Fig. 7A, panels k and l). These results suggest that gGlcT1 activity is responsible for the influx of majority of cellular lipids in Giardia.
      Figure thumbnail gr7
      FIGURE 7gGlcT1 overexpression increases the influx of fluorescent lipids in trophozoites. A, trophozoites (control or +gGlcT1) were labeled with various fluorescent lipid probes for 1 h at 37 °C following the methods described under “Experimental Procedures.” Cells were then processed and examined under a confocal microscope. Characteristic localizations of all lipid probes in control (transfected with empty plasmid) and their changes in gGlcT1-overexpressing (+gGlcT1) trophozoites are apparent. The arrows indicate ER/perinuclear regions, and arrowheads denote plasma membranes. All images were captured at the same magnification and resolution gain. Bar, 5 μm. B, fluorescence intensity of each lipid probe in control and overexpressed cells was measured by Zeiss 2009 ZEN confocal software. At least 10 cells were counted from a single field, and a total of 100 cells were counted. The data shown are mean values ± S.D. of three separate experiments (*, p < 0.05). PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; SM, sphingomyelin; CER, ceramide; FAST Dil, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarboxyanine.

      gGlcT1 Overexpression Elevates the Intracellular Levels of Cholesterol and Fatty Acid in Trophozoites

      Like other lipid molecules, Giardia also has a limited ability to synthesize cholesterol and fatty acids de novo, which are obtained from the growth medium. Interestingly, both cholesterol and fatty acids have been shown to be involved in regulating encystation and cyst formation by this waterborne pathogen (
      • Lujan H.D.
      • Mowatt M.R.
      • Nash T.E.
      Lipid requirements and lipid uptake by Giardia lamblia trophozoites in culture.
      ,
      • Luján H.D.
      • Mowatt M.R.
      • Byrd L.G.
      • Nash T.E.
      Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.
      ,
      • Yichoy M.
      • Nakayasu E.S.
      • Shpak M.
      • Aguilar C.
      • Aley S.B.
      • Almeida I.C.
      • Das S.
      Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an early-divergent protozoan, Giardia lamblia.
      ). As gGlcT1 overexpression increased the internalization of fluorescent lipid probes from the medium (Fig. 7), we thought it would be important to examine whether gGlcT1 expression also changes the cellular fatty acid and cholesterol levels. Cholesterol and free fatty acids in control and gGlcT1-overexpressing cells were analyzed by GC-MS. The sterol analysis showed that cholesterol is the major sterol in both control and gGlcT1-overexpressing cells (Fig. 8A), and no other sterols, including cholesteryl esters or ergosterol, were detected (
      • Ellis J.E.
      • Wyder M.A.
      • Jarroll E.L.
      • Kaneshiro E.S.
      Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia.
      ). It is interesting that gGlcT1 overexpression increased the intracellular level of cholesterol by ∼2-fold. Fatty acid analysis (Fig. 8B) revealed that palmitic acid, stearic acid, oleic acid, and linoleic acid were the major fatty acids present in Giardia trophozoites and that gGlcT1 overexpression increased the level of these fatty acids by ∼20, ∼22, ∼25, and ∼38%, respectively. Thus, as with other lipids as shown in Fig. 7, gGlcT1 expression also increased the uptake of cholesterol and fatty acids by Giardia (Fig. 8, A and B)
      Figure thumbnail gr8
      FIGURE 8gGlcT1 overexpression increases cholesterol and fatty acid levels but does not alter membrane fluidity. Control and gGlcT1-overexpressing Giardia were subjected to Folch's partition and subsequent fractionation to separate sterols and fatty acids. A, sterols were extracted in a solvent containing chloroform, methanol, and water as described under “Experimental Procedures” followed by GC-MS analysis. Stigmasterol was added as an external standard to quantify cholesterol. The experiment was carried out in triplicate, and data are shown as relative quantity ± S.D. of two separate experiments (**, p < 0.01). B, fatty acids extracted from Giardia were subjected to methylation and analyzed by GC-MS. The experiment was carried out in triplicate, and data are shown as relative quantity ± S.D. of two separate experiments (***, p < 0.001). C, membrane fluidity was assessed using a fluorescent lipid, pyrene decanoic acid. The monomer/excimer ratio was calculated by normalizing fluorescence intensities. Control (black line, transfected with empty plasmid) and gGlcT1-overexpressed (+gGlcT1) cells (red and green lines, two separate gGlcT1-overexpressing trophozoite samples) are shown. D, changes of membrane fluidity in cold-shock trophozoites. The black line indicates control trophozoites incubated at 25 °C for 30 min, and the red line denotes cold-shock trophozoites kept at 4 °C also for 30 min before conducting the assay as described under “Experimental Procedures.” In both cases (C and D) experiments were repeated several times with different cell preparations, and the results shown here came from a single experiment.
      Because the changing of membrane fluidity may increase lipid uptake by trophozoites, we investigated the possibility that the membrane fluidity could be modified due to the up-regulation of cholesterol and fatty acids by GlcT1, allowing the cells to internalize excess fluorescent lipids as shown in Fig. 7. The membrane fluidity was measured using a fluorescent lipid probe, pyrene decanoic acid, as detailed under “Experimental Procedures.” This pyrene probe usually forms excimer by interacting with membrane components that cause a shift of emission spectrum to a longer wavelength. Therefore, the ratio of monomer to excimer shift could be utilized to measure the change of membrane fluidity (described by the manufacturer, Marker Gene Technologies). Fig. 8C shows that gGlcT1 overexpression did not produce pyrene excimers from monomers. However, the trophozoites treated at 4 °C (cold-shock) showed significant changes from monomer to excimer of the pyrene probe (Fig. 8D). This suggests that the fluidity of trophozoite plasma membranes is not affected by gGlcT1 overexpression, although it increased the uptake of lipid, cholesterol, and fatty acids. We speculate that the increased lipid uptake by gGlcT1-overexpressed trophozoites occurs via receptor- or raft-mediated lipid endocytosis (
      • Lujan H.D.
      • Mowatt M.R.
      • Nash T.E.
      Lipid requirements and lipid uptake by Giardia lamblia trophozoites in culture.
      ,
      • Hernandez Y.
      • Castillo C.
      • Roychowdhury S.
      • Hehl A.
      • Aley S.B.
      • Das S.
      Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.
      ) rather than to the change of membrane fluidity.

