Uptake and Interconversion of Fluorescent Lipid Analogs in the Protozoan Parasite, Perkinsus marinus, of the Oyster, Crassostrea virginica

Experimental Parasitology 95, 240–251 (2000) doi:10.1006/expr.2000.4533, available online at http://www.idealibrary.com on

Uptake and Interconversion of Fluorescent Lipid Analogs in the Protozoan Parasite, Perkinsus marinus, of the Oyster, Crassostrea virginica

F.-L. E. Chu, P. Soudant, A. K. Volety,1 and Y. Huang2 Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, U.S.A.

Chu, F.-L. E., Soudant, P., Volety, A. K., and Huang, Y. 2000. Uptake and interconversion of fluorescent lipid analogs in the protozoan parasite, Perkinsus marinus, of the oyster, Crassostrea virginica. Experimental Parasitology 95, 240–251. Uptake, distribution, and interconversion of fluorescent lipid analogs (phosphatidylcholine, PC; cholesteryl ester, CHE; phosphatidylethanolamine, PE; palmitic acid, C16; sphingomyelin, SM) by the two life stages, meront and prezoosporangium, of the oyster protozoan parasite, Perkinsus marinus, were investigated. Class composition of these two life stages and lipid contents in meront cells were also examined. Both meronts and prezoosporangia incorporated and modified fluorescent lipids from the medium, but their metabolic modes differ to some extent. Results revealed that among the tested analogs, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM). HPLC analysis of meront lipids showed that while the majority of the incorporated PC, CHE, and PE remained as parent compounds, most of the incorporated C16 was in triacylglycerol (TAG) and SM was in ceramide and free fatty acids. The cellular distribution of fluorescent labels varied with lipid analogs and the extent of their metabolism by the parasite. Fluorescence distribution was primarily in cytoplasmic lipid droplets of both life stages after 24 h incubation with PC. After 24 h incubation with SM, fluorescence appeared in the membrane and cytosol. Total lipid contents in meront cultures increased during proliferation and TAG accounted for most of the increased total lipids. Since total lipid content per meront cell did not increase until the day of culture termination, the lipid increase in the meront culture was mainly a result of increased cell numbers. Both life stages contain relatively high levels of phospholipids, 53.8% in 8-day-old meronts and 39.4% in prezoosporangia. PC was the predominant phospholipid. q 2000 Academic Press

Index Descriptors and Abbreviations: Lipid incorporation; lipid metabolism; chromatography; parasitic protozoan; Perkinsus marinus; oyster; Crassostrea virginica; FTM, fluid thioglycollate medium; DMEM, Dulbecco’s modified Eagle’s medium; YRW, York River water; FL SM, 8(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine; FL PC, 2-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; FL PE, 2-(4,4-difluoro-5,7diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine; FL C16; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid; FL CHE, cholesteryl-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene3-dodecanoate; CHE, cholesteryl ester; FFA, free fatty acids; TAG, triacylglycerol, DAG, diacylglycerol; MAG, monoacylglycerol; CHO, cholesterol; FFH, free fatty alcohol; CER, ceramide; CL, cardiolipin; PG, phosphatidyl glycerol; SM, sphingomyelin; PI, phosphatidyl inositol; PS, phosphatidylserine; PC, phosphatidylcholine; LPC, lyso-PC; PE, phosphotidylethanolamine; DMSO, dimethylsulfoxylate; TLC/ FID, thin-layer chromatography/flame ionization detector; HPTLC, high-performance TLC; GLC, gas liquid chromatography; FAME, fatty acid methyl ester; PL, phospholipid; CAEP, ceramide aminoethyl phosphonate.

INTRODUCTION

The success or failure of a parasite in establishing infection in the host depends upon its ability to evade the host defense and acquire the necessary nutrients for development and proliferation within the host. It has been established that host lipids play a vital role for long-term survival and lifecycle completion of endogenous parasites (Vial et al.

1

Present address: Florida Gulf Coast University,10501 FGCU Blvd, Fort Myers, FL 33965-6565. 2 Present address: IIT Research Institute, 6000 Executive Boulevard, Rockville, MD 20852.

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0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

