Giardia lamblia:Incorporation of Free and Conjugated Fatty Acids into Glycerol-Based Phospholipids

Experimental Parasitology 92, 1–11 (1999) Article ID expr.1999.4389, available online at http://www.idealibrary.com on

Giardia lamblia: Incorporation of Free and Conjugated Fatty Acids into Glycerol-Based Phospholipids

George R. Gibson, David Ramirez, Julie Maier, Cynthia Castillo, and Siddhartha Das1 Department of Biological Sciences, University of Texas, El Paso, Texas 79968-0519, USA

Gibson, G. R., Ramirez, D., Maier, J., Castillo, C., and Das, S. 1999. Giardia lamblia: Incorporation of free and conjugated fatty acids into glycerol-based phospholipids. Experimental Parasitology 92, 1–11. Giardia lamblia trophozoites are flagellated protozoa that inhabit the human small intestine, where they are exposed to various dietary lipids and fatty acids. It is believed that G. lamblia, which colonizes a lipidrich environment of the human small intestine, is unable to synthesize phospholipids, long-chain fatty acids, and sterols de novo. Therefore, it is possible that this protozoan has developed a special process for acquiring lipids from its host. We have previously shown that G. lamblia can take up saturated fatty acids and incorporate them into phosphatidylglycerol (PG) and other glycerol-based phospholipids (Stevens et al., Experimental Parasitology, 86, 133–143, 1997). In the present study, an attempt has been made to investigate the underlying mechanisms of transesterification and interesterification reactions of giardial phospholipids by free and conjugated fatty acids. Results show that exogenously supplied, unsaturated, fatty acids were taken up by Giardia and incorporated into various phosphoglycerides, including PG. To test whether this intestinal pathogen can utilize conjugated fatty acids, live trophozoites were exposed to either [3H]phosphatidylcholine (PC), where the fatty acid was 3H-labeled at its sn2 position, or to [14C]lyso-PC (fatty acid was 14C-labeled at the sn1 position) for 90 min, followed by phospholipid analysis using thin-layer chromatography. The results suggest that conjugated fatty acids, like free fatty acids, were incorporated into PG. It was also observed that aristolochic acid, an inhibitor of Ca21-ionophore-stimulated phospholipase A2, decreased the transfer of fatty acids from [3H]PC to PG, indicating that giardial phospholipases were involved in these esterification reactions. Additional experiments, which include culturing trophozoites in serumsupplemented and serum-deprived medium, along with numerous biochemical analyses suggest that (i) PG is a major transesterified and interesterified product, (ii) it is likely that giardial phospholipases are

involved in esterification reactions, (iii) in G. lamblia, PG is localized in perinuclear membranes, as well as intracellularly, but not in the plasma membrane, and (iv) various synthetic analogs of PG inhibit the growth of the parasite in vitro. These studies suggest that PG is an important phospholipid of Giardia and a potential target for lipidbased chemotherapy against giardiasis. q 1999 Academic Press Index Descriptors and Abbreviations: Giardia lamblia; phospholipids; fatty acids; phospholipases; aristolochic acid; NBD, N-7-nitrobenz2-oxa-1,3-diazole; AA, arachidonic acid; PA, palmitic acid; MA, myristic acid; OA, oleic acid; PG, phosphatidylgycerol; PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; FFA, free fatty acid; TLC, thin-layer chromatography; PBS, phosphate-buffered saline; LPC, lipoprotein–cholesterol mixture; POPG, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphorylglycerol; DMPG, 1,2-dimyristoyl-snglycero-3-phosphorylglycerol; DPPG, 1,2-dipalmitoyl-sn-glycero-3phosphorylglycerol; DSPG, 1,2-distearoyl-sn-glycero-3-phosphorylglycerol; PLA1, phospholipase A1; PLA2, phospholipase A2.

INTRODUCTION

Giardiasis is a commonly diagnosed waterborne intestinal disease worldwide (Adam 1991). Infection with Giardia lamblia can be asymptomatic or symptomatic with severe diarrhea and fat malabsorption (Hartong et al. 1979). It has been proposed that the growth of Giardia is sustained by lipids and lipid-related components present in the small intestine, with conjugated bile acids playing a major role (Jarroll et al. 1981; Farthing et al. 1985; Gillin et al. 1986; Mohareb et al. 1991; Das et al. 1997). Fatty acids and bile salts are also important for encystation (Gillin et al. 1987).

1 To whom correspondence should be addressed at Department of Biological Sciences, University of Texas, 500 W. University Avenue, El Paso, TX 79968-0519. Fax: (915) 747-5808. E-mail: [email protected].