      DISCUSSION

      In this study, we show that gGlcT1 activity is important in the production of viable cysts by Giardia and that it acts by regulating ESV biogenesis and controlling the import of lipids and fatty acids from the environment.
      Several strategies were followed to modulate gGlcT1 activity in Giardia. First, gGlcT1-knockdown trophozoites were generated (using anti-gglct1 morpholino oligonucleotide), which reduced the activity of gGlcT1 in both cysts and trophozoites (Figs. 1B and 3A). Second, the gglct1 gene was overexpressed in trophozoites, and this caused an elevation of enzyme activity by ∼3-fold (Fig. 3A). Third, the increased gGlcT1 activity in overexpressed cells was rescued by knocking down the gene using morpholino oligonucleotide, which reduced the excess activity caused by the overexpression of the gglct1 gene (Fig. 3A). We found that the modulation of gGlcT1 activity directly correlated with ESV biogenesis (Fig. 4). When the excess gGlcT1 activity in gGlcT1-overexpressing cells was normalized by introducing anti-gglct1 morpholino oligonucleotide, ESV synthesis was restored and appeared to be normal as judged by confocal microscopy as well as by analyzing the perimeter and area of individual ESVs (Fig. 4). These results suggest that gGlcT1 directly influences the secretion and maturation of ESVs, which are critical for cyst production/viability. This postulation can be further supported by our cyst experiment, in which the rescue treatment generated classical oval-shaped cysts with thick cyst walls that are similar in morphology and viability to control cysts (Fig. 6).
      To identify the possible mechanism how gGlcT1 regulates ESV biogenesis and cyst production, we investigated the overall lipid balance in gGlcT1-overexpressing trophozoites. Our results showed that the elevated gGlcT1 activity caused an increased uptake of mono-hexosylceramides, fluorescent lipids, cholesterol, and fatty acids (Figs. 3C, 7, and 8). The rising levels of mono-hexosylceramide in gGlcT1-overexpressed cells can also be caused by the de novo synthesis of these molecules (catalyzed by an active gGlcT1 enzyme) in addition to the increased uptake from the growth medium (Fig. 3C). Therefore, it is likely that Giardia uses its gGlcT1 enzyme to maintain an overall lipid balance by importing a majority of lipids from outside and synthesizing a selective few (
      • Yichoy M.
      • Duarte T.T.
      • De Chatterjee A.
      • Mendez T.L.
      • Aguilera K.Y.
      • Roy D.
      • Roychowdhury S.
      • Aley S.B.
      • Das S.
      Lipid metabolism in Giardia: a post-genomic perspective.
      ,
      • Gibson G.R.
      • Ramirez D.
      • Maier J.
      • Castillo C.
      • Das S.
      Giardia lamblia: incorporation of free and conjugated fatty acids into glycerol-based phospholipids.
      ,
      • Das S.
      • Castillo C.
      • Stevens T.
      Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.
      ,
      • Luján H.D.
      • Mowatt M.R.
      • Byrd L.G.
      • Nash T.E.
      Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.
      ,
      • Yichoy M.
      • Nakayasu E.S.
      • Shpak M.
      • Aguilar C.
      • Aley S.B.
      • Almeida I.C.
      • Das S.
      Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an early-divergent protozoan, Giardia lamblia.
      ). We propose that the controlled expression of gGlcT1 is critical for lipid internalization in trophozoites because overexpression or knocking down of this enzyme affects ESV biogenesis and cyst viability. The observation that gGlcT1 regulates the overall lipid balance/homeostasis in Giardia was further supported by the report that GlcT1 overexpression influences several body fat storage genes in Drosophila (
      • Kohyama-Koganeya A.
      • Nabetani T.
      • Miura M.
      • Hirabayashi Y.
      Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila.
      ) and also that the interactions between SL and other branches of the lipid metabolic pathways are essential for maintaining lipid homeostasis in mammalian cells (
      • Bektas M.
      • Allende M.L.
      • Lee B.G.
      • Chen W.
      • Amar M.J.
      • Remaley A.T.
      • Saba J.D.
      • Proia R.L.
      Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver.
      ,
      • Pewzner-Jung Y.
      • Park H.
      • Laviad E.L.
      • Silva L.C.
      • Lahiri S.
      • Stiban J.
      • Erez-Roman R.
      • Brügger B.
      • Sachsenheimer T.
      • Wieland F.
      • Prieto M.
      • Merrill Jr., A.H.
      • Futerman A.H.
      A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways.
      ).
      As far as the effect of PPMP on Giardia trophozoites is concerned, it has been reported (
      • Sonda S.
      • Stefanic S.
      • Hehl A.B.
      A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.
      ,
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ) that this inhibitor interferes with the replication and cytokinesis of trophozoites and thereby blocks encystation. This action of PPMP has been attributed to GlcT1 inhibition and intracellular ceramide accumulation (
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ). In contrast, we observed that anti-gglct1 morpholino oligonucleotide treatment did not reduce the growth and replication of Giardia trophozoites (Fig. 5), but it did inhibit ESV biogenesis and cyst production when subjected to encystation. Although we did not measure the intracellular concentration of ceramide after PPMP treatment, there is evidence that another GlcT1 inhibitor, PDMP, can influence the cell cycle regulation by inhibiting cyclin-dependent kinases and not exclusively by inhibiting GlcCer synthesis. Detailed analysis revealed that PDMP treatment caused a reversible decrease in the activity of cyclin-dependent kinases (i.e. cdk2 and p34cdc2), which led to cell cycle arrest (
      • Rani C.S.
      • Abe A.
      • Chang Y.
      • Rosenzweig N.
      • Saltiel A.R.
      • Radin N.S.
      • Shayman J.A.
      Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases.
      ,
      • Norris-Cervetto E.
      • Callaghan R.
      • Platt F.M.
      • Dwek R.A.
      • Butters T.D.
      Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells.
      ,
      • Dijkhuis A.J.
      • Klappe K.
      • Jacobs S.
      • Kroesen B.J.
      • Kamps W.
      • Sietsma H.
      • Kok J.W.
      PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy.
      ). Thus, it is possible that PPMP-induced blocking of trophozoite replication and cytokinesis could be an off-target effect that is caused by the inhibition of giardial cell cycle proteins and kinases (
      • Reiner D.S.
      • Ankarklev J.
      • Troell K.
      • Palm D.
      • Bernander R.
      • Gillin F.D.
      • Andersson J.O.
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      Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point.
      ) rather than the inhibition of GlcT1 and accumulation of ceramide. However, more in-depth experiments should be undertaken to resolve this issue.
      Although in our study it is not clear why cyst stage gGlcT1 is not inhibited by PPMP (Fig. 1), Stefanić et al. (
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ) reported that Giardia synthesizes di- and tri-hexosylceramides in encysting cells and cysts. Hillig et al. (
      • Hillig I.
      • Leipelt M.
      • Ott C.
      • Zähringer U.
      • Warnecke D.
      • Heinz E.
      Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris.
      ) also reported that GCS in plant cells shows broader substrate affinities and functions as a sterol glucoside synthase. Interestingly, gGlcT1 and plant GCS are remarkably similar as far as the amino acid identity and sequence motifs are concerned (