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1982a,b, 1984; Furlong 1991). Advancements have been made in understanding the processes of lipid uptake and metabolism in several mammalian parasites: Plasmodium sp., a pathogen from the phylum Apicomplexa which causes malaria (see review by Vial and Ancelin 1992, 1998); Schistosoma mansoni, a parasite which causes schistosomiasis in humans (Smith et al. 1970; Meyer et al. 1970; Furlong et al. 1995; Brouwers et al. 1997, 1998; Redman et al. 1997); Trypanosoma brucei, a tsetse-trasmitted pathogenic protozoan, which lives in the blood and body fluids of the mammalian host (Coppens et al. 1995); and Giardia lamblia, an intestinal protozoan which causes both epidemic and endemic diarrhea (Lujan et al. 1996; Ellis et al. 1996). These parasites are unable to synthesize cholesterol and fatty acids de novo (Jarroll et al. 1981; Vial and Ancelin 1998). To support growth and differentiation, they require exogenous sources of essential lipids. Incorporation of fatty acids, cholesterol, phospholipids, or lysophospholipids from the host has been reported in these parasites (Lujan et al. 1996; Vial and Ancelin 1998; Brouwers et al. 1997; Redman et al. 1997). They can, however, modify host lipids to some extent (Ellis et al. 1996; Brouwers et al. 1997; Redman et al. 1997; Vial and Ancelin 1998). The protistan, Perkinsus marinus (Dermo), which parasitizes American (eastern) oysters, (Crassostrea virginica) is an apicomplexan in the class Perkinsasida (Levine 1988). It was originally described by Mackin et al. as Dermocystidium marinum (Mackin et al. 1950). This parasite infects eastern oyster populations along the east and Gulf coasts of the United States and has caused severe oyster mortality from the mid-Atlantic to the Gulf since the 1950s. Presently, P. marinus is the most prevalent parasite of the eastern oyster in mid-Atlantic waters. The disease caused by P. marinus is infectious (see review by Chu 1996). Four life stages, meront (trophozoite), prezoosporangium, zoosporangium, and biflagellate zoospore have been identified and described (Perkins 1966; Perkins 1988). Immature meronts (merozoites) usually found in the phagosomes of oyster hemocytes are 2–4 mm and coccoid. Meronts (10–20 mm) are mature merozoites with an eccentric vacuole which often contains a refringent vacuoplast. The mature meront (schizont, 20 to 40 mm) contains 8 to 32 cells. Prezoosporangia, developed from meronts, are sometimes observed in moribund and dead oyster tissues and can enlarge to 150 mm. When tissue associated merozoites/meronts are placed in fluid thioglycollate medium (FTM) for 4 to 5 days, they also develops into prezoosporangia (hypnospores). Prezoosporangia are characterized by having a large vacuole and an eccentric nucleus adjacent to the cell wall. Zoosporulation (production of biflagellate zoospores) usually occurs after incubating

FTM-cultured prezoosporangia in estuarine- or seawater (20–22 psu) for 4–5 days. The three life stages, meront, prezoosporangia, and biflagellate zoospore, are infective (Chu 1996). The merozoite/meront stage is the primary agent for disease transmission (Perkins 1988; Chu 1996). In P. marinus, both meront and prezoosporangium stages are characterized by an abundance of refractive bodies, which are lipid droplets. The lipid droplets are, presumably, lipid/fatty acid reservoirs serving as energy for proliferation, development, and life cycle completion of the parasite. To date, almost nothing is known about the lipid metabolism and biosynthesis of this parasite, although its host is an ecologically and economically important aquatic species. The objectives of the present study are to: (1) characterize the lipid class composition of these two life stages and lipid contents in meront cells; and (2) examine the uptake, cellular distribution, and interconversion of exogenous lipids by meronts and prezoosporangia of P. marinus using fluorescent lipid analogs.

MATERIALS AND METHODS

Growth and lipid class composition of in vitro Perkinsus marinus cell culture. Perkinsus marinus cultures were initiated by inoculation of 10 3 106 cells (meronts/merozoites) in culture flasks (n 5 18 flasks) containing 10 ml modified DMEM:HAM’s F-12 medium (Gibco BRL, Gaithersburg, MD; Gauthier and Vasta 1993) and cultured for 25 days. Lipid analysis conducted on the medium revealed a concentration of 58.9 6 6.0 mg lipid/mL of medium, containing 46.2 mg steryl esters and 13.9 mg phospholipids. Cultures were sampled (n 5 3 flasks/ sampling) at 1, 6, 8, 11, 15, and 25 days postinoculation. Numbers of P. marinus cells in each flask were counted (expressed as 106/mL) and their cell sizes measured under a microscope (Olympus, BX40). The cells were harvested via centrifugation (800g for 20 min) and the supernatant (medium) saved and stored at 2208C for later lipid analysis. Cell pellets were washed with 0.22-mm-filtered estuarine water (York River water, YRW, the water used for oyster maintenance, 18–20 psu), freeze–dried, and stored at 2208C until lipid analysis. Isolation and culture of prezoosporangia. Prezoosporangia, developed from FTM-cultivated tissue associated meront cells, were isolated based on the method described by Chu and Greene (1989). Oysters heavily infected with P. marinus were cleaned with 70% ethanol and then rinsed with 0.22 mm YRW. The cleaned infected tissues and associated meront cells were then cultured in FTM with antibiotics (0.8 mg penicillin and 0.8 mg streptomycin per mL of medium) at 25–288C for 6 days. Prezoosporangia were then isolated and purified through a series of centrifugation and washing steps using 0.22 mm filtered YRW. Purified prezoosporangia were frozen at 2208C until lipid extraction. FTM was made by dissolving 29.3 g thioglycollate powders in 1.0 L of 0.22-mm-filtered YRW. This was autoclaved before the addition of antibiotics. Lipid standards. Fluorescent labeled lipid analogs were purchased