0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

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2 Reports suggest that exogenously obtained lipids and fatty acids may participate in various metabolic events in Giardia. For example, [3H]arachidonic acid is incorporated into a wide variety of neutral lipids and phospholipids, while [3H]palmitic acid is incorporated mainly into glycerides and phosphatidylinositol (PI) (Blair and Weller 1987). Exogenously supplied [3H]palmitic acid is incorporated into GP49, an invariant surface antigen of G. lamblia (Das et al. 1991), as well as into a 90-kDa variant-specific surface protein of G. duodenalis (Papanastasiou et al. 1997). Lujan et al. (1995) reported that various proteins in Giardia undergo isoprenylation. More recently, Ellis et al. (1996) reported the expression of fatty acid desaturase activity in G. lamblia during encystation. In higher eukaryotes, extra- and intracellular sterols, fatty acids, and phospholipids are important for maintaining membrane architecture and assembly (Vial and Ancelin 1992). The concentration of free and esterified cholesterol (as well as related sterols) determines membrane fluidity in yeast (Yang et al. 1996). Similarly, fatty acid composition influences the phospholipid esterification in mouse macrophage cells. Enrichment of phospholipids with saturated or unsaturated fatty acids also regulates macrophage adhesion (Calder et al. 1990). Using fluorescent and radiolabeled lipid probes, we have recently shown (Stevens et al. 1997) that G. lamblia is able to carry out selective uptake, compartmentalization, and stage- or analog-specific localization of exogenous lipids. Radiolabeled fatty acids are esterified into cellular phospholipids of G. lamblia. Detailed analysis revealed that phosphatidylglycerol (PG) is a major esterified product, followed by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and PI, indicating the ability of Giardia to carry out transesterification reactions (Stevens et al. 1997). The present investigation was undertaken to study the transesterification (e.g., exchange of acyl groups between a fatty acid and an ester) and interesterification (i.e., exchange of fatty acyl moieties between two esters) of phospholipids by fatty acids. We found that PG was a major phospholipid, transesterified and interesterified by free and conjugated fatty acids. Moreover, we have observed that various synthetic analogs of PG were toxic to Giardia and inhibited the growth of trophozoites in vitro. These studies along with previous reports (Jarroll et al. 1981; Blair and Weller 1987; Stevens et al. 1997) suggest that G. lamblia, due to its limited lipid synthesis capability, may rely more on exogenous sources of fatty acids and phospholipids to supply precursors, which ultimately comprise an integral structural component of the trophozoite’s plasma and endomembranes.

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MATERIALS AND METHODS Materials. [9,10-3H]Palmitic acid (185 MBq), [9,10-3H]myristic acid (185 MBq), [9,103H(N)]oleic acid (37 MBq), [5,6,8,9,11,12,14,153 H(N)]arachidonic acid (1. 85 MBq), [2-palmitoyl-9,10-3H(N)]phosphatidylcholine (9.25 MBq), lyso-palmitoyl phosphatidylcholine-L-1[palmitoyl-1-14C] (370 MBq), and autoradiographic films were obtained from Dupont–New England Nuclear (Boston, MA). Aristolochic acid was purchased from Biomol (Plymouth Meetings, PA). Phosphatidylcholine, phospholipids, and natural and synthetic phosphatidylglycerols were obtained from Matreya Inc. (Pleasant Gap, PA). Other fine chemicals were purchased from Sigma Chemicals (St. Louis, MO). The lipoprotein–cholesterol mixture was obtained from ICN Biochemicals (Costa Mesa, CA). NBD-phosphatidylglycerol was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). NBD-sphingomyelin was obtained from Molecular Probes (Eugene, OR). Parasite. Giardia lamblia. WB (ATCC 30597) clone C6 trophozoites were grown in tissue culture flasks to late log phase in Diamond’s TYI-S-33 medium (Diamond et al. 1978) with bovine bile (500 mg/ ml) and serum (10%) as previously described (Keister 1983; Gillin et al. 1988). Attached trophozoites were separated from nonattached trophozoites by discarding the growth medium and refilling with labeling buffer (PBS 1 5 mM cysteine 1 2 mM ascorbic acid 1 5 mM glucose, pH 7.1). Chilled cells were harvested by centrifugation at 1500g for 10 min at 48C, washed twice, and resuspended in fresh labeling buffer. Trophozoites were also grown in serum-deprived growth medium, with bovine serum replaced with either PC (42 mM) or a lipoprotein–cholesterol–BSA mixture (LPC, 0.4% v/v, and 0.1% BSA) following the protocols previously described by Gillin et al. (1986) and Reiner et al. (1995). Radiolabeling experiments. Chilled G. lamblia trophozoites were harvested by centrifugation (1500g for 10 min at 48C), washed, and resuspended in labeling buffer as described above. Radiolabeled fatty acids (stock solutions) were dried under a N2 stream and resuspended in a minimum volume of absolute ethanol. Approximately 1 3 108 trophozoites, in duplicates, were incubated with radioactive fatty acids (1-ml final volume). Uptake was initiated by addition of 25 mM (,1 3 106 dpm) of 3H-labeled-fatty acids (saturated and unsaturated) and incubated for 0, 10, 20, 30, 60, and 90 min at 378C with shaking (Balsinde et al. 1995). The radiolabeled cells were isolated by centrifugation (5000g, for 10 min at 48C) in a microfuge and washed three times with cold PBS containing 0.5% BSA (fat-free). The cell pellets were resuspended in the same labeling buffer (100 ml), and the radioactivity was measured. For labeling with phospholipids, trophozoites (,1 3 108) were preincubated for 1 h at 378C and the viability was monitored by trypan blue-exclusion procedure and/or by attachment assay (Das et al. 1988). In preliminary experiments we found that efficient labeling of trophozoites by phospholipids was dependent on the preincubation of cells in lipid-free labeling buffer, which was maximal around 60 min of incubation. We hypothesize that preincubation in lipid-free buffer allows the parasites to use endogenous lipids and thus increases the internalization of radioactive phospholipids. At the end of the preincubation, trophozoites were washed, resuspended in the same labeling buffer (0.5-ml final volume), checked for viability, and incubated with either 2-palmitoyl [3H]phosphatidylcholine (10 mM, 20 mCi/ml) or 1palmitoyl [14C]lysophosphatidylcholine (10 mM, 5 mCi/ml) for 1 h at 378C with shaking. After labeling, cells were centrifuged (5000g for 5 min at 48C), washed, and stored at 2208C until further use.