      • Stefanić S.
      • Spycher C.
      • Morf L.
      • Fabriàs G.
      • Casas J.
      • Schraner E.
      • Wild P.
      • Hehl A.B.
      • Sonda S.
      Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.
      ), and therefore, like plant GCS, gGlcT1 may exhibit broader substrate preferences and catalyze the synthesis of di- and tri-hexosylceramides as shown in Fig. 3C. We observed that knockdown of gGlcT1 activity by anti-gGlcT1 morpholino also elevated the levels of mono-hexosylceramides over control trophozoites, which could probably be due to membrane damage caused by the electroporation that allows cells to uptake excess mono-hexosylceramides from the medium (Fig. 3C). This conclusion is based on our observation that the mono-hexosylceramide level also increases in trophozoites that are transfected with scrambled morpholino oligonucleotides (data not shown).
      Earlier studies identified and characterized an enzyme, i.e. cyst wall synthase (N-acetylgalactosaminyltransferase), which is involved in the formation of insoluble N-acetyl-galactosamine homopolymer, a major component of the giardial cyst wall (
      • Jarroll E.L.
      • Macechko P.T.
      • Steimle P.A.
      • Bulik D.
      • Karr C.D.
      • van Keulen H.
      • Paget T.A.
      • Gerwig G.
      • Kamerling J.
      • Vliegenthart J.
      • Erlandsen S.
      Regulation of carbohydrate metabolism during Giardia encystment.
      ). It is likely that cyst gGlcT1 interacts with cyst wall synthase and facilitates the process of cyst production. However, detailed experiments are necessary to characterize the GlcT1 activity in cysts and the classes of complex glycosphingolipids that are formed during encystation. Moreover, there are instances where GSLs have been shown to be involved in maintaining the growth and morphology of mammalian cells. For example, GSL-deficient B16 melanoma cells exhibit altered morphology and slower growth rates (
      • Ichikawa S.
      • Nakajo N.
      • Sakiyama H.
      • Hirabayashi Y.
      A mouse B16 melanoma mutant deficient in glycolipids.
      ). GlcCer synthesis is also necessary for preserving the virulence of infective fungal cells (
      • Noble S.M.
      • French S.
      • Kohn L.A.
      • Chen V.
      • Johnson A.D.
      Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity.
      ,
      • Rittershaus P.C.
      • Kechichian T.B.
      • Allegood J.C.
      • Merrill Jr., A.H.
      • Hennig M.
      • Luberto C.
      • Del Poeta M.
      Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans.
      ). Thus, based on these observations, we propose that gGlcT1, like fungal cells, is an important pathogenic determinant of this parasite and should be targeted for developing new drugs to control giardiasis. The result that gGlcT1 is critical for maintaining cyst viability shown in this study should open a new possibility for investigation of whether gGlcT1-knockdown cysts with reduced viability can also be utilized as a live vaccine candidate to control giardiasis.

      Acknowledgments

      GC-MS, ESI-MS/MS, confocal microscopy, sequencing, and statistical analyses were carried out in the Biomolecule Analysis, Genomic Analysis, Cytometry, Screening and Imaging, and Statistical Consulting Core Facilities at the Border Biomedical Research Center, University of Texas at El Paso, supported by National Institutes of Health Grant G12MD007592 from the National Institutes of Minority Health and Disparity. We thank Dr. Julia Bader for helping us with statistical analysis and Dr. Armando Varela for help with confocal microscopy. We also thank Dr. Chin-Hung Sun (Taiwan) for providing us with pNT5 expression plasmid.

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