242 from Molecular Probes (Eugene, OR, USA). They included: 8(4,4difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine (Bodipy FL C5-sphingomyelin, FL SM), 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine, (b-Bodipy FL C12-HPC, FL PC), 2-(4, 4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine, b-Bodipy FL 530/550 C12-HPE, FL PE), 4, 4-difluoro-5,7diphenyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (Bodipy FL C16, FL C16), cholesteryl-4,4-difluoro-5,7-diphenyl-4-bora-3a,4adiaza-s-indacene-3-dodecanoate (cholesteryl Bodipy FL C12, FL CHE). Nonlabeled lipid standards, cholesteryl ester (CHE), free fatty acids (FFA), triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), cholesterol (CHO), free fatty alcohol (FFH), ceramide (CER), cardiolipin (CL), phosphatidyl glycerol (PG), PI, PS, PE, PC, sphingomyelin (SM), lyso-PS (LPS), and lyso-phosphatidylcholine (LPC) were obtained from Sigma. Uptake, metabolism, and cellular distribution of fluorescent lipid analogs. To test the uptake, metabolism and cellular distribution of fluorescent lipid analogs, meronts/merozoites in the log growth phase (5–8 days old), or freshly isolated prezoosporangia were washed and resuspended in 10 mL of 0.2-mm-filtered YRW at a concentration of 20–40 3 106 cells/mL (meronts/merozoites) and 0.2–1.0 3 106 cells/ mL (prezoosporangia). Fluorescent lipid analogs (FL C16, 4 3 1024 mmol; FL CHE, 8 3 1024 mmol; FL PC, 20 3 1024 mmol; FL SM, 20 3 1024 mmol; FL PE, 60 3 1024 mmol) dissolved in dimethylsulfoxylate (DMSO) were added to meronts/merozoites or prezoosporangia and incubated in the dark at 288C for 24 h. Following incubation, parasites were washed three times with YRW to removed free (nonincorporated) lipid analogs and resuspended in 10 mL of YRW. Two hundred microliters of the cell suspension was fixed in 4% paraformaldehyde (pH 7.0, in 0.1 M phosphate buffer) for 30 min, washed with an equilibration buffer, and stored in SlowFade (Molecular Probes) at room temperature for later microscopic examination. The rest of the suspension was freeze–dried and stored at 2208C for later lipid analysis. Microscopic examination of uptake and distribution of fluorescent lipid analogs. The cellular distribution of fluorescence in the parasites was examined with an epifluorescent microscope (Olympus BH-2) and photographed with an affiliated camera (Olympus, SC35, Type 12). Lipid analysis. Total lipids were extracted from meronts/merozoites and prezoosporangia with chloroform: methanol:water (2:2:1) according to the procedure described by Bligh and Dyer (1959). For samples labeled with fluorescent lipid analogs, to prevent loss of fluorescence, all extraction steps were performed without direct light and in glassware protected from light. Total lipids from culture media were extracted by addition of proportional chloroform and methanol. Lipid contents and lipid class composition of in vitro culture meronts/ merozoites were analyzed with thin-layer chromatography coupled with flame ionization (TLC/FID) detector using an Iatroscan TH-10, MK-III analyzer (Iatron Laboratiories, Tokyo, Japan) (Chu and Ozkizilcik 1995). Briefly, after activation for 30 min at 1108C, silica gel rods were spotted, using a Hamilton syringe, with lipid samples (1–10 ml/ sample). Silica gel rods were then developed using a solvent mixture containing hexane:diethyl ether:formic acid (85:15:0.04, v/v/v). Following development, silica gel rods were analyzed in an Iatroscan analyzer. Operating conditions were 2000 mL min21 air flow, 0.73 kg cm23 hydrogen pressure, and the scan speed of 3.1 mm/s. Peak area

CHU ET AL.