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Pulse–chase experiment. Approximately 3 3 108 trophozoites were pulse-labeled with [3H]palmitic acid (30 mCi) for 1 h at 378C (3 ml, final volume). After pulse-labeling, cells were washed by centrifugation (5000g for 10 min at 48C), resuspended in cold labeling buffer (48C), and divided into six separate microfuge tubes (0.5 ml, ,5 3 107 cells/ tube), prior to chase for various time points, i.e., 0, 1, 2, 3, 5, and 6 h. After the chase period, cells were separated by centrifugation, washed, and stored at 2208C until further use. Extraction of phospholipids. After radiolabeling with fatty acids or phospholipids, trophozoites were resuspended in 800 ml of 0.1 M KCl and subjected to three freeze–thaw cycles (Jarroll et al. 1981). Lipids were extracted by the addition of 2 ml absolute methanol and 1 ml chloroform to the sample, which was then incubated at 48C for 2 h. The extraction mixture was centrifuged at 2500g for 20 min (0–48C), and the supernatant was saved. The pellets were resuspended in 800 ml of 0.1 M KCl, and the extraction was repeated twice. Supernatants (from three extractions) were pooled and mixed with enough chloroform and 0.1 M KCl to achieve a final methanol:chloroform:KCl ratio of 1:1:0.9 (v/v/v). The chloroform layer was separated by centrifugation (1000g for 2 min), dried under N2, and stored in 250 ml of a chloroform:methanol mixture (19:1; v/v) at 2208C until further use. Separation and identification of phospholipids by thin-layer chromatography. Giardial lipid extracts (amount specified in the text) were used to analyze phospholipids by either one- or two-dimensional thinlayer chromatography (TLC). For two-dimensional chromatography, samples were separated in the first dimension by chloroform:methanol:ammonium hydroxide:water (60:50:1:4; v/v/v/v) and in the second dimension by chloroform:acetone:methanol:acetic acid:water (80:30:26:24:14; v/v/v/v/v), as described by Traynor-Kaplan et al. (1989). For the one-dimensional analysis, samples were separated using chloroform:acetone:methanol:acetic acid:water (80:30:26:24:14; v/v/ v/v/v). Phospholipids were identified by staining with iodine vapor and by comparison to respective standards. Quantitative analyses of 3 H-labeled phospholipids were performed by scraping the identified spots from TLC plates and counting the radioactivity using liquid scintillation spectrophotometry or by densitometric scanning of the autoradiogram. Identification of phospholipids by head group staining. Phospholipids were separated in one or two dimensions on TLC plates and sprayed with stains specific for various head groups. Phospholipids containing free amino groups (i.e., PE) were detected using ninhydrin reagent; choline-containing lipids (i.e., PC) were identified by Dragendorff’s stain, and PG was identified by Schiff’s base reaction (Christie 1982; Higgins 1990). HPLC analysis. Phospholipids were first identified by one-dimensional TLC and isolated by scraping the spot. The isolated PG was extracted in chloroform:methanol:H2O (65:25:4; v/v/v) and injected (20 ml) into HPLC (Hewlett-Packard) with a normal phase diol column for separation. Fractions were isolated by triple-gradient elution (80:19.5:0.5 of CHCl3:methanol:acetone to 60:39.5:0.5 of CHCl3:methanol:acetone to 60:34:5.5:0.5 of CHCl3:methanol:water:acetone by volume) (flow 1 ml/min) and detected by an evaporative lightscattering detector. Treatment with aristolochic acid. Giardia trophozoites were resuspended in labeling buffer preincubated for 1 h at 378C. Preincubated cells were harvested by centrifugation (1500g for 5 min at 48C), resuspended in the same labeling buffer (5 3 107 cells/2ml), mixed with 250 mM aristolochic acid (final volume 2.1 ml), and incubated for 30 min at 378C with shaking. The concentration of aristolochic acid was determined from a separate dose-dependent experiment. Radiolabeled