determinations were performed by computer analysis (T DataScan, RSS Inc., Bemis, TN). Lipid classes were quantified using standard curves (1, 5, 10, and 20 mg) constructed for each lipid class standard. An internal standard (stearyl alcohol) was added to the meronts/merozoites and prezoosporangia samples (1 mg per 50 mg DW) during lipid extraction to assess extraction efficiency. The lipid class composition of freshly isolated prezoosporangia and meront/merozoite culture media was also analyzed. Lipid content in meronts/merozoites is expressed as mg lipid/mL of P. marinus meront culture. Lipid class composition is expressed as percentage of total lipids. To determine the extent of incorporation and interconversion of fluorescent lipid analogs by prezoosporangia and in vitro cultured meronts/merozoites, lipid class composition was analyzed using highperformance thin-layer chromatography (HPTLC) (Olsen and Henderson 1989). In short, after activation for 30 min at 1108C, the plates (HPK silica gel 60, 10 3 10 cm, Whatman Laboratory Division) were spotted, using a Hamilton syringe, with lipid samples (fluorescent labeled or nonlabeled) and analyzed with a double development system in parallel with mixtures of fluorescent lipid analogs or lipid standards containing various lipid classes. Two solvent mixtures were employed in the two-development system. The first solvent mixture consisted of methyl acetate:isopropanol:chloroform:methanol:KCl 0.25% (25:25: 25:10:9) that separated the polar lipids into phosphatidyl inositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingolipids (e.g., sphingomyelin) over a distance of 6 cm from neutral lipids. The second solvent mixture contained hexane:diethyl ether:formic acid (85:15:0.05) which differentiated neutral lipids into different classes (i.e., cholesteryl ester, wax ester, mon-, di-, triacylglycerol, free fatty acids). Following development, fluorescent bands were visualized and photographed under UV light. Lipid classes were identified by comparison with the cochromatography standards (fluorescent labeled or nonlabeled standard mixtures). Because only fluorescent-labeled CHE, C16, PE, PC, and SM were available, other lipid classes were tentatively identified by comparison to the separation of non-fluorescent-labeled lipid standards. The incorporation and bioconversion of fluorescent lipids analogs in meronts/merozoites were further analyzed with a Waters HPLC system (equipped with 717plus autosampler, 600E multisolvent delivery system, 996 photodiode array detector, and 474 scanning fluorescence detector), using a Lichrosorb diol column (5 mm; 250 3 4.6 mm i.d.) (Phenomenex, CA). Lipid classes were separated using two successive solvent gradient systems. Polar lipids and FFA were first separated at 308C with a ternary gradient system: 4 min of solvent A (hexane:isopropanol, 90:10), 6 min of a linear gradient from solvent A to solvent B (hexane:isopropanol:water, 46:52:2), 5 min of solvent B, 15 min of a linear gradient from solvent B to solvent C (hexane:isopropanol:water, 42:52:8), and 20 min of solvent C. The solvent front, containing all neutral lipids except FFA, was collected with a 4-mL test tube prior to routing to detectors. The elution order was FFA, CER, PE, CL/PG, PC, SM, PI/PS, and LPC. The column was then reactivated with solvent A for 25 min. Neutral lipids were separated at 308C with a binary gradient system: 20 min of a solvent D (hexane: isopropanol, 99.7:0.3), 10 min of a linear gradient from solvent D to solvent A (hexane:isopropanol, 90:10), 30 min of solvent A. The elution order was CHE, TAG, FFH, DAG, CHO, MAG. The column was reactivated with 100% hexane for 25 min. Fluorescent lipids were

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detected by fluorescence and nonfluorescent lipids were detected with a UV photodiode array detector. The responses of the fluorescence detector to various fluorescent lipid analogs were tested. A linear response was obtained in amounts from 0.5 pmol to 0.5 nmol for all the tested components. Peak identification was furnished by the comparison of retention times with fluorescent-labeled and nonlabeled lipid standards and each component further confirmed by the HPTLC analysis using a double development system as described previously. Results were expressed as nmol fluorescent lipid/mg meront fatty acid. The fatty acid contents of meronts/merozoites were analyzed using gas liquid chromatography (GLC). Total lipids were transesterified with 14% BF3 in methanol for 15 min at 1008C (Metcalfe and Schmitz 1961). After cooling, the fatty acid methyl esters (FAME) were extracted with carbon disulfide (CS2, Marty et al. 1992). The organic phase was evaporated and redissolved in hexane. Separation of FAME was carried out on a gas chromatograph (Varian 3300) equipped with a flame ionization detector, using a DBWAX capillary column (25 3 0.32 mm; 0.2 mm film thickness). The column was temperature programmed from 150 to 2208C at 38C min21; injector and detector temperatures were 230 and 2508C, respectively; and the flow rates of compressed air and hydrogen were 300 and 30 mL min21 respectively. Helium was used as the carrier gas (1.5 mL min21). Identification of FAMEs was based on the comparison of their retention times with those of authentic standards. Statistical analysis. Results are expressed as mean and standard deviation (SD). Differences in cell size distribution (%), total lipid and phospholipid contents in meront cells between sampling dates were analyzed using one factor ANOVA (SAS program, SAS Inc., NC, USA). The Neuman–Keul multiple comparison test was used to compare means when ANOVA was significant. Percentage data (cell size distribution) were arcsin transformed prior to analysis. Differences were considered statistically significant if P , 0.05.

RESULTS

Growth, total lipid content, and lipid class composition of in vitro culture Perkinsus marinus meronts/merozoites. Cell size and number of P. marinus meronts changed over the culture period (Figs. 1 and 2). While the number of cells $ 11 mm decreased, the number of small-size cells (1–5 mm) increased over time, significantly between day 1 and day 6 (P # 0.05). There was no significant change in cell numbers between 11 and 25 days (P . 0.05). Increases in total lipid contents in P. marinus cells paralleled increases in cell number up to 11 days (Fig. 2). Triacylglycerol accounted for most of the total lipid increase in the P. marinus cells, although it was not detected in the culture medium. This component and phospholipids increased to 25 days and 11 days postinoculation, respectively, in the P. marinus cells (Fig. 3). There was no significant change in total lipid content per cell over time (P . 0.05) except at 25 days postinoculation. The lipid content of meront cells harvested at that day had significantly higher (P # 0.05) lipid content per cell than the other days. Phospholipid per cell decreased significantly (P # 0.05) from day 1 to day 6 and remained relatively similar thereafter (Table I). Eight-day-old P. marinus meronts/merozoites consisted predominantly of phospholipids (Table II). Phospholipids (PL), TAG, CHE, and CHO accounted for 53.8, 39.0, 6.2,

FIG. 1. Size distribution of in vitro cultured P. marinus meront cells, 1–25 days postinoculation. *Significantly different from the others (P # 0.05).

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FIG. 2. Lipid contents (Mean, SD, n 5 3) and number of P. marinus meront cells in 1.0 mL of 1- to 25-day-old in vitro P. marinus cultures.

and 1.0%, respectively, of the total lipids in meronts/merozoites. No free fatty acid was detected in P. marinus meronts/ merozoites. Phosphatidylcholine was the dominant phospholipid (49.8%). The other phospholipids were PI/PS/CL (19.5%), PE (25.2%), and SM (5.6%). The prezoosporangia constituted of 39.4% phospholipids and 60.6% neutral lipids. The latter consisted of 90.2% TAG, 4.6% CHE, and 5.3% CHO. Similar to meronts, phospholipids contained predominantly PC (75.7.0%). The remainder consisted of PE (9.5%), PI/PS/CL (10.5%), and SM (4.2%) (Table II). Uptake, metabolism, and cellular distribution of fluorescent lipid analogs. Both meront/merozoite and prezoosporangia modified lipid precursors obtained from their incubation medium (estuarine water). Results from HPTLC

analysis (Fig. 4a) demonstrated that meronts/merozoites metabolized CHE to FFA and TAG; C16 to TAG, DAG, and PC; PC to PE, FFA, TAG, and CL; PE to PC, TAG, DAG, and an unknown component believed to be a glycolipid; and SM to FFA and CER. Similarly, prezoosporangia transformed CHE to FFA, DAG, and TAG and PC to TAG, FFA, and DAG (Fig. 4b). Most of the PC in prezoosporangia was converted into TAG and FFA and to DAG to a much lesser extent. PE was totally metabolized into TAG, FFA, and DAG. SM was completely converted into CER and FFA. However, only trace amounts of C16 were converted into TAG, PC, and two unknown components. HPLC analysis showed that the highest incorporation

FIG. 3. Triacylglycerol, phospholipid and cholesterol contents of meront cells in 1.0 mL of 1- to 25-day-old in vitro P. marinus cultures.

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TABLE I Total Lipid and Phospholipid Contents per Million Meront Cells, 1–25 Days Postinoculation (DPI) DPI Day Day Day Day Day Day

1 6 8 11 15 25

Total lipids (mg/106 cells; mean (SD)) 3.7 3.3 4.1 3.4 4.0 6.0

Phospholipids (mg/106 cells; mean (SD))

(1.0) (0.2) (0.1) (0.3) (0.3) (0.5)*

3.7 2.0 2.2 1.4 1.4 1.7

(1.0)* (0.1) (0.1) (0.1) (0.1) (0.1)

*Significantly different from the other values listed in the same column (P # 0.05).

(29.6 nmol/mg FA) occurred in meronts/merozoites incubated with CHE for 24 h (Table III). About 98.2% remained as CHE and less than 2% was converted to TAG (0.6%) and FFA (0.7%). The uptake of C16 by meronts/merzoites was 9.3 nmol/mg FA. In contrast to CHE, most of the incorporated C16 (87%) was found in TAG. Only 7% remained FFA. A small proportion was transformed to FFH (3.2%) and DAG (2.4%). Among polar lipid analogs, the highest uptake by meronts/merozoites was PC (about 1 nmol/mg FA). However, 69% of the incorporated PC remained as PC, 14% was incorporated into PE, 10% into TAG, and 2.8% into CL. The rest was found in FFA (3.1%), MAG (0.5%), DAG (0.1%), and FAA (0.1%). The incorporation of SM by the parasite was 0.7 nmol/mg FA; 29% stayed as SM. The majority of the incorporated SM resided in CER (50%)

TABLE II Lipid Class Composition (as Weight Percentage) of Meront Cells (8 Days Old) and Prezoosporangia Isolated from Infected Tissues Cultured in Fluid Thioglycollate Medium for 6 Days Meronts Prezoosporangia (N 5 3) (N 5 4) (Mean (SD)) (Mean (SD)) Percentage of total lipids Steryl ester Triacylglycerol Cholesterol Phospholipids Percentage of total phospholipids Phosphatidylethanolamine Phosphatidyl-serine/inositol/ Cardiolipin Phosphatidylcholine Sphingomyelin

6.2 39.0 1.0 53.8

(0.1) (3.2) (0.1) (3.1)

2.8 54.7 3.2 39.4

(0.5) (1.4) (2.9) (2.0)

25.2 (2.1)

9.5 (0.9)

19.5 (2.1) 49.8 (2.1) 5.6 (3.0)