PC or lyso-PC was added (as specified in the text) to the incubation mixture, and the incubation was continued for 1 h. Inhibitor-treated (as well as control) trophozoites were separated by centrifugation, washed, tested for viability (described below), and stored at 2208C until further use. The viability of trophozoites after preincubation, radiolabeling, and inhibitor teatment was monitored by attachment assay (Das et al. 1988) and/or by the trypan blue exclusion method. Treatment with phospholipid analogs. Four synthetic phosphatidylglycerol analogs, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylglycerol (POPG), 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), and 1, 2-distearoyl-sn-glycero-3-phosphorylglycerol (DSPG), were tested on the in vitro growth of G. lamblia. Analogs were solubilized in absolute methanol. Various concentrations (0–350 nM) of analogs were transferred to microfuge tubes, dried under a N2 stream, and resuspended in absolute ethanol (10 ml) before the treatment. Trophozoites were harvested, washed, and resuspended in labeling buffer before the preincubation (1 h at 378C). Preincubated trophozoites were isolated by centrifugation (1500g for 5 min at 48C), resuspended in labeling buffer (1.5 3 105 cells/0.5 ml), and treated with various concentrations (0, 70, 210, and 350 nM) of analogs in 10 ml of absolute ethanol for 1 h at 378C with shaking. At the end of the incubation the inhibitortreated cells were mixed with Diamond’s TYI-S-33 medium, supplemented with bovine serum and bile (5 ml, final volume), and incubated overnight at 378C. Tubes were chilled in ice-cold water for 45 min to detach the trophozoites before counting. Labeling with fluorescent conjugated phosphatidylglycerol. G. lamblia trophozoites (1 3 107 cells/ml) were harvested, washed, and resuspended in labeling buffer prior to incubation with NBD-PG and NBD-sphingomyelin (SM). NBD (fluorophore 4-nitrobenz-2-oxa-1, 3diazole) conjugated PG and SM were dissolved in PBS, added to the cell suspension (1 mM final concentration), and incubated for 30 min at 378C. Labeled trophozoites were allowed to attach to glass slides for 30 min at 378C and fixed with methanol-free formaldehyde (4% in PBS) for 15 min. Slides were rinsed three times in PBS, and coverslips were mounted with mounting medium (DAKO Corp., Carpenteria, CA). Slides were allowed to dry and examined by epifluorescence and confocal microscopy. Identification of phospholipids by autoradiography. Radiolabeled phospholipids (esterified by radiolabeled fatty acids) were separated by TLC as described above. After air drying, TLC plates were exposed on X-ray film (Du Pont) for 10–12 weeks (at 2208C) prior to development.

RESULTS Uptake/Transport of Free Fatty Acids by Giardia Trophozoites Long-chain, nonesterified, free fatty acids are precursors of triglycerides and other lipid components of cell membranes. They are also involved in transducing intracellular signals for gene expression. The process of exogenous fatty acid movement across the plasma membranes is, however,

4 poorly understood (Berk 1996). Since trophozoites are exposed to high concentrations of fatty acids in the human small intestine (Gillin et al. 1987), we measured the uptake/ transport of saturated and unsaturated fatty acids by G. lamblia following the protocol described by Balsinde et al. (1995). Results show that fatty acids are taken up by G. lamblia (Fig. 1); however, the rate of AA uptake/transport was three- to fivefold higher than that in the other fatty acids tested. The uptake of AA reached a maximum (,50–55 nmol/108 trophozoites) at around 15–20 min of incubation and then declined. The rate of palmitic acid uptake/transport increased slightly faster than that of myristic and oleic acids and reached a maximum (,15 nmol/108 trophozoites) at 20–30 min of incubation of live trophozoites with the radioactive fatty acids. Incorporation of Free Fatty Acids into Cellular Phospholipids Since phospholipids are major components of plasma and endomembranes and transesterification of phospholipids by

FIG. 1. Relative uptake/transport of saturated and unsaturated fatty acids by G. lamblia. Trophozoites were harvested, washed, and resuspended in labeling buffer prior to preincubation as described under Materials and Methods; Preincubated trophozoites (,1 3 108) were then mixed with 3H-labeled fatty acids (25 mM; ,1 3 106 dpm), and reactions were carried out for various time points (0–90 min) at 378C before the radioactivity was counted. v indicates arachidonic acid, m denotes palmitic acid, . shows myristic acid, and m indicates oleic acid. The data presented are means of four individual experiments. The standard error bars (1–11%) have been omitted for clarity.