10.5 (9.3) 75.7 (9.4) 4.2 (1.6)

and FFA (17%). Assimilation of PE by meronts/merozoites was low (0.2 nmol/mg FA); 46% remained as PE, 20.9% as TAG, 13.1% as FFA, and 6.3% as DAG. Also, some were transformed into PC (2.6%) and an unknown polar lipid (9.6%), believed to be a glycolipid. Microscopic examination revealed the incorporation of fluorescent lipid analogs by both meronts and prezoosporangia after 24 h incubation with FL PC, FL, PE FL C16, FL CHE, and FL SM. Fluorescence was present primarily in cytoplasmic lipid droplets of meronts and prezoosporangia 24 h after incubation with FL PC (Figs. 5a and 5b). Similar results were observed when the meronts and prezoosporangia were incubated with FL PE, FL CHE, or FL C16. The localization of fluorescence in oil droplets was further confirmed by staining of oil droplets with Sudan Black (an oil-soluble dye). However, after 24 h incubation with SM, fluorescence appeared to localize in the membrane and/or cytosol in both meronts and prezoosporangia (Figs. 5c and 5d). No deterioration was noted in the morphology of meronts/ merzoites due to incubation of fluorescent lipid analogs. Also, fluorescent lipid analogs did not appear to cause death in P. marinus cells.

DISCUSSION

Lipids account for about 50% of the membrane mass of most animal cells (Lehninger 1975; Stryer 1988). Phospholipids, glycolipids, and CHO are major lipids in biological membranes while the neutral lipids, TAG, constitute the most abundant energy compounds. To meet the lipid requirements during life stage transition and for reproduction, parasites exploit the host environment. The helminth parasite, S. mansoni, and the protozoan parasites, such as Plasmodium spp, G. lambolia, and T. brucei, are very capable of incorporating and modifying exogenous lipids (Ellis et al. 1996; Brouwers et al. 1997; Redman et al. 1997; Vial and Ancelin 1998). The increase of total lipid contents in P. marinus meronts/ merozoites cultures during proliferation, and the incorporation/interconversion of incubated fluorescent lipid analogs in meronts and prezoosporangia indicate that, like other parasitic eukaryotes, P. marinus actively acquires lipids from its host for membrane synthesis and also for energy reserve. Because there was no significant change in total lipid content per meront cell until 25 days postinoculation, the increased lipid contents in P. marinus cultures was mainly a result of cell number increase. Within its host, lipid supply is not

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FIG. 4. HPTLC separation of lipids from meronts (a) and prezoosporangia (b) incubated 24 h with fluorescent lipid analogs. Lane 1, PE; 2, CHE; 3, SM; 4, C16; 5, PC; 6, fluorescent lipid standards. PE, phosphatidylethanolamine; PC, phosphatidylcholine; CHE, cholesteryl ester; FFA, free fatty acid; SM, sphingomyelin; C16, palmitic acid; CER, ceramide; CL, cadiolipin; GLY, glycolippid; DAG, diacylglycerol. *Tentative identification.

FIG. 5. Incorporation of fluorescent phosphatidylcholine in meront (a, 3 3500) and prezoosporangia (b, 31400) and of fluorescent sphingomyelin in meront (c, 33500) and prezoosporangia (d, 31400). Oil droplets are indicated with arrows.

METABOLISM OF FLUORESCENT LIPID ANALOGS IN Perkinsus marinus

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TABLE III Incorporation and Conversion of Fluorescent Lipid Analogs in in Vitro Cultured P. marinus Cells (Meronts/Merozoites) after 24 Hours Incubation Fluorescent lipid analogs added to P. marinus culture (% of fluorescent lipids) PC

SM

PE

C16

CHE

Lipid classes

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

CHE TAG FFH DAG MAG FFA CERa UNK PE CLa PC PI&PS SM Total Total nmol/mg FA

— 10.5 0.1 0.1 0.5 3.1 — — 14.1 2.8 68.8 — — 100 1.0

— 1.8 0.1 0.0 0.0 2.6 — — 1.0 0.3 1.7 — — — 0.1

— — — — 2.1 17.6 49.9 — — — 1.3 — 29.0 100 0.7

— — — — 0.2 2.8 3.4 — — — 0.9 — 1.4 — 0.2

— 20.9 0.2 6.3 — 13.1 1.2 9.6 46.1 — 2.6 — — 100 0.2

— 7.4 0.2 2.0 — 0.7 0.4 3.1 12.9 — 1.0 — — — 0.1

— 86.9 3.2 2.4 — 7.0 — — — — 0.5 — — 100 9.3

— 1.4 2.0 1.5 — 2.3 — — — — 0.1 — — — 0.7

98.2 0.6 — 0.3 0.2 0.7 — 0.0 — — 0.1 — — 100 29.6

0.3 0.0 — 0.2 0.1 0.5 — 0.1 — — 0.0 — —

a

3.1

Tentative identification.

limited for P. marinus. We found that the eastern oyster plasma contained 56.9 6 2.28 mg lipid/mL of plasma, a concentration similar to the meront/merozoite culture medium (Chu et al. unpublished results). Oyster tissues are also rich in lipids. The total lipid contents in adductor muscle, visceral mass, and mantle-gills in eastern oyster were 35 6 4.91, 86.5 6 15.8, and 64.4 6 3.2 mg lipid/g dry weight (DW), respectively (Soudant and Chu, unpublished results). The sharp decrease of total phospholipids in the medium during proliferation suggests that phospholipids are essential membrane components. Rapid proliferation and increased proportion of small-size cells (Fig. 1) can probably be attributed to the decrease of phospholipids per cell through the log growth phase (day 1 to day 11). The relatively high PL level found in the prezoosporangia also suggests that PLs are important for the transformation of meronts/merozoites to prezoosporangia. Most of the prezoosporangia’s lipid probably derived from the associated oyster tissue. Our fatty acid analysis revealed that the fatty acid composition of prezoosporangia resembled that of its host, but not the culture medium, FTM (data not shown). Cholesterol is not an important membrane constituent of P. marinus, since only trace amount of it was found in P. marinus meronts/merozoites. The membrane of Plasmodium spp. is also deficient in cholesterol (Vial et al. 1990; Vial and Ancelin 1998).

Both life stages of P. marinus not only actively took up lipids from their culture medium, but also converted them to other lipid components (Figs. 4a and 4b). Based on the HPLC analysis of fluorescent-labeled lipids extracted from meronts/merozoites, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM) tested in the present study. However, the majority of assimilated CHE remained unchanged, thus suggesting that hydrolysis of CHE by the parasite is quite slow. Although the hydrolysis of CHE may be slow, the parasite metabolized it efficiently since some of the CHE present in the medium is converted to TAG and FFA in the parasite (Figs. 4a and 4b). Phospholipids are most abundant in all biological membranes. Surprisingly, the assimilation of PC, PE, and SM by the parasite was low. Nevertheless, the parasite actively converted them to other lipids. It appears that the metabolic mode differs between meront and prezoosporangia. Both meront and prezoosporangia effectively assimilated C16 from their incubation medium (Figs. 4a and 4b). However, while the meront actively incorporated C16 into TAG as energy reserves, C16 in prezoosporangia remained intact; only a trace amount was deposited in TAG. At the time of dormancy (stationary phase), accumulation of energy reserves in meronts/merozoites may be necessary for later proliferation, a process which requires not

METABOLISM OF FLUORESCENT LIPID ANALOGS IN Perkinsus marinus

only membrane constituents such as phospholipids, but energy. Thus, when free fatty acids are the only available nutrient source, depot of free fatty acids in TAG is the only choice. The limited deposition of C16 in TAG in prezoosporangia is difficult to explain. It has been documented that prezoosporangia incubated in nutrient limited medium (estuarine- or seawater, 20–22 psu) for 4–5 days develop into zoosporangia and zoosporulation (production of biflagellate zoospores) occurs (Perkins 1966; Chu and Greene 1989). Outside of the host, in an environment with limited nutrients, conservation of energy may be necessary for later development of prezoosporangia to zoosporangia. Thus, rather than spending extra energy for conversion, they metabolize free fatty acids (i.e., C16) for maintenance energy needs. The conversion of most incorporated PC and PE into FFA and TAG found in the present study (Fig. 4b, HPTLC results) also suggests that they not only actively hydrolyze simple lipids (i.e., free fatty acids) for energy, but convert complex lipids to TAG as reserve whenever possible. In meronts only part of the incorporated PC and PE was metabolized to other lipids. Interconversion between PE and PC has been reported in Plasmodium spp. (Vial and Ancelin 1998). In eukaryotes, PE and PC are synthesized via the cytidine diphospho (CDP)-base pathway using a common precursor DAG and their respective CDP-bases. This is considered to be one of the most active pathways in synthesis of phospholipids under normal conditions. Interconversion between PE and PC likely occurred in meront, but probably not in prezoosporangia. In the FL PC incubated meronts, the PE would probably come from this pathway using FL DAG originating from FL PC, although the synthetic process appeared slow and limited. Considering the low turnover of the metabolic byproduct, DAG, however, most the FL PC found in the FL PE incubated meronts was probably derived from direct methylation of the amino group of FL PE, rather than from the CDP–choline and DAG pathway (Smith 1993; Vial and Ancelin 1998). Transfer of fluorescent acyl chain from FL PC to non-FL PE or FL PE to non-FL PC may have also taken place via deacylation–reacylation reactions. This pathway involves the cleavage of the acyl chain by phospholipases. Our preliminary study has shown the presence of phospholipase A2 in P. marinus meront and the parasite contained higher level of this enzyme than its host hemocytes and plasma. The finding of CL as one of the metabolic byproducts of FL PC suggests that removal of fluorescent-labeled acyl chain from sn-2 position by phospholipase A2 and recycling of the fluorescent acyl radicals for CL synthesis using the CDP–DAG pathway may have occurred. If this is true, then the metabolic mode of P. marinus is quite