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saturated and unsaturated fatty acids is important for maintaining membrane architecture, we asked whether exogenous fatty acids, once taken up by Giardia, are incorporated into membrane phospholipids. In this experiment trophozoites were metabolically labeled with radioactive AA, PA, MA, and OA, and phospholipids were extracted and analyzed by one-dimensional TLC following the protocol described under Materials and Methods. Figure 2A shows giardial phospholipids after staining with iodine vapor. The relative migration of individual phospholipids on TLC was identified by comparing with authentic standards, head group staining, and treatment with phospholipases (Stevens et al. 1997). Figure 2B is the autoradiogram of the same TLC shown in Fig. 2A, which demonstrates that both saturated and unsaturated fatty acids are transesterified into various phospholipids. Relatively larger portions of these fatty acids were transesterified into PG, indicating that PG is a major transesterified product in trophozoites. Figure 2B also shows that the extent of fatty acid incorporation into giardial phospholipids is higher for MA, PA, and OA than AA. In a separate experiment, we observed (not shown) that PG is also a major esterified product when trophozoites are cultured in serumdeprived medium supplemented with PC or LPC–albumin

FIG. 2. Transesterification of giardial phospholipids by free fatty acids. Cells were grown in bovine serum and bile-supplemented TYIS-33 medium. Trophozoites (,108 cells) were harvested and incubated with [3H]arachidonic acid (25 mM; ,1 3 106 dpm), [3H] palmitic acid (25 mM; ,1 3 106 dpm), [3H]oleic acid (25 mM; ,1 3 106 dpm), and [3H]myristic acid (25 mM; ,1 3 106 dpm). Phospholipids were extracted, applied (,20 ml) on TLC, and separated using chloroform: acetone: methanol: acetic acid: water (80:30:26:24:14), as described previously by Traynor-Kaplan et al. (1989). (A) Giardial phospholipids after staining with iodine-vapor. (B) The autoradiogram of the same TLC that was exposed to X-ray film for 12 weeks before development. AA; arachidonic acid; PA, palmitic acid; MA, myristic acid; OA, oleic acid. Individual phospholipids were identified by calculating Rf values and head group staining.

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mixture (Gillin et al. 1986; Reiner et al. 1995). This suggests that esterification of PG by fatty acids is independent of serum. Pulse–chase experiments suggest that PG is a stable transesterified product and was not metabolized within 6 h of chase at 378C (not shown). Cardiolipin, a PG dimer, often comigrates with PG on TLC. Therefore, we asked whether giardial PG is cardiolipin. To test this, PG was isolated from a TLC plate by scraping the spot, extracting in chloroform:methanol:H2O (65:25:4; v/v/v), and separating by HPLC, using a standard normal phase diol column. Fractions were collected by a triplegradient elution procedure as discussed under Materials and Methods. Reference standards (and retention times) included PG (11.787 min), cardiolipin (12.727 min), and PE (15.656 min). Giardial PG was eluted at 11.877 min; however, no cardiolipin could be detected (not shown).

Uptake and Cellular Localization of NBD-PG by Giardia PG is a major phospholipid (12–22%) in bacterial membranes (Yeagle 1993). In mammalian cells, PG is found in many intracellular locations as a minor component of cellular phospholipids, representing less than 1% of total lipid phosphorous, except in the lamellar body fraction of the lung, where it represents about 10% of the total phospholipids (Ohtsuka et al. 1993). Schlame et al. (1993) reported that cardiolipin is found primarily in mitochondrial membranes and is synthesized in mitochondria of higher eukaryotes. Since G. lamblia is considered a primitive eukaryote lacking mitochondria and typical eukaryotic organelles (Hashimoto et al. 1994), we investigated the uptake and cellular localization of PG by trophozoites. To test this, G. lamblia trophozoites were incubated with NBD-PG (1 mM) for 30 min at 378C and fixed before microscopic examinations. The labeling of NBD-PG was also compared with NBD-SM. Figure 3 shows that both lipids are taken up by Giardia and localized in perinuclear/nuclear regions as well as intracellularly (Figs. 3A and 3C). Figures 3B and 3D show corresponding DIC images. Confocal microscopy (Fig. 4) demonstrates that a major portion of NBD-PG is localized in the perinuclear/ nuclear regions of nonencysting trophozoites. Interestingly, NBD-SM is concentrated in the plasma membranes (inner leaflet), in nuclear membranes, and in the cytoplasm (Fig. 4B). Figure 4 suggests that the labeling patterns of PG and SM are specific, and lipid moieties (not fluorophores) are crucial for their cellular localization (Stevens et al. 1997).

Incorporation of Conjugated Fatty Acids into Cellular Phospholipids Next, we asked whether G. lamblia trophozoites could utilize conjugated fatty acids to interesterify some of its phospholipids. To test this, live trophozoites were incubated with [3H]PC (where palmitic acid at the sn2 position was radiolabeled) and/or [14C]lyso-PC (where the fatty acid moiety at its sn1 position was 14C-labeled) for 90 min, followed by extraction and separation of phospholipids as described under Materials and Methods. Results (Fig. 5) show that both sn2-palmitic acid of [3H]PC (lane A) and sn1-palmitic acid of [14C]lyso-PC (lane B) are hydrolyzed from original molecules and incorporated into PG. Figure 5 (lane C) also demonstrates that when trophozoites are incubated with a [3H]PC and [14C]lyso-PC mixture, both 3H- and 14C-labeled fatty acids (from sn1 and sn2 positions, respectively) are incorporated into PG. Since phospholipases are involved in modifying the fatty acid compositions in phospholipids and sn2-palmitic acid of [3H]PC is hydrolyzed off and transferred to giardial PG (shown in Fig. 6), we asked whether aristolochic acid (8methoxy-6-nitrophenanthro [3, 4-d]-1, 3-dioxole-5-carboxylic acid) could affect this transfer. In this experiment, trophozoites were incubated with 250 mM aristolochic acid for 30 min at 378C (which did not affect the viability—please see Materials and Methods) with shaking. The dose and duration of aristolochic acid treatment were determined from separate experiments (data not shown). [3H]PC (10 mM, 20 mCi/ml) or [14C]lyso-PC (10 mM, 5 mCi/ml) was added to the incubation mixture, and incubation was continued for 60 min. Inhibitor-treated (as well as control) trophozoites were separated by centrifugation before the extraction and analysis of phospholipids. It is clear from Fig. 7 that aristolochic acid inhibits (,55%) the transfer (interesterification reaction) of sn2-palmitic acid from [3H]PC to giardial PG. Synthetic PG Analogs The final question we asked was whether synthetic PG analogs were toxic to trophozoites. The preincubated trophozoites were treated with PG analogs as described under Materials and Methods. Results show that DPPG, DSPG, and DMPG were toxic to Giardia (LD50 ,200 nM) and inhibited the growth of the parasite in vitro (Fig. 8). At a lower concentration (i.e., 70 nM), DSPG was more effective (,40% inhibition) than DMPG and DPPG. However, at higher doses (i.e., 210 and 350 nM) these inhibitors were equally effective. Approximately 80% inhibition occurred at 350 nM. Interestingly, POPG was least effective (Fig. 8).

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FIG. 5. Esterification of PG by conjugated fatty acids. Trophozoites (,1 3 108 cells) were incubated separately with [3H]PC, [14C]lysoPC, and a mixture of [3H]PC and [14C]lyso-PC as desribed previously. Although in this experiment standard phospholipids show faster migration, the position of giardial phospholipids was identified by calculating Rf values and head group staining. Lane A; interesterified phospholipids after labeling with [3H]PC (20 mCi); Lane B, labeling with [14C]lysoPC (5 mCi); and Lane C, labeling with a mixture of [3H]PC (15 mCi) and [14C]lyso-PC (5 mCi). PC, phosphatidylcholine; PI, phosphatidylinositol; SM, sphingomyelin; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; FFA; free fatty acids.

DISCUSSION

Previous reports suggest that PE, PC, and SM are the major phospholipids in Giardia (Jarroll et al. 1981; Mohareb et al. 1991; Kaneda and Goutsu 1988). In this investigation, we found that a fourth phospholipid, PG, is also present in trophozoites (Fig. 2A). In addition, this study demonstrates that free and conjugated fatty acids are incorporated into PG

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FIG. 6. Proposed pathways showing the possible involvement of giardial phospholipases (PLA1 and PLA2) in transferring sn1 and sn2 fatty acids from [3H]PC and/or [14C] lyso-PC to PG and other glycerolbased phospholipids.

and other cellular phospholipids by G. lamblia trophozoites. These results are in agreement with those of Blair and Weller (1987), who have shown that [3H]palmitic and [3H]arachidonic acids are incorporated into various phospholipids including PI, PE, and PC. Their report, along with the present observation, suggests that Giardia has developed the ability to take up and/or transport free fatty acids across the plasma membrane (Fig. 1) and incorporate them into cellular lipids by transesterification reactions (Aldercreutz 1994). We