249 different from that of Plasmodium spp. which seems unable to retailor lipid molecules although phospholipases were found in this parasite (Vial and Ancelin 1998). A previous study also showed that the intestinal protozoan parasite, G. lamblia, was capable of incorporating exogenous fatty acids into PG, probably via the CDP–DAG pathway (Stevens et al. 1997). Interconversion of SM and CER has been reported in adult male S. mansoni (Redman et al. 1997). S. mansoni is able to convert BODIPY fluorescent C5-ceramide into a fluorescent SM analog and similarly to breakdown BODIPY fluorescent SM to a fluorescent CER analog (Redman et al. 1997). Although P. marinus hydrolyzes of SM to FFAs and CER, it is not certain whether P. marinus has a SM cycle similar to that of S. mansoni. SM is either absent or present in a very low concentration in marine bivalves, including oyster (Vaskovsky 1987). The ceramide-containing compound found in marine bivalves is primarily ceramide aminoethyl phosphonate (CAEP), which was found to constitute up to 16% in the total phospholipids of several bivalve species (Vaskovsky 1987). The cellular distribution of fluorescent labels varied with lipid analogs and the extent of the lipid analog metabolism in the parasite. The fluorescence distributed in the cytoplasmic oil droplets after 24 h incubation with PC, PE, FFA, or CHE was probably mostly derived from the intermediate metabolites of these lipid analogs, rather than the original compounds, except in the case of CHE. Although the highest uptake was in the case of CHE, the majority of it stayed as CHE and only a small portion was metabolized to TAG and FAA after 24 h incubation. It is believed that PC and PE were first incorporated into the membrane of the parasite. Then parts of the parent compounds and their metabolites (FFA, FFH, MAG, DAG, and TAG, etc.) were incorporated into the cytoplasmic oil droplets. However, kinetic studies coupled with microscopic examination are required to verify this hypothesis. Both HPTLC and HPLC analyses revealed the metabolism of PC and PE to various other lipid components (e.g., FFA, FFH, and acylglycerols). Apparently the oil droplets are the lipid storage house for future usage. However, SM has a different fate in the parasite: fluorescentlabeled parent compound and its metabolites appear localized in both membrane and cytosol. Results from HPLC analysis somewhat support the microscopic examination: a bulk of the incorporated SM was metabolized to CER (about 50%) and FFA. The study of uptake and cellular localization of fluorescent lipid by G. lamblia (Stevens et al. 1997) using both epifluorescent and high-resolution confocal microscopy showed that PC and SM were accumulated in the plasma membranes, whereas palmitic acid and CER were localized

250 intracellularly (perinuclear membrane) after 1 h incubation. However, after incubation of the parasite with SM or CER for a longer time (6 and 24 h), Stevens et al. (1997) observed that some of the fluorescent labels were localized in oil droplets. In summary, lipid content increased in P. marinus meront/ merozoite cultures during cell multiplication. Our results revealed incorporation and modification of lipids from exogenous sources and meronts differed to some extent from prezoosporangia. The incorporated lipids and their metabolites were distributed in either membrane, cytosol, or cytoplasmic oil droplets depending on the characteristic of the parent compounds and their metabolic by-products. However, further study is needed to verify their cellular and subcellular localization using high-resolution confocal microscopy.

CHU ET AL.

Chu, F.-L. E. 1996. Laboratory investigations of susceptibility, infectivity and transmission of Perkinsus marinus in oysters. Journal of Shellfish Research 15, 57–66 Coppens, I., Levade, T., and Courtoy, P. J. 1995. Host plasma low density lipoproteins particles as an essential source of lipids for the bloodstream forms of Trypanosoma brucei. The Journal of Biological Chemistry 270, 5736–5741. Ellis, J. E., Wyder, M. A., Jarroll, E. L., and Kaneshiro, E. S. 1996. Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia. Molecular and Biochemical Parasitology 81, 13–25. Furlong, S. T. 1991. Unique roles for lipids in Schistosoma mansoni. Parasitology Today 7, 59–62. Furlong, S. T., Thibault, K. S., Morbelli, L. M., Quinn, J. J., and Rogers, R. A. 1995. Uptake and compartmentalization of fluorescent lipid analogs in larval Schistosoma mansoni. Journal of Lipid research 36, 1–12. Gauthier, J. D., and Vasta, G. R. 1993. Continuous in vitro culture of the eastern oyster parasite Perkinsus marinus. Journal of Invertebrate Pathology 62, 321–323

ACKNOWLEDGMENTS

Jarroll, E. L., Muller, P. J., Meyer, E. A., and Morse, S. A. 1981. Lipid and carbohydrate metabolism of Giardia lamblia. Molecular and Biochemical Parasitology 2, 187–196.

This study was supported by a grant from the Metabolic Biochemistry Program, Molecular and Cellular Bioscience Division, National Science Foundation (Grant MCB9728284). The authors thank Ms. Georgeta Constantin and Mr. Lee Steider for technical assistance and Drs. Robert C. Hale and Ken Webb and two anonymous reviewers for the constructive review of the manuscript. Contribution 2301 from the Virginia Institute of Marine Sciences, College of William and Mary.

Lehninger, L. 1975. “Biochemistry: The Molecular Basis of Cell Structure and Function,” 2nd ed. Worth Publishers, New York.

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