FIG. 3. Epifluorescence microscopic examination (FITC channel; 100X oil) and corresponding DIC images of nonencysting G. lamblia trophozoites. Trophozites were labeled with NBD-PG or NBD-SM, allowed to attach to glass slides, fixed with methanol-free formaldehyde, and examined by epifluorescence microscopy as described under Materials and Methods. (A) NBD-PG and (C) NBD-SM. (B) and (D) are corresponding DIC images. Arrows indicate plasma membranes and arrowheads show nuclei. Bar, 5 mm. FIG. 4. Confocal microscopy showing the localization of NBD-PG and NBD-SM in G. lamblia. Trophozoites were harvested, washed, and resuspended in labeling buffer, as described under Materials and Methods. Approximately 1 3 107 cells were incubated with NBD-PG or NBDSM (1 mM final concentration) for 30 min at 378C and allowed to attach to glass slides prior to examination by confocal microscopy (Olympus BX 50; 60X oil). Results show that NBD-PG (A) is localized in perinuclear/nuclear regions (arrowheads) as well as intracellularly. Arrow indicates plasma membrane. Bar, 2.0 mm. (B) Localization of NBD-SM in nonencysting trophozoites. Results document that NBD-SM is localized in the plasma membrane (arrow) and in the cytoplasm as well as in the perinuclear/nuclear regions (arrowheads). Bar, 2.0 mm.

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FIG. 7. Decreased interesterification of [3H]PG by aristolochic acid. Trophozoites were initially treated with aristolochic acid (250 mM) and then incubated with [3H]PC (10 mM) described under Materials and Methods. Phospholipids were extracted from control and treated samples and analyzed by TLC. For quantitation, radiolabeled phospholipids were isolated from TLC by scraping “hot spots” and measuring radioactivity. PC, phosphatidylcholine; PG; phosphatidylglycerol; PE, phosphatidylethanolamine; FA, fatty acid. Although data shown were from one experiment, similar results were obtained in two separate experiments. V indicates untreated and v indicates aristolochic acidtreated cells.

found that both saturated and unsaturated fatty acids were largely incorporated into PG (Fig. 2B). Interestingly, AA (20:4), which was taken up by Giardia rapidly (Fig. 1), showed the lowest level of incorporation into PG and other phospholipids (Fig. 2 B). This can be explained by the fact that AA is incorporated not only into phospholipids but into a wide range of cellular lipids as well. On the other hand, PA (16:0), MA (14:0), and OA (18:1) are transesterified mainly into phospholipids (Blair and Weller 1987). PG was also found to be a major transesterified product in trophozoites cultured in serum-deprived (PC or LPCalbumin supplemented) medium (not shown), suggesting that this glycerol-based phospholipid can be derived from other phospholipids present in the growth medium. This hypothesis can further be supported by our recent findings that PG is not present in bovine serum or bile (not shown). It is possible that PG in Giardia can also be synthesized de novo. Using fluorescence conjugated lipid probes, we have shown previously (Stevens et al. 1997) that encysting and nonencysting trophozoites are able to carry out selective uptake, compartmentalization, and stage- or analog-specific localization of exogenous lipids. For example, in nonencysting cells PC was localized in the plasma membranes,

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FIG. 8. Effects of PG analogs on in vitro growth of trophozoites. Preincubated trophozoites were treated with various concentrations of analogs for 1 h at 378C as described under Materials and Methods. Inhibitor-treated and control cells were inoculated into 5-ml culture tubes containing TYI-S-33 medium, supplemented with bovine bile and serum, and incubated overnight at 378C. Tubes were chilled in ice-cold water for 45 min to detach trophozoites before counting. v indicates POPG; m denotes DMPG; m and . show the inhibition of growth by DPPG and DSPG, respectively. The data presented are means of three separate experiments with standard errors between 5 and 12%.

whereas ceramide was localized intracellularly. Like other lipids, exogenously supplied PG is taken up by Giardia trophozoites and is concentrated mostly in perinuclear/nuclear regions. A substantial amount of NBD-PG is also localized intracellularly (Figs. 3 and 4A). In contrast, NBD-SM is localized in the inner leaflet of plasma membranes and nuclear/perinuclear regions, as well as intracellularly (Figs. 3 and 4B). The localization of NBD-PG and SM in perinuclear/ nuclear regions, which have recently been identified as the endoplasmic reticulum (ER) of Giardia (Soltys et al. 1996), suggests that the ER may be responsible for carrying out intracellular trafficking/metabolism of exogenous phospholipids in nonencysting trophozoites. In a separate experiment we found that Brefeldin A, which induces the rapid redistribution of Golgi into the ER in eukaryotic cells (Shah and Klausner 1993), alters the localization of fluorescent lipids in encysting cells but not in nonencysting trophozoites (not shown), indicating that the mechanism of lipid movement in encysting and nonencysting trophozoites may be different. It is possible that Golgi or Golgi-like organelles induced

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during encystation (Reiner et al. 1990) may be involved in lipid trafficking in encysting Giardia. We also observed that PG, unlike PC, does not support the growth of the parasite in serum-deprived medium (not shown). One possible explanation is that PC is a major phospholipid of the giardial plasma membranes (Stevens et al. 1997), whereas PG is found mainly in perinuclear and other endomembranes (Figs. 3 and 4). Figure 5 shows that G. lamblia can utilize conjugated fatty acids to esterify some of its phospholipids, including PG, most likely by interesterification reactions (Aldercreutz 1994). It is clear from Fig. 5 that when a mixture of [14C]lysoPC and [3H]PC was used, both 14C- and 3H-labeled fatty acids could be cleaved and transferred to giardial PG, indicating both sn1 and sn2 fatty acids from lyso-PC and PC can be hydrolyzed off and transferred to PG as demonstrated in Fig. 6. Gillin et al. (1986) found that lecithin with 1-palmitic2-linoleic or 1-palmitic-2-oleic (present in human bile) in culture medium is potentially useful to trophozoites and supports the growth of G. lamblia in serum-deprived medium. It is likely that PC and other exogenous phospholipids are used directly as an energy source, as well as for interesterification of cellular phospholipids. The importance of an ongoing deacylation/reacylation cycle of membrane phospholipids in mammalian cells has recently been demonstrated by Balsinde et al. (1995) and by Balsinde and Dennis (1997). In this cycle, a preexisting phospholipid is cleaved by an intracellular PLA2 to generate a 2-lysophospholipid, which in turn generates a new phospholipid (Lands and Crawford 1976). Therefore, we asked whether, in Giardia, PLA2 is also involved in esterification reactions. To investigate this, we have used aristolochic acid, an inhibitor of calcium ionophore-stimulated PLA2 activity in human neutrophils (Rosenthal et al. 1989). Results show that this inhibitor was able to inhibit the incorporation of sn2 fatty acids from PC to PG (Fig. 7), indicating that giardial PLA2 is involved in hydrolyzing the sn2 fatty acid from [3H]PC. A similar observation was also reported by Balsinde et al. (1995), who found that a Ca21-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids, which could be inhibited by bromo-enol-lactone (an inhibitor of Ca21-independent PLA2 activity). Next, we asked whether synthetic analogs of PG were effective against trophozoites. DMPG, DPPG, and DSPG were found to be toxic, inhibiting growth (LD50 5 ,200 nM) of trophozoites in vitro (Fig. 8). Interestingly, POPG was not a potent inhibitor, suggesting that the type and chain length of fatty acids in PG analogs are important for these effects. Although, at present, we are unable to explain the

mechanism of inhibitor action, it is possible that PG analogs bind (competitively or noncompetitively) to phosphatidylglycerolphosphate (PGP) synthase, one of the major enzymes of the PG biosynthetic pathways (Ohtsuka et al. 1993), as well as other enzymes that are involved in phospholipid remodeling and/or de novo synthesis. Alternatively, fatty acids released from PG analogs by phospholipases may also be responsible for these toxic effects. However, it remains to be seen whether G. lamblia expresses PGP synthase or other enzymes of lipid metabolic pathways. While PG is a major phospholipid in bacterial membranes (Yeagle 1993), in higher eukaryotes its dimer, i.e., cardiolipin, is only found associated with mitochondrial membranes (Schlame et al. 1993). Thus, the finding of PG in Giardia, an amitochondriate eukaryote (Hashimoto et al. 1994), raises an interesting evolutionary question. Does the PG in Giardia derive from a common ancestor between bacteria and this early diverging eukaryote (Sogin et al. 1989) or is it a remnant of an early mitochondrion that has long since been lost by this obligate parasite (Roger et al. 1998)? Investigating the synthesis and function of PG in Giardia trophozoites, therefore, may be helpful to answer these questions. This will also allow us to understand whether G. lamblia has some ability to carry out de novo phospholipid synthesis or whether it is dependent entirely on a remodeling process that chemically alters phospholipids obtained by this parasite from its host.

ACKNOWLEDGMENTS

This work was supported by Grants AI 136597 and GM 08012 from the National Institutes of Health. We are grateful to Dr. J. Moore (Avanti Polar lipids Inc., Alabaster, AL) for performing HPLC analysis of phospholipids and Laura Dader (UTEP) for technical support. DIC and confocal microscopy experiments were carried out at the Analytical Cytology and Confocal Microscopy Core Facilities at the University of Texas at El Paso, funded by RR 08124 (NIH) and MRI-NSF grants. Ms. C. Castillo was supported by the MARC (NIH/GM 08048-13) program.

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